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. Activation of CRH transcription requires phospho-CREB as well as the CREB co-activator, Transducer Of Regulated CREB activity (TORC) and its recruitment by the CRH promoter. Activation of TORC depends on cyclic AMP/PKA inactivation of the TORC kinases, salt induced kinases (SIK) 1 and SIK2. The overall evidence suggests that regulation of the SIK/TORC system 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). However, studies using chromatin immunoprecipitation were unable to show a direct interaction of the glucocorticoid receptors and the CRH promoter suggesting that transcriptional repression of CRH by glucocorticoids is indirect, involving protein-protein interactions or/and modulation of afferent inputs to the PVN. 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. Consistent with the lack of interaction of GR with the CRH promoter, the genes undergoing downregulation following corticosterone injection did not include CRH. 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. This was confirmed by qRT-PCR and by in situ hybridization. These data uncovered glucocorticoid target genes in the hypothalamic PVN area and provides a foundation for further studies on the mechanisms by which glucocorticoids regulate CRH expression in the PVN. A major target of glucocorticoid feedback for HPA axis activity is inhibition of ACTH secretion at the pituitary corticotroph. While transcriptional repression is mediated by genomic actions of glucocorticoid receptors (GR), rapid inhibition of ACTH secretion during ultradian variations may involve non-genomic effects. This hypothesis was tested in rat anterior pituitary cells cultured in micro-carrier beads perifused with corticotrophin releasing hormone (CRH) and 30min pulses of the natural glucocorticoid, corticosterone. ACTH immunoassay in 5-min perifusion fractions revealed concentration-dependent effects of CRH, with sustained elevations with expected basal concentrations (30pM), and higher elevations but rapid desensitization with 100pM CRH concentrations. The effects of corticosterone on 30pM CRH-induced ACTH secretion depended on pulse concentrations, and previous degree of glucocorticoid exposure. Cells exposed to 20nM cortisol during culture followed by only 2 h clearance, respond to only stress levels of corticosterone (1M) with prolonged inhibition of CRH-stimulated ACTH secretion delayed by 25 min. In contrast, 6h clearance increased feedback sensitivity, with basal corticosterone levels (10nM) inhibiting CRH-stimulated ACTH secretion within 5 min, and recovering 5min after corticosterone withdrawal. The possibility that this rapid effect depends on membrane association of the classical GR was examined by Western blot. The data showed ligand-dependent rapid association of GR to membrane fractions, in a time and corticosterone concentration-dependence paralleling the inhibition of ACTH secretion. Consistent with membrane effects of glucocorticoids, corticosterone stimulated Src phosphorylation and had biphasic effects on CRH-stimulated Src phosphorylation, with early inhibition and potentiation. Src inhibitors caused marked inhibition of CRH-stimulated ACTH secretion suggesting that suppression of early CRH induced Src phosphorylation by corticosterone may contribute to rapid glucocorticoid feedback. The data supports the hypothesis that rapid glucocorticoid feedback at the pituitary corticotroph is part of the mechanism of ultradian pulse generation. The estrogen receptor associates to membrane proteins through palmitoylation of cysteine 447 and participation of two nearby leucines located in a conserved region in several steroid receptors, including the glucocorticoid receptor (GR). The role of this cysteine and surrounding region on membrane association of the glucocorticoid receptor (GR) was studied in 4B cells, expressing endogenous GR, and Cos-7 cells transfected EGFP-GR constructs. GR immunoprecipitation and autoradiography in cells preloaded with radiolabeled palmitic acid revealed no radioactivity incorporation into GR. Further, mutation C683A (corresponding to the ER palmitoylation site) did not affect corticosterone-induced membrane or nuclear GR trafficking. However, mutations L687-690A, L682A, E680G and K685G, within helix 8 of the ligand binding domain, prevented membrane and nuclear localization and 3H dexamethasone binding. The L687-690A mutation also reduced association of GR with heat shock protein 90 and transcriptional activity of the receptor. The data demonstrate that palmitoylation does not mediate membrane association of GR, but that the region 680-690 (helix 8), is critical for ligand binding and receptor function. 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). The increases in transcription are preceded by nuclear translocation of TORC 1, 2 and 3. Studies using ACTH-responsive cell lines suggest that TORC3 is important for transcriptional initiation of StAR, while TORC 2 influences the maintenance of the transcriptional response. We have also shown that StAR transcription depends on cyclic AMP, and that multiple cyclic AMP- mediated signaling pathways, including PKA and MAPK are required for full transcriptional activation of StAR.
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