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Neuroendocrinology of Stress
- Greti Aguilera, MD, Head, Section on Endocrine Physiology
- Ying Liu, MD, Research Associate
- Mark Olah, MD, PhD, Visiting Fellow
- Lorna I. Smith, BS, Graduate Student
The goal of the laboratory is to understand the neuroendocrine mechanisms underlying the stress response, with emphasis on the regulation of the hypothalamic pituitary adrenal (HPA) axis. Normal HPA axis activity, leading to the secretion of glucocorticoids by the adrenal gland, is essential for normal metabolic activity and for survival during challenging situations. Our previous studies defined the role of the hypothalamic peptides corticotrophin releasing hormone (CRH) and vasopressin (VP) in the regulation of pituitary ACTH and contributed to elucidating the regulation of CRH and VP expression during stress, and to understanding the 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 master regulator of HPA axis activity in acute and chronic conditions. Following CRH release, rapid but transient activation of CRH transcription is required to restore mRNA and peptide levels. Appropriate control of CRH transcription and release is essential to prevent pathology associated with chronic alterations of CRH and glucocorticoid production.
The laboratory studies the molecular mechanisms leading to activation and termination of transcriptional responses of CRH and steroidogenic proteins in the adrenal. Elucidation of the mechanisms responsible for the normal circadian and ultradian patterns of glucocorticoid secretion by the adrenal, as well as the consequences of exposure of the developing organism to altered glucocorticoids levels are important aspects of the research of the laboratory. Elucidation of the mechanisms regulating the production of stress hormones is critical for understanding the mechanisms leading to HPA axis dysregulation and for developing diagnostic, preventive, and therapeutic tools for stress-related disorders.
Mechanisms for cyclic AMP–dependent regulation of CRH transcription
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. CRE is essential for activating the CRH promoter, and DNA methylation at the internal CpG of this site reduces CREB binding to the promoter, affecting CRH expression. However, evidence from our laboratory demonstrates that phospho-CREB alone is not sufficient to drive CRH transcription. The finding led to the discovery that transcriptional activation requires the CREB co-activator TORC (transducer of regulated CREB) and its recruitment by the CRH promoter. Under resting conditions, the co-activator is found in a phosphorylated, inactive state in the cytoplasm. Its activation and nuclear translocation require protein kinase A (PKA)–mediated inhibition of protein kinases mediating TORC phosphorylation. There are three TORC isoforms encoded by different genes: TORC1, TORC2, and TORC3. Of the isoforms, TORC2 appears to be the most important, though complete blockade of CRH transcription requires knock-down of both TORC2 and TORC3.
During the past year, we obtained evidence that activation/inactivation of TORC in the CRH neuron involves the TORC kinases SIK1 and SIK2. The evidence shows marked induction of SIK1 concomitantly with the declining phase of CRH transcription and 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 revealed differential effects of both isoforms, suggestive that SIK2 is responsible for TORC sequestration in the cytoplasm under basal conditions, while induction of SIK1 probably inactivates TORC in the nucleus and limits CRH transcriptional responses. Overall, the 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, to identify signaling pathways regulating TORC/SIK activity, and to examine the importance of TORC1 and TORC3 during physiological regulation of CRH transcription in vivo.
Glucocorticoids and CRH transcription
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 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. During the past year, we examined in vitro and in vivo effects of glucocorticoids on GR binding to the CRH promoter and CRH transcription in rats. In intact rats, in situ hybridization experiments showed marked inhibition of restraint stress–induced CRH heteronuclear RNA (hnRNA) in the PVN following corticosterone injection (1mg, i.p.). Although the inhibition could be attributed to direct GR–dependent repression of the CRH gene, corticosterone injection had no effect in adrenalectomized rats. To determine whether GR interacts with the CRH promoter in the PVN, we performed chromatin immunoprecipitation (ChIP) assays in a microdissected region of the hypothalamic PVN following glucocorticoid injection. In both intact and adrenalectomized rats, ChIP 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.). In contrast, there were 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. Consistent with the lack of GR recruitment by the CRH promoter, 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). Using a reporter gene assay in the 4B hypothalamic cell line, the same treatment with corticosterone caused only minor inhibition of CRH promoter activity. Using 4B cells, we then examined the possibility that glucocorticoids inhibit CRH transcription by preventing the activation of CREB and its co-activator TORC. Western blot analyses demonstrated 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. Our current studies are using RNA seq ChIP seq technology to identify glucocorticoid-responsive genes in the PVN region.
TORC and adrenal steroidogenesis
In addition to its well recognized diurnal or circadian variations, an important characteristic of glucocorticoid secretion is its episodic nature, with rapid and transient increases during stress superimposed on a basal ultradian pattern with one secretory pulse per hour. In collaboration with Stafford Lightman, we showed that secretory pulses induced by ACTH are associated with episodes of transcription of genes encoding critical proteins for steroidogenesis. Because of mounting evidence for the importance of pulsatility in regulating glucocorticoid-responsive gene transcription, we continued to study mechanisms determining pulsatile secretion at the adrenal level.
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 hnRNA in the adrenal zona fasciculata of rats subjected to restrain stress or ACTH injection. Restraint stress increased StAR hnRNA levels to near maximal by 7 min, with levels starting to decline by 60 min, parallel to the decreases in plasma ACTH. The same effect was seen after ACTH injection (5mg, ip), a dose that reproduces 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 7 min, (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. We found an identical correlation pattern 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 proposal that the coactivator TORC participates in the transcriptional initiation of StAR and other steroidogenic proteins. In addition, the studies indicate that episodic transcription of the rate-limiting proteins is necessary to maintain a physiological pattern of glucocorticoid secretion. Ongoing studies aim to elucidate the molecular mechanisms responsible for pulse generation in the adrenal and the consequences of altered glucocorticoid secretion on glucocorticoid-dependent transcription.
- Aguilera G, Liu Y. The molecular physiology of the CRH neuron. Front Neuroendocrinol 2012;33:67-84.
- Liu Y, Poon V, Sanchez-Watts G, Watts A, Takemori H, Aguilera G. Salt inducible kinase is involved in the regulation of corticotropin releasing hormone transcription in hypothalamic neurons in rats. Endocrinology 2012;153:223-233.
- Chen J, Evans AN, Liu Y, Honda M, Saavedra J, Aguilera G. Maternal deprivation in rats is associated with corticotrophin releasing hormone (CRH) promoter hypomethylation and enhanced CRH transcriptional responses to stress. J Neuroendocrinol 2012;24:1055-1064.
- Grontved L, Bandle R, John S, Baek S, Chung H-J, Liu Y, Aguilera G, Oberholtzer C, Hager GL, Levens D. Rapid genome-scale mapping of chromatin accessibility in tissue. Genome Biol 2012;5:10-18.
- Liu Y, Smith L, Huang V, Poon V, Olah M, Spiga F, Lightman S, Aguilera G. Transcriptional control of episodic glucocorticoid secretion. Mol Cell Endocrinol 2012;E-pub ahead of print.
- Lars Grontved, PhD, Laboratory of Receptor Biology and Gene Expression, NCI, Bethesda, MD
- Gordon Hager, PhD, Laboratory of Receptor Biology and Gene Expression, NCI, Bethesda, MD
- Stafford L. Lightman, MD, University of Bristol, Bristol, UK
- Alan G. Watts, PhD, University of Southern California, Los Angeles, CA
For more information, email Greti_Aguilera@nih.gov.