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Neuroendocrinology of Stress

Greti Aguilera, MD
  • Greti Aguilera, MD, Head, Section on Endocrine Physiology
  • Ying Liu, MD, Research Associate
  • Andrew Evans, PhD, Postdoctoral Fellow

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. Not only during early development but also during adult life, the ability of the organism to adapt to acute and chronic stress situations is determined by genetic constitution and life experiences. The organism's degree of adaptability may lead to long-term consequences for the responsiveness of the HPA axis, with altered expression of hypothalamic corticotrophin releasing hormone (CRH) and circulating levels of glucocorticoids—hormones implicated in the pathogenesis of several psychiatric and metabolic disorders. Our laboratory studies the mechanisms of positive and negative regulation of expression of the hypothalamic hormones CRH and vasopressin (VP) and their receptors under different stress situations and the impact of such stress situations on HPA axis regulation. The influence of life experiences, especially during early development, on neuroendocrine regulation and the expression of genes involved in the stress response are important aspects of our research program. Elucidation of the effects of life experiences on the stress response and its regulation is critical for understanding the mechanisms leading to HPA axis dysregulation and for developing diagnostic, preventive, and therapeutic tools for stress-related disorders.

Neuroprotective actions of VP

VP that is secreted within the brain modulates neuronal function by acting as a neurotransmitter. Studies in past years have shown that expression of VP in parvocellular neurons of the hypothalamic paraventricular nucleus increases markedly during prolonged stress, but that VP plays a relatively minor role in the regulation of the HPA axis under chronic conditions. Interestingly, VP was found to mediate trophic effects in the pituitary and to mediate the increases in pituitary corticotrophs observed following adrenalectomy.

Additional studies from this laboratory using the neuronal cell line H32, which expresses endogenous V1 VP receptors, showed that activation of endogenous V1 VP receptors prevents serum deprivation–induced apoptosis. The mechanism by which VP prevents apoptosis involves phosphorylation-inactivation of the pro-apoptotic protein Bad, with consequent decreases in cytosolic cytochrome c and reduction in caspase-3 activation. These actions of VP are mediated by multiple pathways; while PKCalpha and PKCbeta activation, together with extracellular signal-regulated kinase (ERK)/mitogen–activated protein kinase (MAPK) activation, Bad phosphorylation (inactivation), the full protective action of VP requires additional activation of PKB (PI3K/Akt) pathway. The studies in the neuronal cell line suggest that VP has neuroprotective actions in the brain. The hypothesis was tested in primary cultures of hippocampal neurons subjected to high doses of glutamate or removal of the hormonal/growth factor supplement B27 from the incubation medium. The manipulations decreased neuronal viability and increased Tdt-mediated dUTP nick-end labeling (TUNEL) staining and caspase-3 activity, which is consistent with apoptotic cell death. VP significantly inhibited cell death induced by B27 deprivation or glutamate. The anti-apoptotic effects of VP were completely blocked by a V1 (but not a V2) receptor antagonist, indicating that they are mediated via V1 VP receptors. As in the neuronal cell line H32, the anti-apoptotic effect of VP in neurons involves activation of MAPK/ERK and inositol trisphosphate/protein kinase B (Akt) signaling pathways. This was shown by the transient increases in phospho-ERK and phospho-Akt after incubation with VP, revealed by Western blot analyses, and the ability of specific inhibitors to reduce the inhibitory effect of VP on caspase-3 activity and TUNEL staining by 70% and 35%, respectively. The studies demonstrate that VP has anti-apoptotic actions in hippocampal neurons, an effect that is mediated by the MAPK/ERK and phosphatidylinositol-3 kinase/Akt signaling pathways. The ability of VP to reduce growth factor deprivation or glutamate overstimulation–induced neuronal death supports a role for VP as a neuroprotective agent within the brain.

CRH transcription requires the CREB co-activator TORC.

