The goal of this program is to elucidate and characterize the cellular and molecular mechanisms responsible for the CRH mediated activation of POMC gene expression in the anterior pituitary corticotroph. This proposal is supported by our and others previous work showing the Importance of calcium as well as cAMP in mediating CRH signal transduction and our recent Identification of novel cis-acting elements in the POMC promoter (-700/+63) responding to CRH. These studies will be performed in the well established AtT20 tumor cell system. The AtT20 cell line has been utilized extensively for investigating POMC gene expression and Is Ideal for the proposed biochemical experiments.
The first aim concerns elucidating the exact roles of calcium and cAMP In mediating CRH-stimulated signal transduction resulting in transcriptional activation in the nucleus. Intracellular calcium will be quantitated by microfluorescence measurements and compared to levels of POMC transcription measured by primary transcript solution hybridization/nuclease protection and run-on transcription assays. Two basic questions will be addressed. a) Is basal intracellular Ca++ sufficient of is an elevation required for CRH/cAMP stimulated POMC and c-fos gene expression? b) What is the mechanism of Cd++ inhibition of basal and CRH activated POMC transcription and is c-fos involved? The second aim will characterize in detail the major cis-acting elements (promoter region 234/-133) responsible for mediating CRH activation as well as the transacting factors regulated by the signal transduction events investigated in the first part of this program. Four issues will be addressed. a) Determination of the basal and CRH induced factors (c-fos?) which interact with the major (-234/-133) CRH regulatory POMC promoter element. b) Functionally dissect the -234/133 region with specific promoter mutants to determine the relative contribution between basal and regulated cis-elements in mediating the CRH transcriptional response In a heterologous TK/CAT promoter/reporter system. c) Fully delineate the transcriptional complex consisting of the -171/-160 element and the protein(s) that bind to it for their role in regulating CRH effects. d) Do mutations in the -234/-133 promoter element which cause major functional changes (aim 2b) have similar effects in the context of the entire homologous POMC promoter? In addition to furthering our understanding of the regulation of this important neuroendocrine gene, these studies will also enhance our knowledge about the molecular and cellular mechanisms by which genes are regulated by intracellular calcium ion, an issue that Is currently poorly understood.
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