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 previous experiences. Studies in this laboratory have shown that exposure to a repeated somatosensory stress causes hyper-responsiveness of the HPA axis to a novel stress. Given that hyperactivity of the HPA axis has been implicated in the pathogenesis of a number of psychiatric and metabolic disorders, self-limitation of the stress response is critical to avoid deleterious effects of glucocorticoid excess. The 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 and the consequences of regulation on ACTH secretion and adrenal steroidogenesis.
Regulation of hypothalamic CRH expression
Liu, Aguilera
Our studies have illuminated the role of interaction between CRH and VP in regulating pituitary ACTH and 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 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. During the past year, we continued to examine the regulation of CRH expression by using models of experimental stress in rats and the hypothalamic cell line 4B, which exhibits characteristics of the parvocellular neuron.
While CRH is essential for full ACTH and corticosterone responses to stress, excessive CRH production leads to developmental, psychiatric, metabolic, immune, and reproductive disorders. Activation of the HPA axis during stress is accompanied by rapid but transient increases in CRH transcription and probably secretion of the peptide to the pituitary portal circulation. Our work has focused on the mechanisms by which CRH transcription is controlled. A recognized mediator of negative feedback during the HPA axis response is the effect of increased circulating glucocorticoids in the brain and pituitary. Using adrenalectomized rats with constant levels of corticosterone replacement, we previously showed that termination of CRH transcription is independent of the increases in plasma glucocorticoids in response to stress. More recently, we demonstrated that, in contrast to adrenalectomized rats, corticosterone inhibits CRH transcriptional responses to stress in intact rats irrespective of the time of administration of the steroid. Future studies will address molecular mechanisms modulating the effectiveness of glucocorticoid feedback directly in the CRH neuron or indirectly through modulation of neural pathways afferent to the PVN.
Using in situ hybridization and Western blots, we have demonstrated that termination of CRH transcription is associated with increased expression of inducible cAMP early repressor (ICER), a repressor isoform of the cAMP-responsive element modulator (CREM). In keeping with a role of ICER in regulating CRH transcription, we found that CREM mRNA was induced during stress in CRH cells of the PVN. Electromobility gel shift assay (EMSA) and chromatin immunoprecipitation assays showed that, late during stress and concomitant with increases in ICER protein and a return of CRH transcription to basal values, ICER associates with the CRH promoter. The data demonstrate that ICER produced in CRH neurons during stress may contribute to the limitation of CRH transcription during stress.
During the past year, we examined the ability of ICER to inhibit CRH transcription in the hypothalamic cell line 4B, which expresses CRH. Co-transfection of ICER I and II with CREMbeta, all inhibitory isoforms of CREM, with CRH promoter-luciferase constructs in 4B cells blunted basal and forskolin-stimulated CRH promoter activity, an effect abolished by mutation of the CRE of the CRH promoter. Western blot, electromobility gel shift, and super-shift analyses showed increases in endogenous ICER after incubation with forskolin. Consistent with an inhibitory effect of CREM on CRH transcription, chromatin immunoprecipitation assays in cells transfected with ICER I revealed recruitment of CREM by the CRH promoter in conjunction with decreases in Pol II association. The study shows that, following prolonged stimulation with forskolin, or transfection of an ICER expression vector in hypothalamic cell lines expressing CRH, generation of ICER is associated with CREM binding to the CRH promoter and transcriptional repression. The data support the hypothesis that induction of repressor isoforms of CREM is part of an intracellular feedback mechanism contributing to the termination of CRH transcription during stimulation.
Liu Y, Kalintchenko N, Sassone-Corsi P, Aguilera G. Inhibition of corticotrophin releasing hormone transcription by inducible cAMP-early repressor (ICER) in the hypothalamic cell line, 4B. J Neuroendocrinol (in press).
Shepard JF, Liu Y, Sassone-Corsi P, Aguilera G. Role of glucocorticoids and cAMP-mediated repression on the termination of corticotrophin releasing hormone transcription during stress. J Neurosci 2005; 25:4073-4081.
