NEUROENDOCRINOLOGY OF STRESS
, Head, Section on Endocrine Physiology
Jun Chen, PhD, Postdoctoral Fellow
Sivan Subburaju, PhD, Postdoctoral Fellow
Ying Liu, MD, Research Associate
Anna Kamitakahara, BS, Postbaccalaureate 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 an organism to adapt to acute and chronic stress situations is determined by genetic constitution and experience, and adaptation to stress can have long-term consequences for the responsiveness of the HPA axis, such as altered expression of hypothalamic corticotrophin-releasing hormone (CRH) and circulating levels of glucocorticoids—hormones that are implicated in the pathogenesis of several psychiatric and metabolic disorders. The laboratory studies the mechanisms of positive and negative regulation of expression of the hypothalamic hormones CRH and vasopressin (VP) and their receptors under various stress situations and the impact of stress situations on the regulation of adrenocorticotropin (ACTH) secretion and adrenal steroidogenesis. The mechanisms responsible for self-limitation of stress responses are an important aspect of our research program. They are critical for understanding HPA axis dysregulation and for developing diagnostic, preventive, and therapeutic tools for stress-related disorders.
Regulation of hypothalamic CRH expression
Our studies have helped us understand the interaction between CRH and VP in the regulation of pituitary ACTH as well as 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. Despite the fact that selective increases in the expression of parvocellular VP and pituitary VP V1b receptors suggest that VP becomes the predominant regulator during chronic activation of the HPA axis, no evidence currently indicates that CRH 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. While glucocorticoid feedback plays an important role in regulating CRH expression, the relative importance of direct transcriptional repression of the CRH gene by glucocorticoids in the overall feedback mechanism is not clear. In addition to glucocorticoids, intracellular feedback mechanisms in the CRH neuron involving induction of repressor forms of the cAMP response element modulator (CREM) limit CRH transcriptional responses by competing with the positive regulator phospho-CREB (cAMP response element-binding protein). Supporting the hypothesis that induction of inducible cAMP early repressor (ICER) is part of an intracellular feedback mechanism limiting CRH transcription, inhibition of endogenous ICER production using silencing RNA (siRNA) enhanced forskolin-induced CRH promoter activity in reporter gene assays and attenuated the inhibitory phase of CRH transcription in primary hypothalamic neuronal cultures incubated with forskolin. Rapid repression of CRH transcription following stress-induced activation is likely to contribute both to limiting the stress response and preventing disorders associated with excessive CRH production.
While it is well established that cAMP-dependent signaling is the major regulator of CRH transcription, the most recognized receptors mediating stimulation of CRH neurons—alpha-adrenergic and glutamergic receptors—act through other mechanisms than via cAMP. To determine whether synergism between calcium phospholipid signaling and small elevations of intracellular cAMP mediate activation of CRH transcription during stress, we investigated the effects of the phorbol ester PMA and the cAMP stimulator forskolin on CRH transcription and CREB phosphorylation in the hypothalamic cell line 4B and in hypothalamic neuronal cultures. Incubation of 4B cells transfected with a CRH promoter–driven reporter with forskolin increased phospho-CREB (pCREB) levels and CRH promoter activity. PMA alone increased pCREB to similar levels but had no effect on CRH promoter activity. However, PMA potentiated the stimulatory effect of small concentrations of forskolin. The CREB dominant negative A-CREB reduced forskolin-stimulated CRH promoter activity in the absence or presence of PMA, indicating that pCREB is required but not sufficient to activate CRH transcription. PMA also potentiated the stimulatory effect of forskolin on CRH transcription (measured by intronic qRT-PCR) in primary cultures of hypothalamic cells. The potentiating effect of calcium/phospholipid signaling on cAMP-mediated transcription provides a mechanism by which non–cAMP-dependent neurotransmitters mediate activation of CRH transcription in the presence of minor increases in intracellular cAMP. The relevance of these observations to the regulation of CRH expression by neurotransmitters activated during stress is under investigation.
We have shown that stress activates the renin-angiotensin system and increases the number of type 1 angiotensin (AT1) receptors in the PVN. Sustained pretreatment with AT1 receptor antagonists prevents sympathoadrenal and hormonal responses to a 24-hour isolation stress. To examine the mechanism of the antistress effects of AT1 receptor antagonism, we investigated the effect of chronic subcutaneous infusion of candesartan, a non-competitive AT1 receptor antagonist, on HPA axis responses to 24-hour isolation stress in Wistar rats. In rats treated with vehicle only, 24-hour isolation stress increased pituitary ACTH, adrenal corticosterone content, and AT1 receptor binding in the PVN but decreased CRH mRNA and CRH content in the PVN, changes similar to those found in depression. Candesartan pretreatment prevented the effects of stress, suggesting that activation of AT1 receptors is permissive for the HPA axis response to isolation. These results support the view that inhibition of central AT1 receptors limits the CRH response to stress and could serve as a therapeutic tool to preserve homeostasis under chronic stress conditions.
