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. The laboratory has shown that exposure to a repeated somatosensory stress causes hyperresponsiveness of the HPA axis to a novel stress. With hyperactivity of the HPA axis implicated in the pathogenesis of several psychiatric and metabolic disorders, self-limitation of the stress response is critical for avoiding the 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 as well as the consequences of the regulation on ACTH secretion and adrenal steroidogenesis.

Regulation of Hypothalamic CRH and VP Expression
Arima, Aguilera
The laboratory’s studies have been pivotal for understanding the interaction between CRH and vasopressin (VP) in the regulation of pituitary ACTH and the regulation of the expression of these peptides in the 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 paraventricular nucleus (PVN) are differentially regulated during stress or exposure to glucocorticoids, with VP becoming the predominant regulator during chronic stress. Studies during the past year using prolonged osmotic stimulation emphasized the specificity of increases in parvocellular VP (rather than systemically targeted magnocellular VP) as facilitator of HPA responses during chronic stress. In this experimental model with increased plasma VP levels, HPA axis responses to stress were inhibited in correspondence with blunted parvocellular VP responses.

Studies undertaken in collaboration with Harold Gainer, NINDS, using hypothalamic organotypic cultures and intronic in situ hybridization techniques have yielded important information on the trancriptional regulation of VP in the different hypothalamic nuclei. Given that survival of magnocellular neurons is poor in the culture conditions used, it was possible to examine the effect of signaling systems on VP transcription in CRH-expressing parvocellular neurons of the PVN while preserving the cytoarchitecture of the nucleus. Using tetradotoxin to block synaptic transmission, the studies provided the first demonstration that cAMP stimulates VP transcription directly in parvocellular neurons, while protein kinase C stimulation had no effect. In vitro studies using VP promoter luciferase constructs are in progress to elucidate role of AP1-responding elements present in the VP promoter in the transcriptional regulation of the VP gene. One particularly exciting finding was that basal levels of VP transcription in the suprachiasmatic nucleus (SCN) in these long-term cultures exhibit a circadian rhythm, with a peak in the morning and a nadir in the evening. Although VP cells in the SCN show intrinsic diurnal variation, overt rhythmicity depends on amplification of the signals by interneuronal transmission and the presence of a functional MAP-kinase pathway within the SCN.

Self-Limitation of Stress Responses
Shepard, Nikodemova, Aguilera
The laboratory is 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 increase in circulating glucocorticoids in the brain and pituitary. However, stress causes refractoriness to the inhibitory effect of glucocorticoids, leading to ineffectiveness of the feedback mechanism. Studies are under way 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 the self-limitation of HPA axis responses to stress.

While CRH is essential for stress response, studies in the laboratory have shown that increases in CRH transcription during stress are transient even if the stimulus is sustained. Efforts were made last year to elucidate the mechanisms responsible for turning off CRH transcription during stress. Stimulation of cAMP-dependent signaling systems plays an important role in the activation of CRH transcription. Studies in progress show that cAMP may also be involved in self-limiting CRH transcription during stress. In vivo, there is an increase in expression of inducible cAMP early repressor (ICER) in the PVN, which corresponds with the decreasing phase of CRH transcription. In addition, cotransfection of CRH promoter-luciferase constructs and ICER in a hypothalamic cell line inhibits cAMP-stimulated CRH promoter activity in vitro, suggesting that the ICER mediates a cellular feedback mechanism to limit CRH transcriptional responses during prolonged stress. Current and future studies will attempt to elucidate the physiological role and molecular mechanism by which ICER and other feedback control systems limit the stimulation of CRH transcription during stress.

Regulation of Pituitary CRH and V1b VP Receptors
Rabadan-Diehl, Volpi, Nikodemova, Aguilera
Regulation of the number of CRH and VP receptors in the pituitary plays an important role in the control of the HPA axis activity. The laboratory’s studies have shown that CRH receptor content in the pituitary does not depend on the levels of CRHR1 mRNA, indicating that regulation of the number of functional receptors occurs at post-transcriptional sites. Using CRHR1-specific antibodies, the laboratory showed that CRH binding down-regulation during glucocorticoid administration and adrenalectomy is associated with converse changes in receptor protein levels measured by Western blot, which suggests that glucocorticoids inhibit CRHR1 mRNA translation and/or increase receptor protein degradation. The changes in CRHR1 mRNA translation and the cellular events responsible for receptor desensitization in the presence of high receptor protein content are the subject of current investigation.

During the past year, the laboratory focused on the regulation of pituitary VP receptors. We demonstrated that increased pituitary corticotroph responsiveness during chronic stress involves VP receptor up-regulation, and we studied the molecular mechanisms of the receptor’s regulation. Studies on the transcriptional regulation of the V1b VP receptor have identified a region in the proximal promoter containing a large GAGA repeat, which binds to a pituitary protein complex that differs from the GAGA binding protein described in Drosophila. Transfection of Drosophila GAGA-binding protein markedly enhances V1b receptor promoter activity as well as the expression of endogenous V1b receptor in a hypothalamic cell line, suggesting that a GAGA-binding protein contributes to the regulation of V1b receptor transcription. The full characterization of the GAGA-binding protein complex and of the role of other transcription factors in the regulation of the V1b receptor gene is ongoing.

Previous studies conducted by the laboratory suggest that an important site of regulation of the V1b receptor content in the pituitary is mRNA translation to protein. The 5' untranslated region (5'UTR) of the V1b receptor mRNA is involved in controlling translation of the mRNA. The presence of upstream open reading frames in the 5'UTR may play a role in maintaining low translational activity in basal conditions. On the other hand, studies during the past year have identified an internal ribosome entry site (IRES) in the 5'UTR. IRES activity can be stimulated by activation of protein kinase C and PI3 kinase-dependent pathways, providing a mechanism for rapid stimulation of V1b receptor translation to meet physiological requirements. The importance of IRES activation and the upstream ORFs on the physiological control of the V1b receptor is under current investigation.