Studies of this laboratory have been pivotal for understanding the interaction between CRH and vasopressin (VP) in the regulation of pituitary ACTH, and the regulation of the expression of these peptides in the PVN during stress and other alterations of the HPA axis. Both peptides co-expressed in the same parvocellular neuron of the paraventricular nucleus (PVN) are differentially regulated during stress or exposure to glucocorticoids. CRH coordinates behavioral, autonomic and hormonal responses to stress and is the main regulator of ACTH secretion in acute and chronic conditions. Following CRH release, activation of CRH transcription is required to restore mRNA and peptide levels, but termination of the response is essential to prevent pathology associated with chronic elevation of CRH and glucocorticoid production. This laboratory has made important contributions on the understanding of the mechanisms controlling negative and positive transcriptional regulation of CRH. CRH transcription is under positive control by cAMP/phospho-CREB signaling and negatively regulated by glucocorticoid feedback. Research of the laboratory has has demonstrated that in addition to glucocorticoids, intracellular feedback mechanisms in the CRH neuron, involving induction of repressor forms of cAMP response element modulator (CREM) limit CRF transcriptional responses by competing with the positive regulator, phospho-CREB. Rapid repression of CRH transcription following stress-induced activation is likely to contribute to limiting the stress response and to preventing disorders associated with excessive CRH production.? ? Concerning the positive regulation, this laboratory reported during the past year solid evidence that cAMP/phospho-CREB signaling, believed to mediated activation of the CRH promoter, is essential but not sufficient to activate CRH transcription. This finding strongly suggested that transcriptional activation requires a co-activator of CREB. In a number of systems it has been shown that CREB mediated transcription potentially involved in the regulation of CRH transcription is Transducer Of Regulated CREB activity (TORC) is required for CREB mediated transcription. The ability of TORC to regulate CRH transcription was examined in the hypothalamic cell line 4B transfected with a CRH promoter driven luciferase reporter gene. Consistent with the above observations, forskolin, at threshold concentrations for cAMP production and CREB phosphorylation, induced marked CRH promoter activation, while PMA failed to activate CRH promoter in spite of its ability to phosphorylate CREB. However, in cells co-transfected with and expression vector for TORC2, PMA significantly activated CRH promoter activity compared with basal in TORC-transfected cells or PMA in cells transfected with the empty vector. TORC 2 transfection also potentiated the stimulatory effect of sub-maximal but not that of maximal stimulatory concentrations of forskolin. The involvement of endogenous TORC on cAMP-dependent activation of CRH transcription was examined using western blot and blockade of endogenous TORC production by siRNA. In basal conditions, western blot analysis revealed a 200kDa band, corresponding to the phosphorylated form of TORC2, in cytosolic but not in nuclear fractions. Incubation with forskolin resulted in transient nuclear translocation (maximal at 30min) of a lower molecular weight band corresponding to dephosphorylated TORC. In contrast, PMA slightly delayed the migration of the cytosolic band (suggesting hyperphosphhorylation) and had no effect on nuclear TORC levels, suggesting that the inability of phorbolesters to stimulate transcription is due to the lack of nuclear translocation of TORC. Transfection of TORC1 and TORC2 siRNA oligonucleotides 24h prior to incubation with forskolin abolished the immunoreactive TORC bands in the western blot and prevented activation of CRH promoter by forskolin. This study provides strong evidence that the co-activator TORC is required for activation of CRH transcription. Current research is focused on the interactions of TORC with the CRH promoter and on the importance of the co-activator TORC during physiological regulation of CRH transcription in vivo.
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