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

Abstract

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 (ED 50 5 ng/ml) and IL-4 (ED 50 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 5Ј-end of the site) and DP6 (13 bp at the 3Ј-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. Genes and Immunity (2000) 1, 237-250.

Polyunsaturated Fatty Acid Metabolizing 15-Lipoxygenase-1 (15-LO-1) Expression in Normal and Tumorigenic Human Bladder Tissues

“…Indeed, the gene expression of 15-LO-1, considered an anti-inflammatory molecule, is known to be regulated by several proinflammatory molecules. 27,28 Consequently, as bladder disease progresses, 15-LO-1 activity is no longer required and thus its expression is concomitantly reduced.…”

Enhanced Binding of Poly(ADP-ribose)polymerase-1 and Ku80/70 to the ITGA2 Promoter via an Extended Cytosine-Adenosine Repeat

“…This mechanism of poly ADP-ribosylation has been shown to contribute to various cellular processes, including DNA repair [18], transcriptional regulation [19], [20], [21], and cell cycle progression [21]. Ku80 and Ku70 form the heterodimeric DNA binding complex Ku80/70 that associates with PARP-1 and, among other functions, contributes to the regulation of gene transcription [22], [23], [24], [25], [26].…”

Identification of poly(ADP-ribose)polymerase-1 and Ku70/Ku80 as transcriptional regulators of S100A9 gene expression

“…Besides its vital role in DNA repair, reports have implicated Ku in certain other cellular processes, including telomere maintenance, antigen receptor gene arrangements, cell cycle regulation, regulation of heat shock-induced responses, and specific gene transcription and apoptosis. For example, it has been shown that Ku is required for interleukin-13/-4-induction of 15-lipoxygenase-1 gene expression in human epithelial cells [28]. Here, Ku is induced in response to IL-13 and IL-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.…”