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DNA Helicase) Is Required for Interleukins-13/-4-Induction of 15-Lipoxygenase-1 Gene Expression in Human Epithelial Cells

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DNA Helicase) Is Required for Interleukins-13/-4-Induction of 15-Lipoxygenase-1 Gene Expression in Human Epithelial Cells Genes and Immunity (2000) 1, 237–250 2000 Macmillan Publishers Ltd All rights reserved 1466-4879/00 $15.00 www.nature.com/gene Ku autoantigen (DNA helicase) is required for interleukins-13/-4-induction of 15-Lipoxygenase-1 gene expression in human epithelial cells UP Kelavkar, S Wang and KF Badr Center for Glomerulonephritis, Renal Division, Emory University and the Atlanta Veterans Affairs Medical Center, 1639 Pierce Drive, 3304 WMB, Atlanta, GA 30322, USA 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 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. Keywords: 15-Lipoxygenase-1; interleukin-13; interleukin-4; human epithelial cells Introduction to the elucidation of the JAK-STAT (signal transducer and activator of transcription) signaling pathway, which Cytokines mediate their pleiotropic effects on cells by is now known to transduce signals for other cytokines as binding to specific transmembrane-spanning receptors, well.1–3 Interleukin (IL)-13 and −4 are cytokine products whose activation often results in the induction of new of TH2 cells which exert similar profiles of biological acti- ␥ genes. Characterization of the ability of IFN- and vation in a variety of cell types. Like IL-4, IL-13 is a regu- interleukin-4 (IL-4) to rapidly induce new genes has led lator of human B cell and monocyte functions.4 Recently Yu et al5 demonstrated that IL-13 induces distinct STAT6- DNA binding complexes and tyrosine phosphorylation Correspondence: UP Kelavkar, Center for Glomerulonephritis, Renal of STAT6 and Janus kinase 3 (JAK3) in NK and T cells. Division, Emory University and the Atlanta Veterans Affairs Medical Curiel et al6 have identified a Stat-6-responsive element Center, 1639 Pierce Drive, 3304 WMB, Atlanta, GA 30322, USA. (Stat-6RE) in the promoter of the human IL-4 gene, as Ȱ E-mail: kelavkar emory.edu well as two specific IL-4 responsive DNA-protein com- This work was supported in part by National Institutes of Health plexes in nuclear extracts of both human Th1 and Th2 (NIH) grant No. 2R01DK43883 (to K.F.B). We thank M. Ushio-Fukai for help in electroporation experiments. clones. Their results indicate a possible autocrine mech- Received 30 July 1999; revised 12 November 1999; accepted 19 Nov- anism for the regulation of IL-4 gene transcription ember 1999 through Stat-6RE, as well as a possible mechanism for IL- Ku autoantigen and 15-Lipoxygenase-1 induction UP Kelavkar et al 238 13 regulation of the human IL-4 promoter. Several studies phosphorylate several nuclear DNA-binding regulatory have reported that both IL-4 and IL-13 share common transcription factor proteins (eg, Sp1 and p53), which signaling events, such as those observed in human colon suggests that DNA-PKcs may play a role in regulating carcinoma cell lines HT-29 and WiDr.7,8 These studies transcription, replication, recombination as well as DNA demonstrate that a single cytokine can activate different repair.17,18 We have also shown by site-directed combinations of Stat proteins under different physiologi- mutagenesis (creating mutant DP2 plasmids), gel-shift, cal conditions, and also indicate mechanisms by which and transfection experiments, that the entire 29 bp region distinct cytokines can activate the same Stat protein. within the −353 to −304 bp (+1 is adenine in the ATG start IL-4 was the only cytokine known to induce 15-Lipoxy- codon) region of the 15-LO-1 promoter is required for the genase-1 (15-LO-1) in human monocytes/ macro- binding of Ku-autoantigen 70/80 and that this DNA phages.9,10 We demonstrated that IL-13 also induces 15- binding protein/factor is responsible for cytokine- LO-1 mRNA and protein synthesis in those cells leading induced upregulation of 15-LO-1 in A549 cells. to enhanced production of 15-(S)-HETE.11 These effects were inhibited by IFN-␥, as was seen in IL-4-induced 15- LO-1 expression.10 Similar results have been observed by Results Brinkmann et al.12 in a human epithelial cell line, A549 in which induction of 15-LO-1 by IL-13 and IL-4 was dem- Purification and identification of the 15-LO-1 onstrated. promoter core binding proteins in human A549 cells Arachidonate 15-Lipoxygenase-1 (arachidonate:oxygen We previously identified a protein/s binding region 15-oxidoreductase, EC 1.13.11.33) (15-LO-1) is implicated (DP2) of the 15-LO-1 promoter by footprinting and gel in oxidizing arachidonic acid and low-density lipopro- retardation studies in HeLa cells and human mono- tein, reactions of potential relevance to inflammation, cytes.16 In order to purify and identify these IL-13 and membrane remodeling and atherosclerosis.13 Recently, -4 responsive elements in human A549 cells, we prepared we have demonstrated regulation of 15-LO-1 gene proteins from whole-cell extracts of cells grown in control expression by the mutant form of p53 tumor suppressor and experimental conditions and examined the extracts protein.14 Formation of 15-(S)-hydroxyeicosatetraenoic for protein binding to the DP2 core element. The proteins acid (15-(S)-HETE) and lipoxin (LX) A4 in human leuko- were purified by affinity chromatography using biotin cytes, mediated by 15-LO-1 dependent catalysis of arachi- labeled double-stranded oligonucleotides immobilized donic acid, likely represents a component of endogenous on streptavidin-coated Dynabeads (DP2-DNA affinity anti-inflammatory influences that ultimately regulate the chromatography). extent and severity of inflammatory reaction.15 In order Briefly, proteins from whole-cell extracts of A549 to study the transcriptional control of 15-LO-1 expression, (control and experimentally grown) were prepared from we have previously cloned and sequenced the human 15- cells, added to an eppendorf tube containing biotin lab- LO-1 promoter region.16 We have also shown that there eled double-stranded oligonucleotides immobilized on are no STAT binding DNA sequences in this promoter, streptavidin-coated Dynabeads and then eluted as and that no cis-acting elements could account for the described in the scheme of Figure 1b. Proteins specifically upregulation of 15-LO-1 expression.13 recognizing the DP2 core sequence were finally purified We therefore sought to isolate the transcription from the crude proteins from whole-cell extracts. The factor/s that is/are induced in response to IL-13 and 4 individually purified proteins were then analyzed by gel respectively, and which lead to 15-LO-1 expression in electrophoresis, followed by either Coomassie blue stain- human cells. The usefulness of freshly obtained human ing and/or Western blotting. By SDS-gel electrophoresis monocytes for addressing mechanistic questions of 15- (denaturing), two bands due to 70 and 85 kDa proteins LO-1 expression at the molecular level is limited by the were detectable, but none at 150 kDa (Figure 1c, lanes 4 biological variability of these cells, and their relative and 5).
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