Greti Aguilera

ACTH signalling at the adrenal zona fasciculata induces rapid synthesis of glucocorticoids, accompanied by rapid transcription of genes encoding steroidogenic enzymes, including the labile, rate-limiting protein, StAR protein. ACTH induction of StAR transcription has been demonstrated to be predominantly CREB-dependent, and this mechanism is regulated by both CREB phosphorylation and its binding to the CREB co-activator TORC (transducer of regulated CREB activity, also known as CREB regulated transcription coactivator (CRTC)). Here we show that CREB phosphorylation, and nuclear localisation of the TORC2 and TORC3 isoforms follows ACTH treatment in adrenocortical Y1-BS1 and ATC7-L cells, and this precedes a rapid increase in star transcription (StAR hnRNA). Furthermore, by using chromatin immunoprecipitation we demonstrate that both isoforms bind the endogenous StAR promoter by 30 min after ACTH treatment in ATC7-L cells. In order to further establish the roles of these two isoforms during different types of HPA activity in the adrenal, we used two rat in vivo models; firstly, i.v. injection of a low dose of ACTH was used to mimic an ACTH ultradian pulse; secondly, rats were subjected to an immune stressor through an i.v. LPS challenge. Following both treatments, adrenals were harvested at various time-points and processed for RNA and protein measurements. As expected, we found rapid increases in both CREB phosphorylation and StAR hnRNA in both the i.v. pulse and LPS experiment. Interestingly, we found differential activation of the two TORC isoforms, with only significant TORC3 nuclear localisation induced by the i.v. ACTH ultradian pulse, whilst both TORC2 and TORC3 were induced by the immune stressor. These data demonstrate differing sensitivities of TORC2 and TORC3 to ACTH signalling, suggesting different roles for each in their mediation of ACTH-induced StAR transcription.

The hypothesis that rapid glucocorticoid inhibition of pituitary ACTH secretion mediates a feedforward/feedback mechanism responsible for the hourly glucocorticoid pulsatility was tested in cultured pituitary cells. Perifusion with 30 pM CRH caused sustained the elevation of ACTH secretion. Superimposed corticosterone pulses inhibited CRH-stimulated ACTH release, depending on prior glucocorticoid clearance. When CRH perifusion started after 2 hours of glucocorticoid-free medium, corticosterone levels in the stress range (1 μM) caused a delayed (25 min) and prolonged inhibition of CRH-stimulated ACTH secretion, up to 60 minutes after corticosterone withdrawal. In contrast, after 6 hours of glucocorticoid-free medium, basal corticosterone levels inhibited CRH-stimulated ACTH within 5 minutes, after rapid recovery 5 minutes after corticosterone withdrawal. The latter effect was insensitive to actinomycin D but was prevented by the glucocorticoid receptor antagonist, RU486, suggesting nongenomic effects of the classical glucocorticoid receptor. In hypothalamic-derived 4B cells, 10 nM corticosterone increased immunoreactive glucocorticoid receptor content in membrane fractions, with association and clearance rates paralleling the effects on ACTH secretion from corticotrophs. Corticosterone did not affect CRH-stimulated calcium influx, but in AtT-20 cells, it had biphasic effects on CRH-stimulated Src phosphorylation, with early inhibition and late stimulation, suggesting a role for Src phosphorylation on the rapid glucocorticoid feedback. The data suggest that the nongenomic/membrane effects of classical GR mediate rapid and reversible glucocorticoid feedback inhibition at the pituitary corticotrophs downstream of calcium influx. The sensitivity and kinetics of these effects is consistent with the hypothesis that pituitary glucocorticoid feedback is part of the mechanism for adrenocortical ultradian pulse generation.