Studies in this laboratory demonstrating that cAMP/phospho–CREB signaling is essential but not sufficient to activate CRH transcription suggested that transcriptional activation requires a co-activator. Studies in other CREB-regulated genes, such as those encoding glyconeogenic enzymes and steroidogenic proteins such as the steroidogenic acute regulatory (StAR) protein and the side chain cleavage enzyme, showed that initiation of transcription requires nuclear translocation of the CREB co-activator called transducer of regulated CREB activity (TORC). Three subtypes of TORC have been identified: TORC1, TORC2, and TORC3. In basal conditions, TORC is inactive, and in its phosphorylated state remains in the cytoplasm bound to the scaffolding protein 14-3-3. Activation of the cyclic AMP/PKA pathway inhibits the kinases responsible for TORC phosphorylation—AMP dependent kinase (AMPK) and salt inducible kinase (SIK)—thus allowing dephosphorylation and nuclear translocation. Studies using the hypothalamic cell line 4B provided strong evidence that TORC is involved in the regulation of CRH transcription and that, of the three TORC subtypes, TORC2 appears to be the most important. The principal findings in the cell line were 1) a good correlation between TORC2 translocation to the nucleus and activation of the CRH promoter in reporter gene assays; 2) that TORC over-expression (by transfection of expression vectors for TORC) increased basal CRH promoter activity and potentiated the stimulatory effect of forskolin in reporter gene assays; 3) that simultaneous silencing-RNA knockout of TORC2 and TORC3 was sufficient for complete inhibition; and 4) association of CREB and TORC in the nucleus and recruitment of TORC2 by the CRH promoter, following a 30-minute incubation with forskolin, as revealed by co-immunoprecipitation and chromatin immunoprecipitation experiments. The data demonstrate that, acting as a CREB co-activator, TORC2 is required for transcriptional activation of the CRH promoter in the hypothalamic cell line 4B.

We studied the physiological relevance of these findings in primary cultures of hypothalamic neurons in vitro and in hypothalamic tissue from control and stressed rats. In situ hybridization and immunohistochemical studies revealed the presence of all three TORC subtypes in the PVN, with TORC 2 the most abundant. Immunohistochemical studies using TORC2 antibody in rat hypothalamic tissue, performed in collaboration with Valery Grinevich, revealed specific staining in the parvocellular and magnocellular areas of the PVN. Staining was mostly cytoplasmic in controls but, after 30 min restraint stress, localized to the nucleus in a large proportion of neurons in the parvocellular region of the PVN but not in the magnocellular region, which contain VP and oxytocin neurons. Nuclear staining declined to basal levels by 4 h, corresponding with the decline in CRH transcription. Double fluorescent immunostaining and confocal microscopy showed that, in controls, 100% of CRH-immunoreactive cells also stained for TORC2 in the cytoplasm, while after a 30 min stress 60% of CRH neurons showed nuclear localization of TORC2. Chromatin immunoprecipitation studies showed that 30 min of restraint caused TORC2 to become associated with the CRH promoter. Moreover, decreasing TORC2 expression, using silencing RNA in primary cultures of hypothalamic neurons, significantly reduced cyclic AMP–stimulated CRH transcription. The time relationship between activation of CRH transcription and nuclear translocation and association of TORC2 with the CRH promoter supports the involvement of this TORC subtype in CRH regulation. Our current research focuses on the importance of the co-activator TORC during physiological regulation of CRH transcription in vivo and on the role of AMPK and SIK in the mechanisms of activation/inactivation or TORC2 during stress.

TORC and adrenal steroidogenesis

In the adrenal gland, side chain cleavage cytochrome P450 (P450scc) and the availability of active StAR are rate-limiting for steroidogenesis. Transcription their genes depends on CREB activity, and studies in transfected cell lines suggest the involvement of SIK and the CREB co-activator TORC2. In collaboration with Francesca Spiga and Stafford Lightman, we investigated the relationship between TORC activation and ACTH-induced steroidogenesis in vivo by examining the time-course of the effect of ACTH injection on the transcriptional activity of StAR and P450scc genes and nuclear accumulation of TORC2 in rat adrenal cortex. ACTH produced rapid and transient increases in plasma corticosterone, with maximal responses between 5-15 min, which returned to near basal values at 30 min. Fifteen min following ACTH, StAR and P450scc hnRNA levels increased and decreased towards basal levels by 30 min. These increases were preceded by increases in nuclear levels of TORC2, with maximal increase by 5 min, decreasing to basal levels at 30 min. The decline of nuclear TORC2 was paralleled by increases in SIK hnRNA and mRNA by 15 and 30 min after injection, respectively. The early rises in plasma corticosterone preceding StAR and P450scc gene transcription suggest that post-transcriptional and post-translational changes in StAR protein mediate the early steroidogenic responses. Furthermore, the direct temporal relationship between nuclear accumulation of TORC2 and the increase in transcription of steroidogenic proteins implicates TORC2 in the physiological regulation of steroidogenesis in the adrenal cortex. The delayed induction of SIK suggests that increases in SIK activity contribute to the declining phase of steroidogenesis. Our current research focuses on the mechanisms of regulating SIK and TORC activity and the potential role of the SIK/TORC system to mediate pulsatile glucocorticoid secretion by the adrenal.

