Normal activity of the hypothalamic pituitary adrenal axis, leading to the secretion of glucocorticoids by the adrenal gland, is essential for normal metabolic activity and for survival during challenging situations. Previous studies under this project have defined the role of the hypothalamic peptides corticotrophin releasing hormone (CRH) and vasopressin (VP) in the regulation of pituitary ACTH, contributed to elucidating the regulation of the expression of CRH and VP during stress, mechanisms of action, topographic distribution, regulation and physiological role of the receptors for these peptides in the pituitary gland and in the brain. 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, rapid but transient activation of CRH transcription is required to restore mRNA and peptide levels. Termination of the response is essential to prevent pathology associated with chronic elevation of CRH and glucocorticoid production. Current work is aimed to elucidate the mechanisms controlling negative and positive transcriptional regulation of CRH, as well as the mechanisms responsible to the normal circadian and ultradian pattern of glucocorticoid secretion. Transcriptional regulation of the CRH gene depends on cyclic AMP/protein kinase A signaling and binding of phospho-CREB to a CRE at 270 in the CRH promoter. This CRE is essential for activation of the CRH promoter, and DNA methylation at the internal CpG of this site reduces CREB binding to the promoter affecting CRH expression. However, phospho-CREB alone is not sufficient for driving CRH transcription and emerging evidence indicates that transcriptional activation requires the CREB co-activator, Transducer Of Regulated CREB activity (TORC) and its recruitment by the CRH promoter. During the past year we have obtained evidence indicating that activation/inactivation of TORC in the CRH neuron involves the TORC kinases SIK1 and SIK2. This includes marked induction of SIK1 concomitantly with the declining phase of CRH transcription, and the fact that over-expression of both SIK1 and SIK2 reduces nuclear translocation of TORC and CRH transcription, while the non-selective SIK inhibitor, staurosporin stimulates CRH transcription. Selective silencing of SIK1 or SIK2 using shRNA has revealed differential effects of both isoforms, suggestive that SIK2 is responsible for TORC sequestration in the cytoplasm in basal conditions, while induction of SIK1 probably inactivates TORC in the nucleus and limit CRH transcriptional responses. The overall evidence indicates that TORC is essential for activation of CRH transcription, and suggests that regulation of the SIK/TORC system by stress-activated signaling pathways acts as a sensitive switch mechanism for rapid activation and inactivation of CRH transcription. Experiments are planned to test this hypothesis and to identify signaling pathways regulating TORC/SIK activity. While positive regulation of CRH expression is important for HPA axis responsiveness, negative feedback by adrenal glucocorticoids is also essential for preventing the deleterious effects of excessive corticotrophin releasing hormone (CRH) and glucocorticoid production. One target of glucocorticoid feedback is CRH transcription in the hypothalamic paraventricular nucleus (PVN). While studies in transfected cells suggest direct repressive actions of liganded glucocorticoid receptors (GR) on the CRH promoter, effects on the endogenous CRH gene are unclear. We examined the in vitro and in vivo effects of glucocorticoids on GR binding to the CRH promoter and CRH transcription in rats. In situ hybridization experiments showed marked inhibition of restraint stress-induced-CRH hnRNA in the PVN following corticosterone injection (1mg, i.p.). However, incubation of primary hypothalamic neuron cultures with 10 nM corticosterone (added at -30 min or -18 h) had no significant effect on basal or forskolin-stimulated CRH transcription, measured as increases in primary transcript (CRH hnRNA), and the same treatment only caused minor inhibition of CRH transcription in reporter gene assays in hypothalamic 4B cells. In both intact and adrenalectomized rats, chromatin immunoprecipitation assays revealed no increases in GR recruitment by the CRH promoter 30 min or 1 h after an injection of corticosterone/cyclodextrin complex (HBC corticosterone, 1 mg, i.p.), despite marked increases in GR binding to the promoter of Per-1, a recognized glucocorticoid-dependent gene. Similarly, increases in endogenous corticosterone during restraint were associated with increased GR recruitment by the Per-1 but not the CRH promoter. Using 4B cells, we then examined the possibility that glucocorticoids inhibit CRH transcription by preventing the activation of CREB and its co-activator, transducer of regulated CREB activity (TORC). Western blot analyses showed marked translocation of GR to the nucleus following 30 min or 18 h exposure to corticosterone, irrespective of forskolin stimulation. In contrast, corticosterone had no effect on forskolin-induced phospho-CREB levels or TORC2 translocation to the nucleus. The lack of effect of glucocorticoids on CRH transcription in vitro, in conjunction with the lack of recruitment of GR by the proximal CRH promoter, suggests that negative feedback on CRH transcription in vivo is indirect and may occur at the level of afferent inputs to the PVN. At the adrenal levels, an important characteristic of glucocorticoid secretion is its episodic nature, with rapid and transient increases during stress superimposed to a basal ultradian pattern with one secretory pulse per hour. Because of increasing evidence for the importance of pulsatility in regulating glucocorticoid-responsive gene transcription, studies have been continued to uncover mechanisms determining pulsatile secretion at the adrenal level. We have shown that secretory pulses induced by ACTH are associated with episodes of transcription of genes encoding critical proteins for steroidogenesis. Transcription of steroidogenic proteins including steroidogenic acute regulatory protein (StAR), and steroidogenic enzymes involves cyclic AMP/PKA/CREB signaling. To address the involvement of the CREB co-activator, TORC, in the transcriptional initiation of StAR, we examined the time-relationship between nuclear translocation of TORC and induction of StAR transcription, by measuring StAR heteronuclear (hn) RNA in the adrenal zona fasciculata of rats subjected to restrain stress or ACTH injection. Restraint stress increased StAR hnRNA levels to near maximal levels by 7 min, and levels started to decline by 60 min parallel to the decreases in plasma ACTH. This effect was reproduced by ACTH injection (5g, ip) reproducing stress levels of ACTH and corticosterone. For both restraint stress and ACTH injection, the increases in StAR hnRNA were preceded by increases in nuclear TORC 2 (3 min for restraint and 5 min for ACTH). Nuclear TORC2 levels were maximal from 7 to 30 min with stress and 5 min with ACTH injection, before declining to basal by 120 and 60 min, respectively. With restraint, nuclear phospho-CREB levels first increased at 7min, (maximum at 15 min) and slowly declined near basal levels by 120 min, while following ACTH injection levels were already maximal at 5min and had declined to basal by 60 min. An identical correlation pattern was found for TORC translocation to the nucleus and hnRNA levels for cytochrome P450 11A (side chain cleavage enzyme) and the melanocortin receptor 2 accessory protein, MRAP. The time relationship between nuclear translocation of TORC and changes in StAR hnRNA supports the involvement of the co-activator in the transcriptional initiation of StAR, and other steroidogenic proteins.

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