Aguilera G, Kiss A, Liu Y, Kamitakahara A. Negative regulation of corticotrophin releasing hormone expression and limitation of the stress response. Stress 2007;10:153-61.
Armando I, Volpi S, Aguilera G, Saavedra JM. Angiotensin II AT1 receptor blockade prevents the hypothalamic corticotrophin-releasing factor response to isolation stress. Brain Res 2007;1142:92-9.
Liu Y, Kalintchenko N, Sassone-Corsi P, Aguilera G. Inhibition of corticotrophin releasing hormone transcription by inducible cAMP-early repressor in the hypothalamic cell line, 4B. J Neuroendocrinol 2006;18:42-9.
Interaction between oxytocin and estrogens: effect on HPA axis activity
The neuropeptide oxytocin (OT), which is secreted into the peripheral circulation by the neurohypophysis, has well-established roles during parturition and lactation, facilitating uterine contractility and milk ejection. In addition to its peripheral actions, OT is released within the limbic system and other areas of the brain and influences behavioral and neuroendocrine responses to stress, including HPA axis activity. It has been postulated that, during lactation, increases in central OT levels mediate inhibition of HPA axis responses. However, in our experiments using ovariectomized rats with low estradiol replacement alone, intracerebroventricular (icv) OT had no significant effect on basal or restraint-stimulated plasma ACTH and corticosterone or hypothalamic CRH mRNA. Moreover, icv OT had a stimulatory effect on HPA axis activity when rats received replacement with low estradiol together with progesterone. This observation is at variance with reported inhibitory effects of central OT on HPA axis activity and suggests that central effects of OT can be modulated by ovarian hormones. We tested the hypothesis that estrogen modulates the effects of OT on HPA axis activity in seven-day ovariectomized rats receiving diestrus (low), proestrus (medium), or pregnancy (high) replacement levels of estradiol and icv minipump infusion of OT. Estradiol caused dose-dependent increases in basal plasma ACTH and corticosterone levels but decreased ACTH responses to restraint stress. In parallel with the changes in basal plasma ACTH, high estrogen raised basal CRH heterogeneous nuclear RNA and CRH mRNA in the PVN and pro-opiomelanocortin (POMC) mRNA in the pituitary while depressing restraint stress–stimulated levels. Icv OT reduced basal and stress-stimulated plasma ACTH, hypothalamic CRH hnRNA, CRH mRNA, and pituitary POMC mRNA levels parallel to the increases induced by elevating plasma estradiol. The study showed converse effects of estradiol on basal and restraint stress–stimulated basal HPA axis activity and demonstrated that the ability of central oxytocin to inhibit HPA axis activity depends on the levels of circulating estradiol.
Pituitary actions of vasopressin
VP produced by parvocellular neurons of the PVN potentiates the stimulatory effect of CRH on pituitary ACTH secretion acting through plasma membrane receptors of the V1b subtype (V1bR). The hyper-responsiveness of the HPA axis during chronic stress is associated with a predominant increase in hypothalamic VP and pituitary V1b receptors rather than with CRH and its pituitary receptors. To test the hypothesis that a switch occurs from CRH to VP as the main mediator of pituitary corticotroph responsiveness during chronic HPA activation, we examined the effect of pharmacologic VP receptor blockade on ACTH and corticosterone responses of rats repeatedly restrained for 14 days. Despite increased vasopressinergic activity, repeatedly restrained rats showed lower ACTH and corticosterone responses to 10 minutes of white noise compared with handled controls. The non–peptide-selective V1b receptor antagonist SSR149415 given 1 hour before exposure to noise did not change these responses. In contrast to the response to noise stress, plasma ACTH responses to i.p. hypertonic saline injection were enhanced in the repeatedly restrained rats compared with handled controls, but responses were also unaffected by SSR149415 (30mg/kg/orally) administered daily 1 hour before restraint. Given that SSR149415 bioactivity, assessed by corticosterone responses to exogenous VP, was low, we used minipump infusion of the peptide V1 receptor antagonist dGly[Phaa1,D-Tyr(et), Lys, Arg]VP (V1-Ant) for 14 days; the antagonist effectively blocked ACTH responses to exogenous VP. Chronic V1-Ant infusion significantly reduced plasma ACTH responses to i.p. hypertonic saline in handled controls but not in repeatedly restrained rats. The data show that VP contributes to ACTH responses to an acute stressor but that increased vasopressinergic activity is not responsible for enhanced ACTH responses to a novel stress.