Oxytocin and sex hormones and HPA axis responses
Subburaju, Ochedalski, Aguilera
Secreted into the peripheral circulation from magnocellular neurons in the PVN and supraoptic nucleus (SON), the neuropeptide oxytocin 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, and it has been postulated that oxytocin mediates the blunted HPA axis responses to stress during lactation. Given that previous studies in our laboratory showed no inhibitory effect on HPA axis activity of intracerebroventricular (icv) oxytocin in ovariectomized rats receiving low estradiol (E2) replacement, we examined the influence of circulating estradiol on the effects of icv oxytocin infusion on plasma ACTH and corticosterone, as well as on hypothalamic CRH expression responses to restraint stress in ovariectomized rats. We found that oxytocin infusion had little effect on basal or stress-stimulated plasma corticosterone, irrespective of sex hormone levels. In contrast, icv oxytocin blunted ACTH responses to stress. Basal CRH mRNA levels increased with high E2 replacement but decreased with icv oxytocin infusion in high- and low-E2 experiments. CRH mRNA responses to restraint were not significantly different in high- and low-E2 experiments but were suppressed by icv oxytocin only in rats with high estrogen levels. The study shows that the ability of oxytocin to inhibit HPA axis responses to restraint stress in female rats depends on the presence of high E2 levels. The data emphasize the importance of estrogen in the regulation of the HPA axis. These findings may be relevant to the pathogenesis of psychiatric disorders associated with reproduction such as postpartum depression and premenstrual syndrome.
Ochedalski T, Subburaju S, Wynn P, Aguilera G. Estradiol modulates the effect of central oxytocin on hypothalamic pituitary adrenal axis activity in rats. J Neuroendocrinol (in press).
Central actions of prolactin
Blume, Liu, Aguilera
In addition to its role in lactation, prolactin (PRL) produced in the brain can act as a neurotransmitter/ neuromodulator, attenuating the HPA axis and behavioral responses to stress. Given that the expression of PRL and PRL receptors in the PVN increases during lactation, PRL has been implicated in the mechanism of the blunted stress responses observed in during lactation. To determine the mechanisms mediating the central effects of PRL, we examined the signaling pathways stimulated in the hypothalamus after icv injection of PRL in male and female rats in vivo and in vitro in the hypothalamic cell line 4B, cells that express endogenous CRH. Western blot analysis of hypothalamic proteins following PRL injection showed phosphorylation of Stat1 at 10 minutes in female rats and at 30 minutes in male rats while pStat3 was elevated in males and females at 30 minutes. Furthermore, central PRL administration induced phosphorylation of MEK (at 5 and 10 minutes) and ERK1/2 in the nucleus (30 minutes) in male and female rats. Immunohistochemical analysis of pERK1/2 in hypothalamic sections 30 minutes after icv PRL revealed staining in the PVN. Western blot analysis of protein extracts from hypothalamic cells 4B with a PRL receptor antibody showed a 40kDa band consistent with the short form of the PRL receptor. Incubation of the cells with PRL caused rapid and progressive increases in pERK1/2. We examined the effects of PRL on CRH expression in 4B cells transfected with a luciferase reporter gene driven by the CRH promoter. Prolonged incubation of the cells with PRL increased basal and potentiated forskolin-stimulated CRH promoter activity. In addition to conventional Stat signaling, the study shows that PRL activates the ERK1/2 MAP kinase cascade in the hypothalamic PVN. The ability of PRL to induce ERK phosphorylation and to increase CRH promoter activity in a CRH-expressing hypothalamic cell line suggests that PRL can directly modulate CRH neuron function.
