Transcriptional interference between nuclear receptors and activator protein-1

An alternative pathway of transcriptional regulation is the crosstalk between nuclear receptors and other transcription factors, such as NFkappaB, or activator protein-1 (AP-1). In this project, I have focused on the analysis of the mechanisms of transcriptional interference between nuclear receptors and AP-1.; First I studied regulation of AP-1 activity by steroid hormones, such as estrogen and glucocorticoids. To investigate the tissue-specific agonism of antiestrogens like tamoxifen, we compared transcriptional activation of reporter constructs containing classical estrogen response elements (EREs) as well as AP-1 response elements in the breast carcinoma cell line MCF7 and in the endometrial carcinoma cell line Ishikawa. Our results indicate that the presence of EREs in promoters is sufficient to mediate cell-specific agonism of antiestrogens. Activation of AP-1 by estradiol was not observed in stable cell lines propagating an AP-1 reporter vector. AP-1 was activated by high concentrations (micromolar) of TAM in stably transfected Ishikawa. However, these high concentrations of antiestrogens induced cell death rather than proliferation.; Using the same stably transfected Ishikawa cell lines that propagate AP-1 responsive reporter vectors, we found that glucocorticoids do not repress AP-1 activity, although we observed AP-1 repression by glucocorticoids in stably transfected Hela cells that propagate the same AP-1 reporter vector. In addition, results from cDNA arrays indicate that AP-1 target genes are not regulated by estrogen in Ishikawa cells. Collectively, these results suggest that AP-1 is not sensitive to steroid hormones in Ishikawa cells.; To study the regulation of steroid receptor activity by mitogenic signaling pathways, we developed a simple and sensitive reporter system to monitor glucocorticoid response element (GRE)-mediated transcription in response to signaling molecules. We found that MAP kinase activators, such as TPA and EGF, positively modulate GR transcriptional activity in epithelial cells. This effect is early, specific to GR, as under the same conditions we did not observe this effect on ER, and can be inhibited by ERK pathway inhibitor PD 98059 and p38 pathway inhibitor SB203580, respectively.; To analyze the mechanisms of this transcription regulation, we tested four hypotheses: (1) upregulation of GR expression by TPA. (2) upregulation of GR phosphorylation by TPA. (3) upregulation of GR coactivator phosphorylation by TPA. (4) Protein-protein interactions between GR and TPA-induced Jun or Fos leading to enhanced GR transcriptional activity. Our results obtained from Northern blot and Western blot indicate that GR mRNA and protein expression are not regulated by TPA. Our data also demonstrate that TPA has no significant effect on GR phosphorylation, and that GR phosphorylation mutant S226E and S226A did not alter GRE-mediated transcriptional activity in COS-7 and Jurkat cells.; In addition, we mapped the GR domain that is targeted by TPA. Using GR and ER chimeric receptor, GR A/B and Gal4 chimeric protein and a series of GR deletion mutants, we found that the GR DNA binding domain is crucial to mediate enhancement by TPA. GR tau1 or AB region was not essential, but the presence of a ligand binding domain was necessary to mediate TPA effects. (Abstract shortened by UMI.)...