The objective of these studies was to examine the molecular mechanisms involved in transcriptional regulation of the gene for the intracellular structural variant of the IL-1 receptor antagonist (icIL-1Ra) molecule. By reverse transcription-PCR analysis, constitutive expression of endogenous icIL-1Ra mRNA was observed in the epithelial cell lines A431 and HT-29, but not in the macrophage cell lines RAW 264.7 and U937, or in the lymphocyte cell lines Raji and Jurkat. However, icIL-1Ra mRNA expression was observed in response to stimulation with LPS in RAW 264.7 cells and to PMA and LPS in U937 cells. To examine the mechanisms of transcriptional regulation, 4.5 kb of the 5' flanking sequence was isolated from the human icIL-1Ra gene, sequenced, cloned into a luciferase expression vector (pIC4525.Luc), and examined in transfection studies. The pIC4525.Luc construct exhibited a pattern of expression in epithelial and macrophage cell lines similar to that of the endogenous icIL-1Ra gene. To obtain a generalized map of cell type-specific and inducible cis-acting DNA elements, nested 5' deletional mutants of the icIL-1Ra promoter were constructed. Results from transfection studies with these icIL-1Ra promoter/luciferase fusion constructs indicated that constitutive expression in epithelial cells was under the control of three positively acting regions located between bases -4525 to -1438, -288 to -156, and -156 to -49. In contrast, basal expression of pIC4525.Luc in transfected but unstimulated RAW 264.7 cells was under the control of a weak inhibitory region located between bases -4525 to -1438 and a strong positive element between -156 and -49. LPS induction of icIL-1Ra transcription in RAW 264.7 cells was regulated by strong positively acting DNA regions between bases -1438 to -909 and -156 to -49. In summary, the proximal region of the icIL-1Ra promoter, between bases -156 to -49, contains positive cis-acting elements that are needed for expression in both epithelial and monocyte cell lines. However, our results indicate that the ability of this proximal promoter region to control expression is strongly influenced, both positively and negatively, by other upstream cis-acting elements in a cell type-specific manner.
[Show abstract][Hide abstract] ABSTRACT: Asthma is an inflammatory disease of the airways, currently the most common chronic childhood disorder in industrialized countries. Although environmental factors are known to contribute to the development of the disease, epidemiological studies point towards a strong genetic influence. After a genome-wide screen in German affected sib pair families and a subsequent finemapping approach linkage with asthma was identified on chromosome 2q12-2q14. The interleukin-1 cluster on human chromosome 2q12-2q14 harbours various promising candidate genes for asthma and other inflammatory diseases. A systematic association study with SNPs (Single Nucleotide Polymorphisms) located in candidate genes was conducted. Single marker association, two locus and three locus haplotype analyses of SNPs, yielded several significant results (p<0.05 0.0021) for the human IL1RN gene. It encodes the IL-1ra protein, an anti-inflammatory cytokine playing an important role in the maintenance of the balance between inflammatory and anti-inflammatory cytokines. These results were replicated and confirmed in an independent Italian family sample where also significant though weaker association with SNPs and asthma was detected. Further analysis of 13 SNPs, which was not part of this work showed significant association with asthma and IgE (Immunglobuline E) in the IL1RN gene in single as well as in 13 Marker haplotype-analysis in the ECRHS-study (European Community Respiratory Health Survey). Resequencing of the coding region of the human IL1RN gene revealed additional DNA variants, from which a subset was also associated in the German and the Italian sample. Calculation of the linkage disequilibrium in the human IL1RN gene showed almost perfect LD (Linkage Disequilibrium) for nearly all analysed SNPs. Further haplotype analysis indicated that 6 SNPs are sufficient for tagging haplotypes with a prevalence > 1%. The most frequent haplotype constructed from these SNPs was 1,4 fold overtransmitted in the German families. The results of this study provide evidence that specific SNPs and haplotypes in the human IL1RN gene encoding the IL-1 receptor antagonist (IL-1ra) are a risk factor for the etiology of asthma. An additional re-sequencing approach to reveal undiscovered DNA variations in either of the two promoters regulating the IL-RN expression identified additional variants, from which one SNP was located in a NFkB binding site which did not show association with the disease. Finally the IL-1ra concentration was measured in all participants of the German family study and showed higher levels in patients with asthma.
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