Glucocorticoids (GC) produced by the adrenal cortex mediate the body's adaptive response to stress. The synthesis and secretion of these steroids is regulated by the hypothalamic-anterior pituitary system. In response to stressful stimuli, the hypothalamus is induced to secrete corticotropin releasing hormone (CRH). CRH is transported via the blood to the anterior pituitary where it stimulates the secretion of the neurohormone ACTH, which then stimulates the adrenal cortex to synthesize and secrete GC. Feedback inhibition by GC on both the anterior pituitary and the hypothalamus completes the regulatory loop. CRH influences feeding and sexual behavior in rats, and hypersecretion of CRH is thought to be involved in depression. Knowledge of the regulation of CRH gene expression will provide the groundwork for understanding both normal and abnormal responses to stress. The first step will be to establish the basis for tissue-specific activation of the CRH gene in the paraventricular nucleus of the hypothalamus and other localized regions of the brain. Transgenic mice bearing the rat CRH gene and varied amounts of flanking DNA will be constructed and tissues analyzed by in situ hybridization in order to locate the cis-acting DNA sequences necessary and sufficient for appropriate expression of the transgene in adult mice. Transcription of the CRH gene is high late in gestation but drops immediately after birth in the stress nonresponsive period. The ability of transgenes to recapitulate this modulation of CRH gene expression during development will be monitored. The regulation of CRH gene expression will be further studied in transgenic mice following adrenalectomy, to identify the DNA sequences critical for feedback inhibition by GC. Immortalized cell lines producing gene products characteristic of differentiated hypothalamic cells would be invaluable for studying CRH gene regulation. To achieve this, tissue-specific elements from the CRH gene will be engineered onto a gene encoding an oncogene (SV40 T-antigen) as well as a selectable marker (neomycin) and the hybrid genes used to produce transgenic mice. Immortalized CRH producing cell lines will be derived from these animals using the selective agent. CRH transgenic mouse studies will shed light on the intricacies of cell-specific expression in the brain and hypothalamus, develop cell lines necessary to study regulation of gene expression, and thus provide the basis for understanding clinically important disorders such as depression.
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