The low impact of vasopressinergic blockade on HPA axis activity during chronic stress suggests that VP plays additional roles, such as controlling the number of pituitary corticotrophs. We studied the role of VP in mediating pituitary corticotroph mitogenesis in adrenalectomized rats by examining the effect of V1-Ant on the number of cells incorporating bromouridine (BrdU). Long-term adrenalectomy increased the number of both BrdU-labeled cells and ACTH-stained cells. Osmotic minipump infusion of V1-ant for 28 days prevented adrenalectomy-induced increases in BrdU incorporation but not changes in the number of ACTH-stained cells. Unexpectedly, co-localization of BrdU uptake in ACTH-positive cells was minor and unaffected by adrenalectomy or V1-antagonist infusion. We observed no BrdU-stained nuclei in luteinizing hormone, thyrotropin, prolactin, growth hormone, or folliculo-stellate cells or nestin-labeled progenitor cells. The pituitary corticotroph–exclusive transcription factor Tpit colocalized in more than 80 percent of ACTH-containing cells but in only in 5 percent of BrdU-labeled nuclei in controls and 10 percent in adrenalectomized rats. In V1bR knockout mice, the number of cells incorporating BrdU following adrenalectomy was lower than in wild type, with no major colocalization of BrdU and ACTH. The data demonstrate that VP mediates mitogenic activity in the pituitary during long-term adrenalectomy. The lack of colocalization of ACTH and BrdU suggests that recruitment of corticotrophs during adrenalectomy occurs from undifferentiated cells. The data suggest that one of the functions of the marked increases in parvocellular vasopressinergic activity during adrenalectomy (and probably chronic stress) is to regulate cell proliferation and remodeling pituitary tissue.
Rabadan-Diehl C, Martínez A, Volpi S, Subburaju S, Aguilera G. Inhibition of vasopressin V1b receptor translation by upstream open reading frames in the 5¢ untranslated region. J Neuroendocrinol 2007;19:309-19.
Subburaju S, Aguilera G. Vasopressin mediates mitogenic responses to adrenalectomy in the rat the anterior pituitary. Endocrinology 2007;148:3102-10.
Volpi S, Liu Y, Aguilera G. Vasopressin increases GAGA binding activity to the V1b receptor promoter through transactivation of the MAP kinase pathway. J Mol Endocrinol 2006;36:581-90.
Interaction between CRH and V1b receptors
Increasing evidence indicates that G protein–coupled receptors (GPCRs), including members of the VP receptor family, can act as homo- and heterodimers. It is clear that regulated expression and interaction of pituitary VP V1b receptor (V1bR) and CRH receptor type 1 (CRHR1) are critical for HPA axis adaptation, but it is not known whether physical interaction between these receptors is involved. Bioluminescence resonance energy transfer (BRET) experiments using V1bR and CRHR1 fused to either Renilla luciferase (Rluc) or yellow fluorescent protein (YFP) at the N-terminus, but not at the C-terminus, revealed specific interaction that was inhibited by untagged V1b or CRHR1 receptors, suggesting homo- and heterodimerization. We confirmed the BRET data in co-immunoprecipitation experiments that used fully bioactive receptors tagged at the N-terminus with c-myc and Flag epitopes, demonstrating specific homodimerization of the V1b receptor and heterodimerization of the V1b receptor with CRHR1 receptors. Given that stimulation with CRH and VP had no effect on co-immunoprecipitation, we concluded that heterodimerization between V1bR and CRHR1 is not ligand-dependent. In membranes obtained from cells cotransfected with CRHR1 and V1bR, incubation with the heterologous nonpeptide antagonist did not alter the receptor’s binding affinity or capacity. The data demonstrate that V1bR and CRHR1 can form constitutive homo- and heterodimers and suggest that heterodimerization does not influence the binding properties of these receptors.
Young SF, Griffante C, Aguilera G. Dimerization between vasopressin V1b and corticotropin releasing hormone type-1 receptors. Mol Cell Neurobiol 2007;27:439-61.
1 Sharla Young, PhD, former Postdoctoral Fellow
COLLABORATOR
Tomasz Ochedalski, MD, PhD, University of Lodz, Lodz, Poland
For further information, contact aguilerg@mail.nih.gov.