Negative glucocorticoid feedback is essential for preventing the deleterious effects of excessive hypothalamic pituitary adrenal axis axis activation, with an important target being CRH transcription in the hypothalamic paraventricular nucleus. The aim of these studies was to determine whether glucocorticoids repress CRH transcription directly in CRH neurons, by examining glucocorticoid effects on glucocorticoid receptor (GR)–CRH promoter interaction and the activation of proteins required for CRH transcription. Immunoprecipitation of hypothalamic chromatin from intact or adrenalectomized rats subjected to either stress or corticosterone injections showed minor association of the proximal CRH promoter with the GR compared with that with phospho-CREB (pCREB). In contrast, the Period-1 (Per1, a glucocorticoid-responsive gene) promoter markedly recruited GR. Stress increased pCREB recruitment by the CRH but not the Per1 promoter, irrespective of circulating glucocorticoids. In vitro, corticosterone pretreatment (30 minutes or 18 hours) only slightly inhibited basal and forskolin-stimulated CRH heteronuclear RNA in primary hypothalamic neuronal cultures and CRH promoter activity in hypothalamic 4B cells. In 4B cells, 30 minutes or 18 hours of corticosterone exposure had no effect on forskolin-induced nuclear accumulation of the recognized CRH transcriptional regulators, pCREB and transducer of regulated CREB activity 2. The data show that inhibition of CRH transcription by physiological glucocorticoids in vitro is minor and that direct interaction of GR with DNA in the proximal CRH promoter may not be a major mechanism of CRH gene repression. Although GR interaction with distal promoter elements may have a role, the data suggest that transcriptional repression of CRH by glucocorticoids involves protein-protein interactions and/or modulation of afferent inputs to the hypothalamic paraventricular nucleus.

Exposure to stress during early development causes long‐lasting alterations in behaviour and hypothalamic pituitary adrenal (HPA) axis activity, including increased expression of corticotrophin‐releasing hormone (CRH). To determine whether early‐life stress causes epigenetic changes in the CRH promoter leading to increased CRH transcription, 8‐week old female and male rats, subjected to maternal deprivation (MD) between days 2 and 13 post‐birth, were studied for HPA axis responses to stress and CRH promoter methylation in the hypothalamic paraventricular nucleus (PVN) and central nucleus of the amygdala (CeA). Plasma corticosterone and PVN CRH heteronuclear (hn)RNA responses to acute restraint stress were higher in MD rats of both sexes. DNA methylation analysis of the CRH promoter revealed a significantly lower percentage of methylation in two CpGs preceding (CpG1) and inside (CpG2) the cyclic AMP‐response element (CRE) at −230 bp in the CRH promoter in the PVN but not the CeA of MD rats. Gel‐shift assays, using nuclear proteins from forskolin‐treated hypothalamic 4B cells and CRH promoter CRE oligonucleotides, unmethylated or methylated at CpG1, revealed a strong band that was supershifted by phospho‐cAMP response element‐binding antibody. This band was 50% weaker using oligonucleotides methylated at CpG2 (intra‐CRE), or methylated at both CpG1 and CpG2. These findings demonstrate that HPA axis hypersensitivity caused by neonatal stress causes long‐lasting enhanced CRH transcriptional activity in the PVN of both sexes. Hypomethylation of the CRH promoter CRE, a region critical for CRH transcriptional activation, could serve as a mechanism for the increased transcriptional responses to stress observed in MD rats.

Activation of CRH transcription requires phosphorylation of cAMP response element-binding protein (CREB) and translocation of the CREB coactivator, transducer of regulated CREB activity (TORC) from cytoplasm to nucleus. In basal conditions, transcription is low because TORC remains in the cytoplasm, inactivated by phosphorylation through Ser/Thr protein kinases of the AMP-dependent protein kinases (AMPK) family, including salt-inducible kinase (SIK). To determine which kinase is responsible for TORC phosphorylation in CRH neurons, we measured SIK1 and SIK2 mRNA in the hypothalamic paraventricular nucleus of rats by in situ hybridization. In basal conditions, low mRNA levels of the two kinases were found in the dorsomedial paraventricular nucleus, consistent with location in CRH neurons. One hour of restraint stress increased SIK1 mRNA levels, whereas SIK2 mRNA showed only minor increases. In 4B hypothalamic neurons, or primary cultures, SIK1 mRNA (but not SIK2 mRNA) was inducible by the cAMP stimulator, forskolin. Overexpression of either SIK1 or SIK2 in 4B cells reduced nuclear TORC2 levels (Western blot) and inhibited forskolin-stimulated CRH transcription (luciferase assay). Conversely, the nonselective SIK inhibitor, staurosporine, increased nuclear TORC2 content and stimulated CRH transcription in 4Bcells and primary neuronal cultures (heteronuclear RNA). Unexpectedly, in 4B cells specific short hairpin RNA knockdown of endogenous SIK2 but not SIK1 induced nuclear translocation of TORC2 and CRH transcription, suggesting that SIK2 mediates TORC inactivation in basal conditions, whereas induction of SIK1 limits transcriptional activation. The study provides evidence that SIK represses CRH transcription by inactivating TORC, providing a potential mechanism for rapid on/off control of CRH transcription.

