SEP | SGM | UGD | UGE | SEGEN | UGO | Main Page

neuroendocrinology of stress

Greti Aguilera, MD, Head, Section on Endocrine Physiology Ying Liu, MD, Research Associate Natalya Kalintchenko, MD, Postdoctoral Fellow Sivan Subburaju, PhD, Postdoctoral Fellow Simona Volpi, PhD, Postdoctoral Fellow Sharla Young, 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. The ability of the organism to adapt to acute and chronic stress situations is determined by genetic constitution and earlier experiences. Studies in our laboratory have shown that exposure to a repeated somatosensory stress causes hyper-responsiveness of the HPA axis to a novel stress. Since hyperactivity of the HPA axis has been implicated in the pathogenesis of several psychiatric and metabolic disorders, self-limitation of the stress response is critical to avoid deleterious effects of glucocorticoid excess. Our laboratory studies the mechanisms by which the expression of the hypothalamic hormones corticotropin releasing hormone (CRH) and vasopressin (VP) and their pituitary receptors are regulated under different stress situations as well as the consequences of such regulation on adrenocorticotropic hormone (ACTH) secretion and adrenal steroidogenesis. Regulation of hypothalamic CRH expression Kalintchenko, Liu, Aguilera Our laboratory has completed studies that have been pivotal for understanding the interaction between CRH and VP in the regulation of pituitary ACTH and the regulation of the expression of these peptides in the paraventricular nucleus (PVN) during stress and other alterations of the HPA axis. Previous studies showed that CRH and VP, which are co-expressed in the same parvocellular neuron of the PVN, are differentially regulated during stress or exposure to glucocorticoids. VP becomes the predominant peptide expressed in parvocellular neurons of the PVN during chronic stress. However, our studies suggest that, despite the prevalence of VP, ACTH secretion depends primarily on rapid but limited increases in CRH secretion and transcription. During the past year, studies have focused on elucidating molecular mechanisms of regulation of CRH expression, using recently characterized hypothalamic cell lines with a parvocellular neuron phenotype and models of experimental stress in rats. We are conducting in vivo and in vitro studies to determine mechanisms responsible for the termination of the stress response. A recognized mediator of negative feedback during the HPA axis response is the effect of increased circulating glucocorticoids in the brain and pituitary. However, stress causes refractoriness to the inhibitory effect of glucocorticoids, leading to ineffectiveness of the feedback mechanism. We are conducting studies to elucidate molecular mechanisms modulating the effectiveness of glucocorticoid feedback as well as the role of neurotransmitters such as GABA and autoregulatory mechanisms in hypothalamic neurons in limiting HPA axis responses to stress. While CRH is essential for stress response, we have shown that the increases in CRH transcription during stress are transient even if the stimulus is sustained. Using adrenalectomized rats with constant levels of corticosterone replacement, we showed that termination of CRH transcription is independent of the increases in plasma glucocorticoids in response to stress. We also demonstrated that termination of CRH transcription is associated with increased expression of inducible cAMP early repressor (ICER), a repressor isoform of cAMP-responsive element modulator (CREM), colocalized in CRH cells of the PVN. This paralleled formation of ICER-CRH CRE complexes, as demonstrated by electromobility gel shift assay (EMSA), suggests that endogenous levels of ICER can interact with the CRH CRE. Chromatin immunoprecipitation assays revealed stress-inducible binding of CREM to the CRH promoter CRE and decreases of Pol II association with the CRH promoter at a time when CRH transcription has declined, indicating that induction of ICER contributes to the limitation of CRH transcription during stress. Kasckow JW, Aguilera G, Mulchahey JJ, Sheriff S, Herman JP. In vitro regulation of corticotropin-releasing hormone. Life Sci 2003;73:769-781. Nikodemova M, KasckowJ, Liu H, Manganiello V, Aguilera G. cAMP regulation of CRH promoter activity in AtT-20 cells and in a transformed hypothalamic cell line. Endocrinology 2003;144:1292-1300. Oxytocin and HPA axis responses Subburaju, Ochedalski,^a Aguilera The neuropeptide oxytocin, secreted into the peripheral circulation by magnocellular neurons in the PVN and the supraoptic nucleus (SON), plays a major role in reproduction, controlling uterine contractility and milk ejection. In addition, oxytocin released within the brain is responsible for maternal behavior and can modulate behavioral and hormonal responses to stress. Physiological conditions under which oxytocin secretion is high are associated with decreased responsiveness of the HPA axis to stress. To determine whether oxytocin can mediate this effect, we studied the effects of intracerebroventricular (icv) oxytocin on HPA axis responses to restraint stress in ovariectomized