Interleukin-5 (IL-5) has been implicated in the pathogenesis of asthma. Also, corticosteroids which have been a mainstay in asthma therapy, inhibit IL-5 gene expression. The molecular events involved in the activationof IL-5 gene transcription in T helper subtype 2 (Th2) cells, those that prevent its production in Th1 cells and the mechanisms that underlie inhibition of IL-5 gene expression by glucocorticoids are poorly understood. We have demonstrated that: (a) The CLE0 element and the GATA-3-binding sequence in the IL-5 promoter are both crucial for IL-5 gene transcription. (B) In a Th2 clone, antigen- and mitogen-stimulated IL-5 gene expression involves GATA, CLE0 and additional cis- response elements in the IL-5 promoter. ~ Glucocorticoids inhibit IL-5 gene transcription despite the lack of glucocorticoid response elements within about 1 kb of the promoter. This has led us to hypothesize that : 1. Activation of GATA-3 plays a major role in IL-5 gene expression in Th2 cells. 2. The transcription factor GATA-3 is a target for steroid-inhibition of IL-5 gene transcriptoin via protein interactions between the glucocorticoid receptor (GR) and GATA-3. To test this hypothesis, we will use the following ex vivo and in vivo approaches:
Aim #I : Characterize the key regulatory DNA sequences that mediate regulation of IL-5 gene expression in murine Th1 and Th2 cell clones. Dnase I hypersensitivity assays, footprinting assays, methylation studies, electrophoretic mobility shift assays (EMSAs), and transfection experiments will be performed to characterize the molecular mechanisms that permit IL-5 gene expression in Th2 cells buty not in Th1 cells. Th0 clones before and after switching to a Th1 or Th2 phenotype will be also similarly explored.
Aim #II : Determine the mechanisms by which glucocorticoids inhibit IL-5 gene expression in T cells. (a) The effect of glucocorticoids on IL-5 production by T cells will be examined at both transcriptional and post- transcriptional levels. (b) Transcriptional interference between GR and GATA-3 will be determined by EMSAs, transfection experiments and co-immunoprecipitation techniques.
Aim # III: Determine the in vivo relevance of the cis-elements defined in Aim # I including their steroid responsiveness. Transgenic mice harboring wild-type or mutated IL-5 promoter sequences driving a reporter gene will be generated. The expression of this gene will be determined in antigen and virus models of airway inflammation in the presence or absence of glucocorticoids.
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