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, and contributed to understanding the regulation of CRH and VP expression during stress. Studies under this project also led to the characterization of the receptors for these peptides in the pituitary gland and in the brain, the identification of their topographic distribution and mechanisms of regulation and action of these receptors. Limitation of the stress response is essential to prevent pathology associated with chronic elevation of CRH and glucocorticoid production. Current work is aimed to elucidate the molecular mechanisms controlling activation and inactivation of HPA axis activity, as well as the mechanisms responsible for normal episodic patterns of glucocorticoid secretion. Regulation of CRH transcription depends on cyclic AMP/protein kinase A (PKA) signaling and binding of phospho-CREB to a cyclic AMP response element (CRE) at 270 in the CRH promoter. This CRE is essential for activation of the CRH promoter, and epigenetic 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 requires the CREB co-activator, Transducer Of Regulated CREB activity (TORC) and its recruitment by the CRH promoter. We have also provided evidence that activation/inactivation of TORC in the CRH neuron involves the TORC kinases salt induced kinases (SIK) 1 and SIK2. 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. While positive regulation of CRH expression is important for HPA axis responsiveness, negative feedback by adrenal glucocorticoids is also essential for preventing 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). To elucidate the unanswered question of whether glucocorticoid receptors (GR) directly interact with the proximal CRH promoter, we examined the effects of glucocorticoids on GR recruitment by the hypothalamic CRH promoter using chromatin immunoprecipitation. In contrast to the marked GR recruitment by promoters of glucocorticoid dependent genes, such as Per 1, stress or corticosterone injection had no significant effect on GR binding to the proximal CRH promoter. Stress increased pCREB recruitment by the CRH promoter, irrespective of circulating glucocorticoids. In the hypothalamic cell line 4B, glucocorticoids had no effect on forskolin-induced nuclear accumulation of phospho-CREB or TORC2. These data suggest that transcriptional repression of CRH by glucocorticoids involves protein-protein interactions or/and modulation of afferent inputs to the PVN rather than direct interaction with the proximal promoter. In order to identify target genes for glucocorticoid feedback in the PVN region, in collaboration with Drs Keiichi Itoi (Sendai University, Japan) and David Klein, NICHD, we conducted genome wide analysis of the transcriptome by RNA-Seq in microdissected PVN region of rats subjected to changes in circulating glucocorticoids. Chronic glucocorticoid withdrawal (7-days adrenalectomy) or elevation (s.c. corticosterone pellets) induced upregulation or downregulation of a relative small number of genes compared with the large number (near 3,000) significantly induced one hour after an acute corticosterone injection increasing plasma levels in the stress range. A small number of genes were downregulated following acute injection. Unexpectedly, early response genes during stress such as Fos, Egr, and Nur77, increased after 1 corticosterone injection and returned to values significant below basal after 3h. CRH mRNA levels increased after acute corticosterone injection, despite exhibiting the expected changes following adrenalectomy and high corticosterone exposure. In addition, a number of ion channels, neuropeptide and neurotransmitter receptors were highly regulated by glucocorticoids suggesting that they are target genes for glucocorticoid feedback. These data uncover a number of genes as target of glucocorticoid regulation in the hypothalamic PVN area and provides a foundation for further studies on the mechanisms by which glucocorticoids regulate CRH expression in the PVN. Another important target of glucocorticoid feedback is the pituitary corticotroph where the steroid inhibits ACTH secretion and proopiomelanocortin (POMC) transcription. The rapid inhibition of ACTH secretion by glucocorticoids has suggested the involvement of non-genomic mechanisms. Rapid non-genomic effects of glucocorticoids at the pituitary level have been postulated to drive a pituitary-adrenal feedforward/feedback mechanism responsible for hourly (ultradian) rhythmicity of glucocorticoid secretion. Ongoing studies provide evidence that the classical glucocorticoid receptor (GR) could mediate rapid effects through a ligand-dependent association to membrane fractions. Perifusion experiments in trypsin-dispersed anterior pituitary cells shows that while inhibition of POMC transcription parallels nuclear translocation of the GR, inhibition of ACTH secretion is associated with membrane localization of GR. On the other hand, the fact that only high glucocorticoid concentrations, in the stress range, were able to inhibit ACTH secretion suggests that glucocorticoid feedback at the pituitary levels is not responsible for ultradian pulse generation. Current efforts in this area are aimed to identify the target proteins of GR in the plasma membrane. At the adrenal level, 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 including steroidogenic acute regulatory protein (StAR), side chain cleavage enzyme and melanocortin receptor associated protein (MRAP). Although a number of transcription factors have been implicated, the precise mechanisms regulating StAR transcription are unclear. We showed that increases in transcription are preceded by nuclear translocation of TORC, supporting role for the CREB co-activator in transcriptional initiation. However, studies using the ACTH-responsive clone of the adrenocortical cell line Y1, showed that suppression of TORC by siRNA had only minor effects of StAR transcription. Cyclic AMP analogs but not stimulation of protein kinase C by phorbol esters, or MAP kinase pathway by EGF mimicked ACTH stimulation of StAR transcription. However, simultaneous inhibition of both protein kinase A and MAP kinase were necessary to block the effect of ACTH, indicating that the effect of ACTH on StAR transcription requires cAMP-dependent activation of both PKA and MAPK pathways. Interestingly, the combination of PKA and MAP kinase inhibitors had no effect on TORC translocation to the nucleus. These data show that although cyclic AMP is essential, multiple cyclic AMP- mediated signaling pathways are required for full transcriptional activation of StAR. In addition, it appears that the CREB co-activator TORC is not essential for initiation of StAR transcription.

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