rats receiving slow-release implants containing estradiol or estradiol plus progesterone. Hormone replacement resulted in plasma estradiol levels lower than in normal estrus and lower early proestrus levels of progesterone. In keeping with the positive feedback of oxytocin on its own expression, icv oxytocin infusion increased oxytocin mRNA in the PVN and SON independently of progesterone replacement, confirming the effectiveness of the icv infusion. Central oxytocin infusion did not influence basal plasma corticosterone or ACTH while, in rats receiving estradiol plus progesterone, central oxytocin administration augmented the responses and their duration. Consistent with the plasma hormone responses, central oxytocin had no effect on the increases in CRH mRNA in the PVN or pituitary proopiomelanocortin mRNA, as shown by in situ hybridization in rats receiving estradiol alone, but did significantly enhance responses in estradiol plus progesterone–replaced rats. The study shows that central oxytocin can either inhibit or enhance HPA axis activity depending on the levels of circulating sex steroids. The mechanism of the interaction is under current investigation. The findings may be relevant to the pathogenesis of psychiatric disorders associated with reproduction such as post-partum depression and premenstrual syndrome. Neuroendocrine immune interactions Grinevich,^b Aguilera; in collaboration with Jezova Studies of this laboratory have led to important findings on the effects of immune challenge on neuroendocrine responses in normal rats and in an experimental model of autoimmune arthritis. Acute or repeated administration of lipopolysaccharide (LPS) leads to marked activation of the HPA axis with activation of parvocellular neurons of the PVN and increases in plasma ACTH and corticosterone. Endotoxemia also leads to alterations in cardiovascular and fluid homeostasis, and in some conditions there is deficient urine-concentrating capacity despite normal or elevated plasma VP levels, suggesting refractoriness of the kidney to VP. To test this hypothesis, we examined the effect of LPS injection on plasma VP, urine osmolality, and the expression of V2 receptors and aquaporin-2 in the kidney. LPS injection caused prolonged decreases in urine osmolality without significant changes in plasma levels of sodium or VP, an effect associated with marked decreases in V2 VP receptor mRNA, as measured by in situ hybridization, and VP binding to kidney medulla membranes. As measured by immunohistochemistry and Western blot, aquaporin-2 was also reduced in the kidney inner medulla. These changes paralleled marked increases in cytokine expression in the kidney medulla. In addition, in vitro incubation of kidney medulla slices with IL-1 beta reduced VP binding, suggesting that cytokines are at least in part responsible for the marked downregulation of V2 VP receptors and aquaporin-2 of the kidney inner medulla observed during LPS-induced endotoxemia. These data indicate that inflammatory response to acute endotoxemia downregulates V2 VP receptors and aquaporin-2 of the kidney inner medulla, resulting in prolonged impairment of the renal capacity to concentrate urine. Chronic immune challenge also resulted in changes in sympathoadrenal and renin-angiotensin-aldosterone responses to novel stressors. Repeated treatment of rats with increasing doses of LPS resulted in a decrease in plasma epinephrine and aldosterone levels and in reduced renin activity responses as compared with those after acute administration. Repeated LPS administration was associated with decreased plasma aldosterone responses to a different stressor (immobilization) despite preserved or even elevated responses of plasma renin activity and catecholamines. Studies using in situ hybridization and dispersed adrenal glomerulosa cells demonstrated that decreased aldosterone responses are the result of reduced aldosterone synthase expression and activity, alterations that may contribute to deficient cardiovascular adaptation during chronic inflammatory states. Grinevich V, Knepper MA, Verbalis J, Reyes I, Aguilera G. Acute endotoxemia in rats induces down-regulation of V2 vasopressin receptors and aquaporin-2 content in the kidney medulla. Kidney Int 2004;65:54-62. Moncek F, Aguilera G, Jezova D. Insufficient activation of adrenocortical but not adrenomedullary hormones during stress in rats subjected to repeated immune challenge. J Neuroimmunol 2003;142:86-92. Regulation of pituitary CRH and V1b VP receptors Volpi, Young, Aguilera; in collaboration with Sandberg Regulation of the number of CRH and VP receptors in the pituitary plays an important role in the control of the HPA axis activity. Studies in our laboratory have shown that CRH and V1b receptor content in the pituitary depends on transcriptional and post-transcriptional events. We have demonstrated that increased pituitary corticotroph responsiveness during chronic stress is associated with VP receptor upregulation. Studies on the transcriptional regulation of the V1b VP receptor have identified a region in the proximal promoter containing a large GAGA repeat that is