The major goal of this project is to investigate regulation of genes expressed in the mammalian hypothalamus. Initial studies will focus on Corticotropin Releasing Factor, (CRF), a hypothalamic releasing hormone produced in neurosecretory cells of the paraventricular nucleus (PVH). The production of CRF is a critical first step in the synthesis of glucocorticoids, which are essential for life and an integral component of mammalian carbohydrate metabolism. Glucocorticoids also are a principal part of the response to physiologic stress such as hemorrhage, trauma, and infection. Furthermore, glucocorticoid therapy is widely used for treatment of auto-immune, inflammatory, and rheumatic diseases, often resulting in long-lasting effects on endogenous glucocorticoid expression and regulation. Production of CRF is modulated by levels of glucocorticoids, the end product of this complex multi-organ pathway, as well as exhibiting circadian and tissue specific patterns of gene expression. Two major hypotheses regarding CRF expression will be tested using existing heterologous cell lines and transgenic mice which express human CRF transgenes. The hypothesis that stress-induced transcription factors related to Fos dictate CRF expression that is resistant to glucocorticoid feedback regulation will be tested using transfection of heterologous cell lines. The hypothesis that primate-specific cis elements dictate hCRF expression in placenta will be tested by examining the placentas of transgenic mice for expression of the hCRF transgenes. At present, many techniques used to study the molecular details of cellular regulation, rely on the use of tissue culture systems. However, certain types of differentiated tissues simply do not exist in cell culture, and therefore are not available for study. Among these cell types which do not exist are many cells of neuroendocrine organs. A third goal of this project is to develop an immortal, regulated cell line expressing CRF. This will be done by targeting an oncogene to the CRF producing cells of the hypothalamus in transgenic mice, or by using somatic cell fusion. All cell lines developed will be tested for expression of CRF, neuron specific marker genes, and retention of regulated expression, particularly negative regulation by glucocorticoids. The development of a cell line that faithfully represents CRF-producing neuroendocrine cells would provide a powerful tool for a host of future studies. Data obtained testing the regulatory hypotheses in this project should contribute to a greater understanding of hypothalamic regulation and expression of CRF, including glucocorticoid regulated gene expression, tissue-specific gene expression, and other components of integrated neuroendocrine regulation.
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