Susheng Wang

Human 15-Lipoxygenase gene promoter: analysis and identification of DNA binding sites for IL-13-induced regulatory factors in monocytes

In order to study the transcriptional control of 15-LO expression, we have cloned and sequenced the human 15-LO promoter region. The 15-LO promoter is associated with a CpG island at the 5′-end of the gene, and sequence analysis reveals putative Sp1 and Ap2 binding site/s and absence of TATA or CAAT motifs. Transcription is initiated at one major site. Using deletion constructs, we have defined an active promoter region of 1056 bp. Gel-shift assays revealed that transcriptional factor(s) induced only in response to IL-13 treatment of human peripheral blood monocytes bind to the 15-LO promoter DNA. Two regions, DP1 (−140 to −92 bp) and DP2 (−353 to −304 bp) of the promoter were essential for transcription in HeLa cells and human peripheral monocytes. Hela nuclear extracts contained a specific nuclear factor(s) binding to 15-LO promoter DNA which are distinct from those derived from IL-13-treated human peripheral monocyte nuclear extracts. In addition, fluorescent in situ hybridization (FISH) results refined the previous localization of 15-LO to human chromosome 17p13.3.

Ku autoantigen (DNA helicase) is required for interleukins-13/-4-induction of 15-lipoxygenase-1 gene expression in human epithelial cells.

As reported previously in human monocytes, a human lung epithelial cell line, A549, showed de novo induction of 15-Lipoxygenase-1 (15-LO-1) in response to interleukins-13 (IL-13) and −4 (IL-4). In this cell line, 15-LO-1 expression, by RT-PCR and western blotting, was observed following 6 and 24 h of exposure to human IL-13 (ED50 5 ng/ml) and IL-4 (ED50 0.2 ng/ml). We have previously shown that no cis-acting regulatory elements exist within the 15-LO-1 promoter region. To define IL-13 and IL-4 responsive trans-acting elements, we identified a region (DP2: −353 to −304 bp site) within the 15-LO-1 promoter (by footprinting experiments) to which IL-13-responsive elements (or factors) bind specifically (Kelavkar et al, 1998, Mol Biol Rep 25, 173–182). To further delineate this region, we constructed (by site-directed mutagenesis) several deletion mutants in the ‘LOPB5’ region containing the 29 bp within the −353 to −304 bp of the DP2 core element. These were: DP3 (site totally deleted), DP4 (5 bp deleted at the center of the site), DP5 (8 bp at the 5′-end of the site) and DP6 (13 bp at the 3′-end of the site). Cotransfection of these deletion constructs (driving luciferase reporter genes) was associated with 90% (DP4, DP5 and DP6) or 100% (DP3) abrogation of promoter activity at 24 h. Purification of nuclear protein extracts from IL-13 and IL-4-stimulated A549 cells, using a DP2 core containing affinity column, identified a 150 kDa protein under non-denaturing conditions, and two, 70 and 85 kDa proteins under denaturing conditions. These were not detectable by Coomassie blue staining in control nuclear protein extracts. Matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) of the tryptic digests of these proteins, identified one as the 86 kDA Lupus KU autoantigen protein P86 and the second as the 70 kDa Lupus KU autoantigen protein P70. Gel shift and supershift experiments using monoclonal antibodies toward Ku antigen and its individual subunits, and utilizing DP2 and other mutant oligonucleotides with purified nuclear protein extracts from control and cytokine-treated A549 cells, confirmed our findings. Furthermore, electroporation of neutralizing anti-Ku70, Ku 80 and Ku70/80 antibodies into A549 cells totally suppressed IL-13 and IL-4-stimulated 15-LO-1 induction in these cells. Further, immunoprecipitation experiments data suggests that IL-4 and IL-13 activate Ku antigens and 15-LO-1 expression through distinct signaling events. In summary, in A549 cells, Ku antigen is induced in response to the cytokines, IL-13 and −4, and a 29 bp region within the −353 to −304 bp region of the 15-LO-1 promoter is required for its binding and subsequent induction of 15-LO-1 gene expression. The findings may provide an important link between the established dysregulated function of Ku antigen in auto-immune diseases, such as systemic lupus erythematosus and thyroiditis, and the increasingly recognized ‘anti-inflammatory’ role of 15-LO-1.

Identification and characterization of an enhancer sequence in the promoter region of human 15-lipoxygenase (15-LO) gene

Lipoxygenases are lipid-peroxidating enzymes that are implicated in the pathogenesis of a variety of inflammatory disorders 1-3, membrane remodeling 4-6 and atheroma formation 7-9. Formation of 15-(S)-hydroxyeicosatetraenoic acid [15-(S)-HETE] and lipoxin (LX) A4 in human leukocytes, mediated by 15-LO dependent catalysis of arachidonic acid, likely represents a component of endogenous pro- and anti-inflammatory influences that ultimately regulate the extent and severity of inflammatory reactions 1-4, 10.

KU lupus autoantigen is the transcription factor induced by interleukins (IL)-13 and IL-4 leading to induction of 15-lipoxygenase-1 (15-LO) in human cells

Cytokines such as Interleukin-4 (IL-4) and IL-13 mediate their pleiotropic effects on cells by binding to specific transmembrane-spanning receptors, whose activation often results in the induction of genes via the JAK-STAT (signal transducer and activator of transcription) signaling pathway, which is now known to transduce signals for other cytokines as well1-3. Interleukin (IL)-13 and -4 are cytokine products of TH2 cells, which exert similar profiles of biological activation in a variety of cell types. Like IL-4, IL-13 is a regulator of human B cell and monocyte functions4. Recently studies by Yuet al. 5 and Curielet al. 6 indicate a possible autocrine mechanism for the regulation of IL-4 gene transcription through Stat-6RE, as well as a possible mechanism for IL-13 regulation of the human IL-4 promoter. Also, several studies have reported that both IL-4 and IL-13 share common signaling events7’8 and suggests on one hand that a single cytokine can activate different combinations of Stat proteins under different physiological conditions, and on the other hand distinct cytokines can activate the same Stat protein.