Pituitary actions of vasopressin
Volpi,1 Subburaju, Liu, Syed, Aguilera
Vasopressin produced by parvocellular neurons in the PVN modulates the stimulatory effect of CRH on pituitary ACTH secretion acting through plasma membrane receptors of the V1b subtype. The expression of parvocellular VP as well as that of pituitary V1b receptors (V1bR) increases during chronic stimulation of the hypothalamic pituitary adrenal axis, suggesting that VP plays a critical role in long-term adaptation to stress. Recent studies using VP antagonists and VP and V1bR-deficient animal models have shown little impact of vasopressinergic blockade on HPA axis activity during chronic stress, suggesting that VP has additional roles, such as controlling the number of pituitary corticotrophs. We studied such a possibility by using immunohistochemistry to examine the effects of prolonged (6 to 28 days) adrenalectomy (ADX) and VP V1 receptor blockade by minipump infusion of the peptide antagonist dGly[Phaa1,D-tyr(et),Lys,Arg]VP on the number of cells incorporating bromodeoxyuridine (BrdU) and of cells stained for ACTH in the anterior pituitary. ADX elevated the number of BrdU-labeled cells about three-fold compared with controls while infusion of the V1 antagonist completely prevented the effect of ADX. The number of ACTH-stained cells also increased four weeks after ADX; however, in contrast to BrdU incorporation, the increase was not affected by the V1 antagonist. Unexpectedly, we observed only minor co-localization of BrdU uptake in ACTH-positive cells, with co-localization unaffected by ADX or V1 antagonist infusion. Incubation of primary pituitary cell cultures with VP for 48 hours increased the number of cells incorporating BrdU by about 30 percent. The studies show that VP has several roles in pituitary function, including upregulation of the V1b receptor, potentiation of CRH-stimulated ACTH secretion, and trophic actions. While in acute conditions VP facilitates ACTH secretion, in chronic situations VP contributes to mitogenic activity in the pituitary without increasing the number of ACTH-containing corticotrophs. The lack of co-localization of ACTH in mitotic cells suggests that recruitment of corticotrophs during adrenalectomy occurs from undifferentiated cells.
Interaction between CRH and V1b receptors
Young, Aguilera
Although classically thought to function as monomers, a growing body of evidence supports the notion that G protein–coupled receptors (GPCRs) can form oligomers. Regulation of ACTH secretion involves strong interaction between activation of V1b receptors and corticotrophin-releasing hormone receptor type 1 (CRHR1), but whether a physical interaction occurs between the receptors is currently unknown. We first addressed this issue by using bioluminescence resonance energy transfer (BRET) to study dimerization in living cells. The V1bR and CRHR1 fused to either Renilla luciferase (rluc) or yellow fluorescent protein (YFP) at the carboxy-terminus of the receptor were fully bioactive when transiently transfected into Chinese hamster ovary cells. Using a multiplate reader, we determined dimerization by measuring excitation of receptor-YFP by energy transferred from receptor-rluc. Energy transfer from V1bRrluc to V1bYFP, CRHR1YFP, or the unrelated bradykinin 2 receptor-YFP (B2RYFP) was very low, with levels unchanged by co-transfection with wild-type V1bR or following incubation of the cells with the ligands CRH or VP. However, used as a positive control, the OTRrluc/OTRYFP pair displayed the expected BRET levels and lack of interaction with B2RYFP. In contrast to the BRET data, co-immunoprecipitation using receptors tagged with c-myc and Flag epitopes demonstrated specific homodimerization of the V1b receptor and heterodimerization of the V1b receptor with both the OTR and CRHR1 receptors, suggesting that the position of the rluc and YFP tags impaired the BRET signal. Studies in progress focus on the functional significance V1b and CRHR1 receptor dimerization.
Feng N, Young S, Aguilera G, Puricelli E, Adler-Wailes D, Sebring NG, Yanovski JA. Co-occurrence of two partially inactivating polymorphisms of the melanocortin 3 receptor (MC3R) is associated with pediatric-onset obesity. Diabetes 2005;54:2663-2667.
1Simona Volpi, PhD, former Postdoctoral Fellow
Collaborators
Annegret Blume, PhD, Universität Regensburg, Regensburg, Germany
Tomasz Ochedalski, MD, PhD, University of Lodz, Lodz, Poland
For further information, contact aguilerg@cc1.nichd.nih.gov.