Glucocorticoids autoregulate their own glucocorticoid receptor (GR) mRNA, protein, and hormone-binding activity levels. In the mouse AtT-20 pituitary tumor cell line, the hormone coordinately regulates both GR and c-jun mRNA levels. Preliminary results indicate the involvement of primarily transcriptional components in the autoregulation of GR and c-jun mRNA. The first specific aim of this proposal is to determine the mechanism by which the GR and c-jun mRNA levels are coordinately regulated by glucocorticoid treatment. Preliminary studies in the mouse AtT-20 cell line have demonstrated an oscillatory regulation of both of these mRNAs, while studies on F9 teratocarcinoma and CEM-C7 leukemia cells have shown that both of these mRNAs are coordinately up-regulated when the cells are treated with retinoic acid and glucocorticoids, respectively. These observations will be extended in the nontumorigenic mouse L929 fibroblasts and in the human T-lymphoid cell line, CEM-C7. Confirmation of coordinate GR and c-jun gene expression will lead to experiments to determine (by nuclear run-on assays) if the transcription rates of both genes show corresponding coordinate regulation. Using an immunological analysis, the second specific aim will determine if the GR and Jun proteins form a heteromeric complex that results in transcriptional interference of AP-1 (Fos/Jun) activity. These experiments would be among the first to demonstrate in vivo cross-talk between these two intracellular signaling pathways at physiological levels of GR and Jun. The third specific aim will focus on the promoter elements in the c-jun and GR genes that are involved in the coordinate down-regulation of GR and c-jun mRNA and protein levels. The glucocorticoid response element (GRE) in the c-jun promoter will be mutated, as will the AP-1 sites in the c-jun and GR promoters. Cotransfection/ reporter gene expression experiments will then be used to determine if cross-talk, and hence coordinate regulation, has been abolished by these mutations. Intracellular Jun levels will be lowered using antisense approaches to directly determine if Jun is involved in GR gene regulation. In addition to focusing on the AP-1 sites in the promoters of these two genes, additional promoter analyses will be performed to insure that other potentially important cis-acting sequences are identified. Particular attention will be paid to the GR promoter, as no definitive functional studies have yet been performed to characterize this promoter. Important promoter elements involved in both the basal and hormone-regulated expression of the GR gene will thus be identified. These studies will reveal the molecular mechanism of basal and steroid- mediated alterations in GR and c-jun mRNA gene expression and the cross- talk that occurs between these two important intracellular signaling pathways. This could lead to novel therapeutic approaches for the treatment of steroid-hormone regulated cancers by modulating the functional glucocorticoid receptor and Jun status in the cell.
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