The 5-Lipoxygenase Promoter Is Regulated by DNA Methylation

Abstract

5-lipoxygenase (5-LOThe enzyme 5-lipoxygenase (5-LO, arachidonate:oxygen 5-oxidoreductase, EC 1.13.11.34) 1 catalyzes the conversion of arachidonic acid to (5S)-hydroperoxy-6-trans-8,11,14-cis-eicosatetraenoic acid (5-HPETE) and further to leukotriene A 4 ((5S)-6-oxido-7,9-trans-11,14-cis-eicosatetraenoic acid) (1, 2).5-LO is expressed in a variety of immune competent cells including B-lymphocytes, granulocytes, monocytes, mast cells, and dendritic cells (3). Depending on the cell type, several cytokines have been shown to be inducers of the 5-LO pathway. In granulocytes 5-LO expression is stimulated by granulocytemacrophage colony-stimulating factor (GM-CSF) (4), whereas interleukin-3 regulates the development of the 5-LO pathway in mouse mast cells (5). In the human myeloid leukemic cell lines HL-60 and Mono Mac 6, cell differentiation by 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ) and transforming growth factor-beta (TGF␤) leads to a strong induction of the 5-lipoxygenase pathway (6, 7). In Mono Mac 6 cells, the induction of 5-LO protein expression and activity by TGF␤ and 1,25(OH) 2 D 3 was accompanied by a 64-fold up-regulation of mature 5-LO mRNA and an up to 5-fold increase in 5-LO primary transcripts (7, 8), whereas no significant induction of 5-LO transcription was found in nuclear run-off assays (9). The human 5-LO gene promoter was first characterized by Hoshiko et al. (10). Several features of the putative 5-LO promoter region (such as the lack of TATAA or CCAAT boxes and repeated (GϩC)-rich elements) are characteristic for so-called housekeeping genes. Previous data suggest that the transcription factors Egr-1 and/or Sp1 are required for basal 5-LO transcription and that they functionally interact with the 5-LO promoter and activate it via repeated response elements located between positions Ϫ212 and Ϫ88 bp, relative to the translational start site (10, 11). Interestingly, naturally occurring mutations were found in the 5-LO promoter consisting of the deletion of one or two, or the addition of one Sp1 binding site (12). These mutations only slightly alter 5-LO promoter activity in reporter gene assays but have a significant impact on the response of asthma patients to 5-LO inhibitors (13).As yet, no data are available on the mechanisms involved in the cell type-specific activation of the 5-LO promoter in response to cell differentiation signals and inflammatory stimuli. Expression of several genes with (GϩC)-rich promoters has been shown to be regulated by DNA methylation (14). Whereas promoters of (GϩC)-rich housekeeping genes are usually unmethylated, methylation of promoters of tissue-specific genes is usually linked with silencing of the respective genes.Therefore, it was of interest to study the role of DNA methylation in the regulation of 5-lipoxygenase expression. The human myeloid cell lines Mono Mac 6 and HL-60 show prominent 5-LO gene expression and 5-LO activity after differentiation by TGF␤ and 1,25(OH) 2 D 3 (6, 7), whereas U937 cells and the HL-60TB cell line, a subline of the HL-60 ce...

Transcellular biosynthesis of cysteinyl leukotrienes in vivo during mouse peritoneal inflammation

“…LT production is regulated at different levels (8), including methylation of the 5LO promoter (21) and posttranslational control of 5LO activity through phosphorylation, interaction with FLAP (11,12), and translocation induced by calcium or free AA (22). Additionally, experimental evidence suggests that cells cooperate in the synthesis of LTB 4 and LTC 4 .…”

Inhibition of class I HDACs abrogates the dominant effect of MLL-AF4 by activation of wildtype MLL

“…For the purpose of our studies, we used a promoter fragment (−778 to +53) of ALOX5 that was fused to a luciferase reporter gene. [9][10][11] In order to understand transcript initiation in more detail, we investigated the chromatin signatures at the endogenous promoter and observed that TSA treatment leads to a profound upregulation of the canonical H3K4me3 signature. Therefore, we started to investigate the function of the H3K4 histone methyltransferase MLL and its role in the control of ALOX5 transcription.…”

“…[9][10][11] Here we analyzed the TSAmediated induction of 5-LO mRNA expression in a timedependent manner in MM6 and HL-60 cells. Cells were grown in the presence or absence of TSA (330 nM) for the indicated time points (Figure 1a).…”

“…9,10 The pSG5-VDR and pSG5-RXR expression plasmids for the human VDR and RXRα, and the control plasmid p(DR3)4tk-Luc were obtained from C Carlberg (Kuopio, Finland). The pCGN-Smad3 and pCGN-Smad4 expression plasmids were obtained from XF Wang (Durham, NC, USA).…”

Oxidative Stress Stimulates the Synthesis of the Eosinophil Chemoattractant 5-Oxo-6,8,11,14-eicosatetraenoic Acid by Inflammatory Cells

“…These cells contain levels of 5-HEDH comparable with circulating neutrophils and monocytes, but do not convert either 5-oxo-ETE or 5-HETE to -oxidation products, as do neutrophils. Furthermore, they lack both 5-lipoxygenase activity (36) and the NADPH oxidase system (37), which, if present, could complicate the interpretation of the data.…”