Neuroendocrinology of Stress

Greti Aguilera, MD, Head, Section on Endocrine Physiology
Ying Liu, MD, Research Associate
Jun Chen, PhD, Postdoctoral Fellow
J. Alice Kim, BS, Predoctoral 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). 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.

Regulation of hypothalamic CRH expression

Liu, Kim, Aguilera

Appropriate responses of the HPA axis to stress, with adequate control of adrenal glucocorticoid secretion, is essential for homeostasis. Our studies have played a critical role in understanding the interaction between the peptides CRH and VP in the regulation of pituitary adrenocorticotropic hormone (ACTH) and the regulation of the expression of these peptides in the paraventricular nucleus (PVN) during stress and other alterations of the HPA axis. Co-expressed in the same parvocellular neuron of the PVN, both peptides are differentially regulated during stress or exposure to glucocorticoids.

CRH coordinates behavioral, autonomic, and hormonal responses to stress and is the main regulator of ACTH secretion in acute and chronic conditions. Following CRH release, activation of CRH transcription is required to restore mRNA and peptide levels, but termination of the response is essential to prevent pathology associated with chronic elevation of CRH and glucocorticoid production. Our laboratory has furthered the understanding of the mechanisms of negative and positive transcriptional regulation of CRH. We demonstrated that CRH transcription is under positive control by cAMP/phospho–cyclic AMP response element binding protein (CREB) signaling and negatively regulated by glucocorticoid feedback. Our research led to the demonstration that, in addition to glucocorticoids, intracellular feedback mechanisms in the CRH neuron, which involve induction of repressor forms of cAMP response element modulator (CREM), limit CRF transcriptional responses by competing with the positive regulator phospho-CREB. Rapid repression of CRH transcription following stress-induced activation is likely to help limit the stress response and work to prevent disorders associated with excessive CRH production.

With respect to positive regulation, the conventional view holds that activation of CRH transcription is initiated by binding of phosphorylated CREB to the CRH promoter. During the past year, our laboratory challenged this view by reporting that cAMP/phospho-CREB signaling is essential but not sufficient to activate CRH transcription. Our finding strongly suggests that transcriptional activation of the CRH gene requires a co-activator of CREB. In a number of systems, it has been shown that CREB-mediated transcription involves the co-activator TORC (transducer of regulated CREB activity), which could be the missing link required for CREB-induced regulation of CRH transcription. We examined the ability of TORC to regulate CRH transcription in a hypothalamic cell line 4B transfected with a CRH promoter–driven luciferase reporter gene. Consistent with the above observations, at threshold concentrations for cAMP production and CREB phosphorylation, forskolin induced marked CRH promoter activation while phorbol-12-myristate-13-acetate (PMA) failed to activate the CRH promoter despite PMA’s ability to phosphorylate CREB. However, in cells co-transfected with an expression vector for TORC2, PMA significantly activated the CRH promoter compared with baseline activity in TORC-transfected cells or PMA administration to cells transfected with the empty vector. TORC2 transfection also potentiated the stimulatory effect of submaximal, but not that of maximal, concentrations of forskolin. We examined the involvement of endogenous TORC in cAMP-dependent activation of CRH transcription by using Western blot and blockade of endogenous TORC production by siRNA. Under basal conditions, Western blot analysis revealed, in cytosolic but not nuclear fractions, a 200 kDa band that corresponded to the phosphorylated form of TORC2. Incubation with forskolin resulted in transient nuclear translocation (maximal at 30 minutes) of a lower–molecular weight band corresponding to dephosphorylated TORC. In contrast, PMA slightly delayed the migration of the cytosolic band (suggesting hyperphosphorylation) and had no effect on nuclear TORC levels, suggesting that the inability of phorbolesters to stimulate transcription is attributable to the lack of nuclear translocation of TORC. Transfection of TORC1 and TORC2 siRNA oligonucleotides 24 hours before incubation with forskolin abolished the immunoreactive TORC bands in the Western blot and prevented activation of the CRH promoter by forskolin. Our study provides strong evidence that the co-activator TORC is required for activation of CRH transcription. Our current research focuses on the interactions of TORC with the CRH promoter and on the importance of the co-activator TORC during physiological regulation of CRH transcription in vivo.

