GC-rich sequences in the 5-lipoxygenase gene promoter are required for expression in Mono Mac 6 cells, characterization of a novel Sp1 binding site

Dishart, David; Schnur, Nicole; Klan, Niko; Werz, Oliver; Steinhilber, Dieter; Samuelsson, Bengt; Rådmark, Olof

doi:10.1016/j.bbalip.2005.11.008

Cited by 22 publications

(7 citation statements)

References 41 publications

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“…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]. There are also 5 GC boxes that bind SP1 and have been shown to be important in regulating expression of ALOX5 [60] however, ablation of all GC-boxes only reduces ALOX5 promoter activity 47%, implicating other transcription factors such as hypoxia- inducible factor (HIF) in ALOX5 regulation. The oxygen-sensing molecule, HIF-1α [61] regulates the adaptive response by activating genes associated with energy metabolism, erythropoiesis, vasomotor tone, and angiogenesis [62], and recent work demonstrates that HIF-1α mediates hypoxia-induced FLAP expression in human pulmonary microvascular endothelial cells [63].…”

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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Section: Discussionmentioning

confidence: 99%

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

et al. 2014

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“…Expression of endogenous 5-LO (in Mono Mac 6 cells) was reduced by mithramycin, a drug that blocks GC-boxes. Also, a novel Sp1 binding site was found, beside an initiator-like sequence, which includes the major transcription initiation site (TIS) (10). In early studies, the proximal GC-rich part of the promoter was crucial for the expression of reporter genes.…”

Section: Gc-boxes In the 5-lo Genementioning

confidence: 99%

5-Lipoxygenase: mechanisms of regulation

Rådmark

Samuelsson

2009

Journal of Lipid Research

143

115

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5-Lipoxygenase (5-LO) catalyzes two steps in biosynthesis of leukotrienes (LTs), a group of lipid mediators of inflammation derived from arachidonic acid (AA). LT antagonists are used in treatment of asthma; more recently a potential role also in atherosclerosis has raised considerable interest. Furthermore, possible effects of 5-LO metabolites in relation to tumorigenesis have emerged. Thus, an understanding of the biochemistry of this lipoxygenase has potential implications for treatment of various diseases.-Rådmark, O., and B. Samuelsson. 5-Lipoxygenase: mechanisms of regulation. J. Lipid Res. 2009. 50: S40-S45.

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“…The proximal promoter region contains numerous consensus binding sites for several known transcription factors, including a GϩC-rich region containing 5 tandem binding motifs for the zincfinger transcription factors Sp-1 and Egr-1 that has been termed the core promoter region (21,24). However, in Mono Mac 6 monocyte-like cells, the core promoter region appears to be responsible for basal 5-LO expression, whereas functional elements for the TGF-␤ effectors smad3 and smad4, as well as vitamin D response elements located in intronic sequences and in the distal part of the 5-LO gene, are responsible for enhanced 5-LO expression following TGF-␤-and 1,25dihydroxy-vitamin D 3 -induced differentiation (25)(26)(27)(28). The ALOX5 gene is also subject to regulation by methylation of CpG motifs within the core promoter region, since myeloid HL-60TB and U-937 cell lines that do not express 5-LO possess a hypermethylated core promoter region and the methylation of 5-LO core promoter region constructs abolishes reporter gene expression (29).…”

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confidence: 98%

Novel 5‐lipoxygenase isoforms affect the biosynthesis of 5‐lipoxygenase products

et al. 2010

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5-Lipoxygenase (5-LO) is the essential enzyme for the biosynthesis of leukotrienes, important mediators of inflammation. This study investigated whether variants of 5-LO exist in human leukocytes. 5-LO mRNA isoforms that are consistent with alternative splicing were identified by RT-PCR in a cell line or cell type-specific pattern. All evaluated cells expressed mRNA containing all 14 exons of 5-LO with the expected splicing sites. Individual isoforms that retained intron 10 (α-10), lacked exon 13 (Δ-13), and lacked exons 10 and 13 (Δ-10,13) or that lacked the first 96 base pairs of exon 10 (Δ-p10) were identified. Immunoreactive bands coeluting with the cloned α-10 and Δ-13 isoforms were measured in primary neutrophils and in Raji cells. When expressed in HEK293 cells, alternative proteins were without catalytic activity. However, when coexpressed with the active full-length 5-LO, alternative isoforms significantly decreased the biosynthesis of 5-LO products by up to 44%, as assessed by reverse-phase HPLC analysis. Additionally, in stimulated neutrophils the full-length active 5-LO was detected by immunoblot in both nuclear and non-nuclear compartments, while the Δ-13 isoform was only detected in the nuclear fraction. These alternative 5-LO isoforms may represent a new mechanism for the regulation of the 5-LO pathway and lipid mediator biosynthesis.

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GC-rich sequences in the 5-lipoxygenase gene promoter are required for expression in Mono Mac 6 cells, characterization of a novel Sp1 binding site

Cited by 22 publications

References 41 publications

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

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

5-Lipoxygenase: mechanisms of regulation

Novel 5‐lipoxygenase isoforms affect the biosynthesis of 5‐lipoxygenase products

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