Adrenocorticotropic hormone (ACTH) stimulates the secretion of glucocorticoids by acting upon melanocortin type 2 receptors (MC2R) located in the adrenal zona fasciculata. However, translocation of MC2R from the endoplasmic reticulum to its active state in the cell membrane requires its interaction with the melanocortin receptor associated protein (MRAP). To determine whether adrenocortical activation during stress involves regulation of MRAP, we examined the effects of restraint stress and ACTH injection on HPA axis activity, MC2R mRNA, and the levels of MRAP primary transcript (hnRNA) and mRNA. Blood levels of ACTH and corticosterone showed peaks at 15 min and 1 hour restraint, respectively, and both return to basal levels by 2 hours. Real time PCR analysis of MC2R mRNA levels showed a progressive decrease up to 2h, and returned near basal levels by 4h after restraint. Conversely, there were rapid increases in MRAP hnRNA and mRNA levels, with peaks at 15 min and 1h, respectively. Similar changes in MC2R mRNA and MRAP hnRNA and mRNA levels were observed after a single injection of ACTH (4ng) in methylprednisolone-suppressed rats, suggesting that the effects of stress are mediated by ACTH. Studies in collagenase dispersed rats adrenal fasciculata cells revealed a dose dependent effect of ACTH on MRAP hnRNA levels with an ED50 of about 0.1nM. Using 10nM ACTH, the maximal stimulation was at 30 min and levels remained elevated up to 2h. The PKA inhibitor, H89, prevented the stimulatory effect of all ACTH concentrations on MRAP hnRNA while the MAP kinase inhibitor, UO126 inhibited only the effect of low concentrations of ACTH. The data show rapid changes in MRAP transcription during stress or ACTH exposure. This effect is mediate by ACTH in a cyclic AMP and PKA-dependent manner. The rapid and transient increases in MRAP transcription during adrenal fasciculata activation suggest that regulation of MRAP levels plays an important role controlling ACTH receptor activity in the adrenals. Sources of Research Support: Intramural Research program, NICHD. Nothing to Disclose: YL, AGC, VH, FS, SL, GA

The activity of the hypothalamic-pituitary-adrenal axis is characterized by an ultradian pulsatile pattern of glucocorticoid secretion. Despite increasing evidence for the importance of pulsatility in regulating glucocorticoid-responsive gene transcription, little is known about the mechanism underlying the pulsatility of glucocorticoid synthesis and release. We tested the hypothesis that pulsatile ACTH release is critical for optimal adrenocortical function. Hypothalamic-pituitary-adrenal activity was suppressed by oral methylprednisolone, and ACTH (4 ng/h) was infused for 24h either as a constant infusion or in 5-min pulses at hourly intervals. Control methylprednisolone-treated rats had very low plasma corticosterone (CORT) levels with undetectable pulses and also had steroidogenic acute regulatory protein (StAR) and cytochrome P450 side-chain cleavage (P450scc) heteronuclear RNA levels reduced to approximately 50% of that seen in untreated animals. Pulsatile but not constant ACTH infusion restored pulsatile CORT secretion, and this was accompanied by parallel rises in StAR and P450scc heteronuclear RNA levels during the rising phase of the CORT pulse, which then fell during the falling phase. The pulsatile pattern of StAR and P450scc was paralleled by pulsatile transcription of the melanocortin 2 receptor accessory protein. Pulsatile ACTH activation of the adrenal cortex not only is critical for the secretion of CORT but also induces episodic transcription of the rate-limiting enzymes necessary for physiological steroidogenic responses. Because constant infusion of identical amounts of ACTH did not activate CORT secretion, pulsatility of ACTH provides a more effective signaling system for the activation of adrenocortical activity.