The goal of the research in this proposal (Phase I) is to determine the involvement of the glucocorticoid sensitivity of the proopiomelanocortin (POMC) gene promoter in diseases associated with overproduction of adrenocorticotropin hormone (ACTH). This will be accomplished by analyzing the POMC promoter usage and function in normal human pituitary and ectopic POMC-expressing tumors. Specific attention will be focused on the role of upstream transcription start sites in these tissues and their potential regulation by glucocorticoids. Hybrid constructs of the human promoter containing 767 bases of the 5' flank spanning into the middle of exon 1 will be fused to the beta-globin reporter gene and then transfected into various cell lines (including AtT20 cells, CV-1 cells, and ectopic tumor cell lines) in order to study promoter usage and hormonal sensitivity. Later experiments will then concentrate on POMC promoter usage (transcription start sites) and their glucocorticoid sensitivity in pituitary and ectopic POMC-expressing tumors. Human pituitary and ectopic tissue will be obtained at surgery and then either extracted for nuclear and cytoplasmic RNA, or cultured for short periods of time in media with and without Dexamethasone. Primary POMC transcripts will then be characterized using a 5' flank/exon 1/intron A human POMC gene probe. These studies will both characterize the promoter start sites and quantitate their usage and glucocorticoid sensitivity. It is anticipated that these studies will establish the molecular mechanisms by which glucocorticoids modulate (or fail to modulate) transcription from upstream start sites in the human POMC gene, and will shed insight to the pathophysiology of ACTH overproduction in humans.
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