Early life stress and CRH transcription

During the past year, we placed considerable emphasis on the long-term consequences of early life stress on the function of the HPA axis. It is well recognized that stress exposure during early development causes long-lasting alterations in behavior and HPA axis activity, including increased levels of CRH mRNA in the PVN. The aim of this study was to test the hypothesis that early life stress causes epigenetic changes in the CRH promoter leading to increased CRH transcription. We evaluated ACTH and corticosterone as well as CRH primary transcript or hnRNA levels (as an index of CRH transcription) in groups of 8-week old female and male rats that had been subjected to maternal deprivation (MD) between days 2 and 10 after birth, with or without 30 or 60 min restraint stress. Groups of control and MD rats were also used for methylation analysis of the CRH promoter in the PVN and amygdala. Adrenal weight, basal levels of plasma corticosterone, and hypothalamic CRH hnRNA were elevated in MD females but not in males. However, plasma corticosterone and CRH hnRNA responses to acute restraint stress were elevated in MD animals of both sexes. DNA methylation analysis of the CRH promoter revealed, in both sexes, a lower percent of methylation specifically in two CpGs located immediately preceding (CpG1) and inside (CpG2) the cAMP-responsive element (CRE) at −230, in the PVN and amygdala. This CRE has been shown to be an absolute requirement for activation of the CRH promoter. In contrast to the PVN, the percentage of methylation of CpG1 and CpG2 in the amygdala was identical in control and in rats subjected to maternal deprivation. These findings demonstrate that HPA axis hypersensitivity caused by neonatal stress results in long-lasting enhanced CRH transcriptional activity in the PVN of both sexes.

In gel-shift assays, we examined the molecular consequences of CRE methylation on CREB binding, using CRH-promoter CRE DNA oligos, unmethylated or methylated either at CpG1(Met-C1) or CpG2 (Met-C2), or both Met-C1 and Met-2. When we incubated labeled oligos with nuclear protein extracts from hypothalamic 4B cells and resolved them on an acrylimide gel, we observed a strong band corresponding to bound phosphorylated CREB (p-CREB) for both the unmethylated and Met-C1 oligos. We verified the identity of this shifted band by supershift following incubation with a p-CREB antibody. Methylation of the intra-CRE CpG Met-C2 significantly decreased pCREB binding (by 50%), and a similar decrease in binding was observed for the Met-1,2 oligo. These results demonstrate that the methylation state of the intra CRE CpG of the CRH promoter significantly affects transcription factor binding. Hypomethylation of the −230 CRE in the CRH promoter is likely to serve as a mechanism for the increased transcriptional responses to stress that are observed as a consequence of maternal deprivation in rats.


  • Liu Y, Aguilera G. Cyclic AMP inducible early repressor (ICER) mediates the termination of corticotropin releasing hormone transcription in hypothalamic neurons. Mol Cell Neurobiol 2009;29:1275-1281.
  • Blume A, Torner L, Liu Y, Subburaju S, Aguilera G, Neumann ID. Prolactin induces Egr-1 gene expression in cultured hypothalamic cells and in the rat hypothalamus. Brain Res 2009;1302:34-41.
  • Liu Y, Coello AG, Grinevich V, Aguilera G. Involvement of transducer of regulated cAMP response element-binding protein activity on corticotropin releasing hormone transcription. Endocrinology 2010;51:1109-1118.
  • Chen J, Aguilera G. Vasopressin protects hippocampal neurones in culture against nutrient deprivation or glutamate-induced apoptosis. J Neuroendocrinol 2010;22:1072-1081.
  • Aguilera G. HPA axis responsiveness to stress: Implications for healthy aging. Exp Gerontol. 2010;Epub ahead of print.
  • Arima H, Baler R, Aguilera G. Fos proteins are not prerequisite for osmotic induction of vasopressin transcription in supraoptic nucleus of rats. Neurosci Lett 2010;Epub ahead of print.


  • Valery Grinevich, MD, PhD, DSc, Max Plank Institute, Heidelberg, Germany
  • Stafford L. Lightman, MD, University of Bristol, UK
  • Francesca Spiga, PhD, University of Bristol, UK
  • Alan G. Watts, PhD, University of Southern California, Los Angeles, CA


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