INTRACELLULAR IL-1 RECEPTOR ANTAGONIST PROMOTER - CELL-TYPE-SPECIFIC AND INDUCIBLE REGULATORY REGIONS

The objective of these studies was to examine the molecular mechanisms involved in transcriptional regulation of the gene for the intracellu lar structural variant of the IL-1 receptor antagonist (iclL-1Ra) mole cule. By reverse transcription-PCR analysis, constitutive expression o f endogenous iclL-1Ra mRNA was observed in the epithelial cell lines A 431 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, iclL-1Ra m RNA 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 mechanis ms of transcriptional regulation, 4.5 kb of the 5' flanking sequence w as isolated from the human iclL-1Ra gene, sequenced, cloned into a luc iferase expression vector (pIC4525.Luc), and examined in transfection studies. The pIC4525.Luc construct exhibited a pattern of expression i n epithelial and macrophage cell lines similar to that of the endogeno us icIL-1Ra gene. To obtain a generalized map of cell type-specific an d inducible cis-acting DNA elements, nested 5' deletional mutants of t he icIL-1Ra promoter were constructed. Results from transfection studi es with these icIL-1Ra promoter/luciferase fusion constructs indicated that constitutive expression in epithelial cells was under the contro l of three positively acting regions located between bases -4525 to -1 438, -288 to -156, and -156 to -49. In contrast, basal expression of p IC4525.Luc in transfected but unstimulated RAW 264.7 cells was under t he control of a weak inhibitory region located between bases -4525 to -1438 and a strong positive element between -156 and -49. LPS inductio n 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, betwee n bases -156 to -49, contains positive cis-acting elements that are ne eded for expression in both epithelial and monocyte cell lines. Howeve r, our results indicate that the ability of this proximal promoter reg ion to control expression is strongly influenced, both positively and negatively, by other upstream cis-acting elements in a cell type-speci fic manner.