essential for transcriptional activation of the V1b receptor promoter and that binds to a protein complex found in pituitary nuclear extracts. GAGA binding activity of pituitary nuclear extracts increases rapidly during stress, a condition associated with VP release into the pituitary portal circulation and V1b receptor upregulation. Using the hypothalamic cell line H32, which expresses endogenous VP receptors, we showed that VP rapidly augmented binding of nuclear proteins to radiolabeled GAGA oligonucleotides, as assessed by electromobility shift assays, through activation of either V1a or V1b receptors. This effect was mimicked by epidermal growth factor (EGF) and blocked by the EGF receptor inhibitor AG1478 or by MAP kinase inhibitors, suggesting that VP activates GAGA binding through transactivation of the EGF receptor (EGFR). The effect of VP was preceded by transient phosphorylation of extracellular signal-regulated protein kinase 1 and 2 (pERK). ERK phosphorylation by VP was mediated by transactivation of the EGF receptor, as demonstrated by the ability of VP to induce transient phosphorylation of tyrosine 1173 (Tyr 1173) of the EGF-R and by the prevention of ERK phosphorylation by EGF receptor inhibitors. MAP kinase transactivation by the V1 VP receptor is mediated by the EGFR but, in contrast to other G protein–coupled receptors, the transactivation is independent of metalloprotease-induced cleavage of EGF-like peptides, or activation of Src or Pyk2, but does involve differential effects of protein kinase C (PKC) subtypes alpha and beta mediating the stimulatory and inhibitory phases of the effect of VP. Transfection of a PKC alpha dominant negative into H32 cells inhibited VP-stimulated EGFR Tyr 1173 and ERK phosphorylations. VP promoted rapid serine phosphorylation of the adapter protein Shc and its association with the EGFR, effects that were also reduced by the PKC alpha dominant negative. On the other hand, a dominant negative for PKC beta-1 prevented the decline in EGFR Tyr 1173 and ERK phosphorylations. The data suggest that, while MAP kinase transactivation by VP depends on sequential PKC alpha–induced phosphorylation of Shc and its association with the EGFR, PKC beta-1 mediates the declining phase. These effects of VP have important functional implications not only in the regulation of the VP receptor but also in potentially mediating the mitogenic and trophic actions of VP in the brain and pituitary corticotroph. We have previously shown that the 5´UTR of CRH-R1 mRNA inhibits CRH-R1 protein expression, an effect probably attributable to inhibition of mRNA translation. One of the mechanisms by which the 5´UTR can regulate protein translation is through binding of cytosolic proteins. In collaboration with Kathryn Sandberg, we examined pituitary cytosolic proteins that form RNA protein complexes with the 5´UTR of the CRF-R1 and compared them with the complexes formed between such proteins and the 5´UTR of the type 1 angiotensin II receptor (AT1aR). Competition studies and UV cross-linking analysis suggest that formation of CRF-R1 and AT1aR 5´UTR RNA protein complexes require at least some proteins that are common to both receptor mRNAs. Pituitaries isolated from male rats six days after adrenalectomy showed significant increases in CRH-R1 5´UTR RNA binding protein activity compared with sham-operated rats. The effect of adrenalectomy was prevented by glucocorticoid replacement. In contrast, no differences in the number of AT1aR binding sites or AT1aR 5´UTR binding protein activity were observed between sham-operated and adrenalectomized animals, indicating that the effect of adrenalectomy on RNA protein complex formation was specific for CRF-R1 mRNA. The data show that alterations of the hypothalamic-pituitary-adrenal axis specifically regulate mRNA binding proteins that interact with the 5´UTR of the CRF-R1, suggesting an involvement in translational regulation of CRF-R1 mRNA. Aguilera G, Nikodemova M, Wynn PC, Catt KJ. Corticotropin releasing hormone receptors: two decades later. Peptides 2004;25:319-329. Rabadan Diehl C, Nikodemova M, Volpi S, Aguilera G. Translational regulation of the vasopressin V1b receptor involves an internal ribosome entry site. Mol Endocrinol 2003;17:1959-1971. Volpi S, Rabadan-Diehl C, Aguilera G. Regulation of vasopressin V1b receptors and stress adaptation. Ann NY Acad Sci 2004;1018:293-301. Volpi S, Rabadan-Diehl C, Aguilera G. Vasopressinergic regulation of the hypothalamic pituitary adrenal axis and stress adaptation. Stress 2004;7:75-83. Wu Z, Ji H, Hassan A, Aguilera G, Sandberg K. Regulation of pituitary corticotropin releasing factor type-1 receptor mRNA binding proteins by modulation of the hypothalamic-pituitary-adrenal axis. J Neuroendocrinol 2004;16:214-220. ^aTomasz Ochedalski, MD, PhD, former Visiting Fellow ^bValery Grinevich, MD, DSc, former Visiting Fellow COLLABORATORS Daniela Jezova, PhD, Slovak Academy of Science, Bratislava, Slovak Republic John Kasckow, MD, University of Cincinnati, Cincinnati, OH Kathryn Sandberg, PhD, Georgetown University, Washington, DC For further information, contact aguilerg@mail.nih.gov