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Neuroendocrinology of Stress

Greti Aguilera, MD
  • Greti Aguilera, MD, Head, Section on Endocrine Physiology
  • Ying Liu, MD, Research Associate
  • Qiong Deng, BS, Graduate Student
  • 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 the expression of CRH and VP during stress, the mechanisms of action, topographic distribution, and 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. Our current studies focus on the molecular mechanisms leading to activation and termination of transcriptional responses of CRH, on steroidogenic proteins in the adrenal, and on glucocorticoid feedback. 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 this laboratory. Knowledge 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.

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 cyclic AMP-response element (CRE) at 270 in the CRH promoter. The 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 to drive 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. Our studies also provided evidence that activation and inactivation of TORC in the CRH neuron involve the TORC kinases SIK1 and SIK2, i.e., 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 short hairpin RNA (shRNA) revealed differential effects of both isoforms, suggesting that SIK2 is responsible for TORC sequestration in the cytoplasm in basal conditions, while induction of SIK1 probably inactivates TORC in the nucleus and limits 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.

CRH transcription and glucocorticoids

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. Studies completed during the past year 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 hnRNA in the PVN following corticosterone injection. Although this could have been 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 the microdissected hypothalamic PVN region following glucocorticoid injection. In both intact and adrenalectomized rats, ChIP assays revealed no increases in GR recruitment by the CRH promoter after an injection of corticosterone/cyclodextrin complex. 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 promoter but not by that of CRH. Consistent with the lack of GR recruitment by the CRH promoter, incubation of primary hypothalamic neuron cultures with corticosterone had no significant effect on basal CRH transcription or that stimulated by the cAMP–activating agent forskolin, measured as increases in primary transcript (CRH hnRNA). Using a reporter gene assay in the hypothalamic cell line 4B, the same treatment with corticosterone caused only minor inhibition of CRH promoter activity. In 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 showed marked translocation of GR to the nucleus following 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.

The above findings strongly suggest that negative feedback on CRH transcription involves indirect mechanisms. To test this hypothesis, studies were conducted in collaboration with Keiichi Itoi and David Klein to identify target genes for glucocorticoid action, using genome-wide analysis of the transcriptome by RNA-Seq in microdissected PVN region of rats. Glucocorticoid withdrawal by seven-day adrenalectomy resulted in significant changes in mRNA levels of 757 genes, of which 233 were upregulated (normally suppressed by glucocorticoids) and 524 downregulated (glucocorticoid dependent expression). In contrast, high glucocorticoid levels for seven days resulted in upregulation of 221 genes and downregulation of 132. Interestingly, a group of 23 genes showed more than 30% upregulation with both adrenalectomy and high glucocorticoid exposure, suggesting that the glucocorticoid-inducible genes are suppressed by basal levels of glucocorticoids. Unlike the relative small number of genes regulated by chronic changes of glucocorticoids in the PVN region, about 3,000 genes (900 were above 30% different from basal) showed significant changes following an acute injection of corticosterone providing plasma levels in the stress range. The majority of the genes showed transient induction; of 847 genes upregulated by 1h only 24 remained high by 3h. A small number of genes (63) showed more than 30% downregulation following acute injection, 47 of which decreased only by 3h. Unexpectedly, early response genes during stress such as Fos, Egr, and Nur77, increased after a single corticosterone injection and returned to values significantly below basal after 3h. CRH mRNA levels were not significantly affected by acute corticosterone injection, despite exhibiting the expected changes following adrenalectomy and high corticosterone exposure. In addition, several ion channels and neuropeptide and neurotransmitter receptors were highly regulated by glucocorticoids, suggesting that they are target genes for glucocorticoid feedback. The data reveal that several genes are targets of glucocorticoid regulation in the hypothalamic PVN area and provide a foundation for further studies on the mechanisms by which glucocorticoids regulate CRH expression in the PVN. For our current studies, we are using RNA seq ChIP seq technology to identify glucocorticoid-responsive genes in the PVN region.