Liu Y, Kamitakahara A, Kim AJ, Aguilera G. Cyclic AMP responsive element binding protein phosphorylation is required but not sufficient for activation of CRH transcription. Endocrinology 2008;49:3512-3520.

Anti-apoptotic actions of VP

Chen, Aguilera

VP secreted within the brain modulates neuronal function by acting as a neurotransmitter. We previously showed that the 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 hypothalamic pituitary adrenal axis under chronic conditions. These observations led to the search for new roles of vasopressin during stress adaptation. Based on the observation that VP prevents serum deprivation–induced cell death in the neuronal cell line H32, which expresses endogenous V1 receptors, we tested the hypothesis that VP has anti-apoptotic properties. Flow cytometry experiments showed that 10nM VP prevented serum deprivation–induced cell death and annexin V binding. Serum deprivation increased caspase-3 activity in a time- and serum concentration–dependent manner while VP prevented these effects through interaction with receptors of the V1 subtype. The signaling pathways mediating the anti-apoptotic effect of VP involve mitogen-activated protein (MAP) kinase and extracellular signal–regulated kinases (ERK), Ca²⁺/calmodulin-dependent kinase (CaMK), and protein kinase C (PKC). Western blot analyses revealed time-dependent decreases of Bad phosphorylation and increases in cytosolic levels of cytochrome c following serum deprivation, effects prevented by 10nM VP. These data demonstrate that activation of endogenous V1 VP receptors prevents serum deprivation–induced apoptosis through phosphorylation inactivation of the pro-apoptotic protein Bad and consequent decreases in cytosolic cytochrome c and caspase-3 activation. The data suggest that VP has anti-apoptotic activity in neurons and may act as a neuroprotective agent in the brain.

Using the cell line H32, we performed additional studies on the role of protein kinase C signaling in the anti-apoptotic effects of VP in neurons. Serum deprivation for 6 hours induced caspase-3 activity and dephosphorylation of Bad while co-incubation with VP reversed these changes. The selective PKC α/β subtype inhibitor Gö 6976 and PKCα and PKCβ dominant negatives reduced the effect of VP in caspase activation while PCKδ inhibition or a PCKδ dominant negative reduced serum deprivation–induced caspase-3 activity without affecting the protective action of VP. Consistently, serum deprivation increased PKCδ but not PKC α or PKCβ activity, and VP increased PKCα and PKCβ activity without affecting PKCδ activity. VP-induced Bad phosphorylation was reduced by PKCα and PKCβ or MEK inhibitors but was completely blocked by both inhibitors combined. However, total inhibition of caspase-3 activation required additional inhibition of the PI3K/Akt pathway. The data demonstrate distinct roles of PKC subtypes, with PKCδ mediating the apoptotic effect of serum deprivation and PCKα and PCKβ together with the MAP kinase and PI3K/Akt pathways mediating the antiapoptotic actions of VP.

Aguilera G. Signal transduction: receptors and G-proteins. In: Izzo JL, Black HR, eds. Hypertension Primer. American Heart Association, 2007;1-3.
Aguilera G, Subburaju S, Young S, Chen J. The parvocellular vasopressinergic system and responsiveness of the hypothalamic pituitary adrenal axis during chronic stress. Prog Brain Res 2008;170:29-39.
Chen J, Volpi S, Aguilera G. Anti-apoptotic actions of vasopressin in H32 neuronal cells involve MAP kinase transactivation and Bad phosphorylation. Exp Neurol 2008;211:529-538.
Chen J, Young S, Subburaju S, Shepard J, Kiss A, Atkinson H, Wood S, Lightman S, Serradeil-Le Gal C, Aguilera G. Vasopressin does not mediate hypersensitivity of the hypothalamic pituitary adrenal axis during chronic stress. Ann NY Acad Sci, in press.

For further information, contact aguilerg@mail.nih.gov.