The purpose of the research is to study the cellular and molecular mechanisms of adaptation to stress with emphasis on the regulation of the various components of the hypothalamic pituitary adrenal (HPA) axis. This includes the expression of hypothalamic corticotropin releasing hormone (CRH) and vasopressin (VP), pituitary CRH and V1b VP receptors, and adrenal steroidogenesis. At the hypothalamic level, past studies showed that CRH and VP co-expressed in the same parvocellular neuron of the paraventricular nucleus (PVN) are differentially regulated during stress or exposure to glucocorticoids. Stimulation of cAMP-dependent signaling systems is largely responsible for activation of CRH transcription during stress, and termination of this response is critical to prevent deleterious effects of prolonged stress. Studies during the past year using hypothalamic organotypic cultures showed that cAMP directly stimulates CRH and VP transcription in parvocellular neurons. During prolonged stimulation, cAMP induces the transcription repressor factor, inducible cAMP early repressor (ICER), in the PVN, and ICER inhibits cAMP-stimulated CRH promoter activity in vitro, suggesting the ICER mediates a cellular feedback mechaninsm to limit CRH transcriptional responses during prolonged stress. In the suprachiasmatic nucleus (SCN), the transcription of VP shows a circadian rhythm, which was maintained in vitro in long-term organotypic culture. This system was used to demonstrate that the VP neuron exhibits an intrinsic rhythmicity but full expression of this rhythm depends upon both ongoing neural activity as well as a functional MAP kinase activity. Cis- and trans-acting factors and signaling systems controlling the differential expression of CRH and VP are under current investigation. Important effort also was placed on the regulation of pituitary CRH and VP receptors. These studies show that transcriptional mechanisms are important to maintain high levels of receptor mRNA but that the content of CRH and VP receptors in the pituitary corticotroph depends largely on translational and post-translational mechanisms. Studies on the transcriptional regulation of the V1b VP receptor have identified a region in the proximal promoter containing a large GAGA repeat, which bind a Drosophila GAGA binding protein and a protein complex found in the pituitary. Transfection of Drosophila GAGA binding protein markedly enhance V1b receptor promoter activity as well as the expression of endogenous V1b receptor in a hypothalamic cell line, suggesting that a GAGA binding protein contributes to the regulation of V1b receptor transcription. The 5'untranslated region (5'UTR) of the V1b receptor mRNA plays an important role controlling translation of the mRNA. The presence of upstream open reading frames in the 5'UTR may play a role maintaining low translational activity in basal conditions. On the other hand, studies during the past year have identified an internal ribosome entry site (IRES) in the 5'UTR. IRES activity can be stimulated by activation of protein kinase C and PI3 kinase dependent pathways, a mechanism which may mediate stimulation of V1b receptor translation according to the physiological requirement. The importance of upstream ORF and IRES activity on the physiological control of the V1b receptor are under current investigation.
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