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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 |
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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, John
Kasckow, MD, Kathryn Sandberg, PhD, For further
information, contact aguilerg@mail.nih.gov |