Intracellular IL-1 receptor antagonist promoter: cell type-specific and inducible regulatory regions.

Division of Rheumatology, Department of Medicine, University of Colorado Health Sciences Center, Denver 80262, USA.
The Journal of Immunology (Impact Factor: 5.36). 02/1997; 158(2):748-55.
Source: PubMed

ABSTRACT 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.

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    01/2002: pages 169-188; Academic Press., ISBN: 9780120781416
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    ABSTRACT: IL-1R antagonist (IL-1Ra) exists in two well-characterized forms, 17-kDa secretory IL-1Ra (sIL-1Ra) and 18-kDa intracellular IL-1Ra (icIL-1Ra), that arise by alternative transcription of the same IL-1Ra gene. A third, lower molecular mass form (;16 kDa) was detected by immunoblot within lysates of a variety of cells, including human monocytes and myelomonocytic cell lines. The 16-kDa isoform was designated icIL-1RaII, and the previously established 18-kDa form was designated icIL-1RaI. Intracellular IL-1RaII bound type I IL-1R up to fivefold less avidly than did sIL-1Ra and icIL-1RaI. Microsequencing of cyanogen bromide fragments of purified icIL-1RaII provided evidence consistent with initiation of protein translation at the second start site in either IL-1Ra mRNA. The results of site-directed mutation experiments established that icIL-1RaII could be derived by alternative translation initiation. In vitro transcription and translation of intact sIL-1Ra cDNA in rabbit reticulocyte lysates led to both pro-sIL-1Ra and icIL-1RaII proteins, whereas transcription and translation of icIL-1RaI cDNA produced both icIL-1RaI and icIL-1RaII proteins. Mutation of the first 5* ATG in sIL-1Ra cDNA led to translation of only icIL-1RaII, while only sIL-1Ra was observed after mutation of the second ATG. These results indicate that icIL-1RaII is a third member of the IL-1Ra family and is a 16-kDa, 143-amino acid intracellular protein derived by alternative translation initiation from either sIL-1Ra mRNA or icIL-1Ra mRNA. The role in biology of either intracellular form of IL-1Ra remains unknown. The Journal of Immunology, 1998, 161: 1997-2003.
    The Journal of Immunology 08/1998; 161(4). · 5.36 Impact Factor
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