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Abstract
The role of eosinophilia in allergic disorders indicates hIL-5 as a potential target for therapy. The conservation of hIL-5 gene proximal elements suggests they are important in controlling expression. Corticosteroids are important in the treatment of allergy, and are powerful inhibitors of IL-5 expression. This study aimed at understanding the role of hIL-5 conserved proximal elements, and elucidating the target of corticosteroid activity, in hIL-5 gene expression. Methods used include transient transfection of PBMC and PER117 cells with hIL-5 deletion constructs, EMSA, Western Blotting, and RT-PCR.
The conserved proximal CLE0/TATA elements driving a reporter gene gave similar or higher expression than a 500 bp promoter in primary human T cells and a T-cell line. Two and three copies of IL-5 CLE0 upstream of the silent IL-4 minimal promoter gave 30–45 fold increases in expression in forward orientation, but little activity in reverse orientation. Consequently, CLE0 is a powerful activator but not a classical enhancer. Deletion analysis identified CLE0 as the key element in the inhibition of IL-5 reporter constructs by dexamethasone, and RT-PCR analysis indicated that GILZ expression correlated with dexamethasone-induced inhibition of IL-5. Ectopic expression of GILZ, confirmed by western blotting, gave a 90% inhibition of promoter constructs in absence of dexamethasone. CLE0 is a powerful activator sufficient for the inducible expression of IL-5, and functions when moved upstream in a heterologous promoter. CLE0 is also the main target for IL-5 inhibition by dexamethasone, and we present evidence consistent with a role of GILZ in this.
| Abbreviations | ||
| cAMP | = | Cyclic Adenosine-3′, 5′-Monophosphate |
| CLE0 | = | Conserved Lymphokine Element 0 |
| Dex | = | Dexamethasone |
| GAPDH | = | Glyceraldehyde-3-phosphate dehydrogenase |
| GC | = | Glucocorticoid |
| GILZ | = | Glucocorticoid-Induced Leucine Zipper |
| GRE | = | Glucocorticoid Response Element |
| h | = | Human |
| His | = | Histamine |
| Ig | = | Immunoglobulin |
| NFAT | = | Nuclear Factor of Activated T cells |
| PBMC | = | Peripheral Blood Mononuclear Cells |
| PMA | = | Phorbol-12-Myristate-13-Acetate |
| PRE | = | Palindromic Regulatory Element |
| w/o | = | without |
| Abbreviations | ||
| cAMP | = | Cyclic Adenosine-3′, 5′-Monophosphate |
| CLE0 | = | Conserved Lymphokine Element 0 |
| Dex | = | Dexamethasone |
| GAPDH | = | Glyceraldehyde-3-phosphate dehydrogenase |
| GC | = | Glucocorticoid |
| GILZ | = | Glucocorticoid-Induced Leucine Zipper |
| GRE | = | Glucocorticoid Response Element |
| h | = | Human |
| His | = | Histamine |
| Ig | = | Immunoglobulin |
| NFAT | = | Nuclear Factor of Activated T cells |
| PBMC | = | Peripheral Blood Mononuclear Cells |
| PMA | = | Phorbol-12-Myristate-13-Acetate |
| PRE | = | Palindromic Regulatory Element |
| w/o | = | without |