The pituitary corticotroph and glucocorticoid feedback

Another important target of glucocorticoid feedback is the pituitary corticotroph, where the steroid inhibits ACTH secretion, as well as the transcription of the ACTH precursor protein, proopiomelanocortin (POMC). The rapid inhibition of ACTH secretion by glucocorticoids has suggested that the effect is mediated by non-genomic mechanisms. In addition, rapid non-genomic effects of glucocorticoids at the pituitary levels have been postulated to be part of a feedforward/feedback mechanism responsible for hourly (ultradian) rhythmicity of glucocorticoid secretion, without the need of pulsatile hypothalamic CRH secretion. Ongoing studies provide evidence that the classical GR associates with membrane fractions in a ligand-dependent manner, suggesting that rapid membrane effects of glucocorticoids are mediated by the classical GR. Perifusion experiments in trypsin-dispersed anterior pituitary cells show that, while inhibition of POMC transcription (measured as changes in primary transcript of heteronuclear RNA) parallels nuclear translocation of the GR, inhibition of ACTH secretion is associated with membrane localization of GR. Interestingly, low concentrations of glucocorticoids within the basal levels caused association of the GR to membrane proteins and mediated rapid feedback, while high glucocorticoid concentrations, in the stress range, lead to a more delayed inhibition, probably involving genomic effects. Ongoing studies will identify interactions of the GR with specific membrane proteins and characterize the involvement of rapid feedback at the pituitary level on ACTH–glucocorticoid pulse generation.

TORC and adrenal steroidogenesis

At the adrenal level, 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. Given mounting evidence of the importance of pulsatility in regulating glucocorticoid-responsive gene transcription, we continued studies to uncover mechanisms determining pulsatile secretion at the adrenal level. 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. 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 raised 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. The effect was duplicated by ACTH injection at a level that reproduced stress levels of ACTH and corticosterone. The increases in transcription of these steroidogenic proteins were preceded by activation and nuclear translocation of the CREB co-activator TORC2, supporting the involvement of the co-activator in this transcriptional initiation. However, in studies using the ACTH–responsive clone of the adrenocortical cell line Y1, suppression of TORC by siRNA had only minor effects of StAR transcription. The effects of ACTH on StAR transcription were mimicked by cyclic AMP analogs and stimulators but not by stimulation of protein kinase C by phorbolesters or of the MAP kinase pathway by epidermal growth factor (EGF). Although only cyclic AMP reproduced the effects of ACTH, inhibition of PKA had little effect on ACTH–stimulated StAR transcription. On the other hand, the combination of protein kinase A and MAP kinase inhibitors prevented StAR transcription, indicating that the full stimulatory 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, which required additional inhibition of the calcium-dependent phosphatase calcineurin, a known mediator of TORC dephosphorylation. The data show that, although cyclic AMP is essential, multiple cyclic AMP–mediated signaling pathways are required for full transcriptional activation of StAR and that the CREB co-activator TORC2 is not essential for initiation of StAR transcription.

Publications

  1. Aguilera G, Liu Y. The molecular physiology of the CRH neuron. Front Neuroendocrinol 2012;33:67-84.
  2. 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.
  3. 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;371:62–70.
  4. Grøntved L, John S, Baek S, Liu Y, Buckley JR, Vinson C, Aguilera G, Hager GL. C/EBP maintains chromatin accessibility in liver and facilitates GR recruitment to response elements. EMBO J 2013;32:1568-1583.
  5. Evans AN, Liu Y, MacGregor R, Huang V, Aguilera G. Regulation of hypothalamic corticotropin releasing hormone transcription by elevated glucocorticoids. Mol Endocrinol 2013;27:1796-1807.

Collaborators

  • 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
  • Keiichi Itoi, PhD, Tohoku University, Sendai, Japan
  • David Klein, PhD, Program on Developmental Endocrinology and Genetics, NICHD, Bethesda, MD
  • Stafford L. Lightman, MD, University of Bristol, Bristol, UK

Contact

For more information, email Greti_Aguilera@nih.gov.

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