Our goal 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 our 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 several psychiatric and metabolic disorders, self-limitation of the stress response is critical for avoiding 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 and VP Expression
Kalintchenko, Nikodemova, Shepard
Studies in the laboratory have been pivotal in 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 co-expressed in the same parvocellular neuron of the PVN are differentially regulated during stress or exposure to glucocorticoids, with VP becoming the predominant regulator during chronic stress. During the past year, our studies have focused on the mechanisms by which prolonged stress results in lowered CRH transcription but sustained increases in VP in the parvocellular neuron. Characterization of a recently produced fetal rat hypothalamic cell line revealed expression of endogenous CRH, VP, and glucocorticoid receptors, suggesting a parvocellular neuron phenotype. Transfection of CRH or VP promoter reporter gene constructs into this cell line revealed that activation of VP promoter required much higher levels of cAMP than activation of the CRH promoter. High levels of cAMP also stimulate the expression of inhibitory isoforms of cAMP-responsive element modulator (CREM). We showed that inhibitory isoforms of CREM cause marked repression of cAMP-stimulated CRH promoter but had no effect on cAMP-stimulated VP promoter activity. This diverse response of the CRH and VP promoters to cAMP-dependent signaling could contribute to the differential regulation of both genes during stress.

Self-Limitation of Stress Responses
Nikodemova, Shepard, Aguilera
We are conducting in vivo and in vitro studies to determine the mechanisms responsible for 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. Studies are under way to elucidate molecular mechanisms modulating the effectiveness of glucocorticoid feedback and the role of neurotransmitters such as GABA and to elaborate on autoregulatory mechanisms in hypothalamic neurons in the self-limitation of 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. Research in the laboratory has focused on determining the mechanisms responsible for turning on and off CRH transcription during stress. Evidence suggests that cAMP-dependent signaling plays an important role in the activation of CRH transcription in non-hypothalamic cells. During the last year, we used the rat hypothalamic cell line 4B to investigate tissue specificity of cAMP-dependent regulation of a 498 bp CRH promoter fragment. The data showed that minimal elevations in intracellular cAMP are sufficient to activate CRH transcription fully irrespective of the cell line. Rapid degradation of cAMP by phosphodiesterases may contribute to the turn-off of the CRH transcriptional response to cAMP. Elevations in intracellular cAMP also appear to be involved in self-limiting CRH transcription during stress by inducing the expression of inhibitory CREMs. in vivo, there is an increase in expression of the CREM isoform, inducible cAMP early repressor (ICER), in the PVN corresponding to the decreasing phase of CRH transcription. In addition, co-transfection 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.

Neuroendocrine Immune Interactions
Grinevich,* Lightman, Tilders, Aguilera
Single exposure to the proinflammatory cytokine interleukin-1 (IL-1) induces sensitization of the ACTH and corticosterone responses to stressors weeks after HPA axis sensitization. Studies were conducted to determine whether sensitization of the HPA responses to a novel stressor (novelty) involves autoexcitatory feedback mediated by CRH and VP receptors in the hypothalamic PVN. Single administration of IL-1 induced sensitization of the HPA to novelty from three to 22 days later and biphasic increases in CRH and CRH-R1 mRNAs in the PVN: an early peak within 24h, followed by a delayed (more than 7 days) increase that peaked after 22 days. Hypothalamic V1a and V1b mRNA levels were unaffected. In contrast, in the pituitary gland, there was an early decrease in CRH-R1 mRNA and V1b receptor mRNA, which returned to control levels from 24h onwards. In contrast to the prolonged activation of CRH and CRH-R1 mRNA expression in the PVN, there were no changes in the expression of the ACTH precursor molecule, POMC, V1b receptor, or CRH R1 mRNAs in the pituitary gland. These studies indicate that long-lasting hypothalamic adaptations that result in enhanced central CRH signaling contribute to the long-term sensitization of the HPA axis to transient exposure to immune events.

One particularly exciting finding was that chronic inflammatory stress in a rat model of autoimmune arthritis causes hypersensitivity of the HPA axis and the immune system to a novel immune challenge. Rats with adjuvant-induced arthritis show hyperactivity of the HPA axis accompanied by paradoxical decreases in CRH expression and increases in VP expression in parvocellular neurons of the PVN. In spite of blunted HPA axis responses to psychosensory stressors, the rats showed normal or exacerbated responses to the novel immune challenge of lipopolysaccharide (LPS) injection compared with control rats. Arthritic rats also showed much higher plasma IL-1 and IL6 levels and IL-1 and IL-6 mRNA in the brain than control rats after LPS injection. The data reveal an increased peripheral and central immunological response to LPS challenge during the chronic inflammatory process of arthritis. The fact that chronic inflammatory disorders can influence the responses to a novel immune challenge and enhance the induction of interleukins in the brain and periphery may be relevant for the pathogenesis of cytokine-mediated chronic degenerative disorders.

Regulation of Pituitary CRH and V1b VP Receptors
Rabadan-Diehl, Volpi
Regulation of the number of CRH and VP receptors in the pituitary plays an important role in the control of HPA axis activity. Studies in our laboratory 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. During the last year, the laboratory has focused on the regulation of the VP receptor V1b, which is the major subtype in the pituitary corticotroph. Our laboratory demonstrated that increased pituitary corticotroph responsiveness during chronic stress is associated with VP receptor up-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 is essential for positive regulation of the V1b receptor promoter and binds to a protein complex found in pituitary nuclear extracts. The protein complex appears to bind to DNA as a dimer of 70 kDa subunits. Transfection of a GAGA binding protein described in Drosophila markedly enhances V1b receptor promoter activity as well as the expression of endogenous V1b receptor in a hypothalamic cell line. In addition, stress increases the GAGA binding protein activity of pituitary nuclear extracts, suggesting that a GAGA binding protein plays a role in the physiological regulation of V1b receptor transcription. Studies of the full characterization of the GAGA binding protein complex and the role of other transcription factors on the regulation of the V1b receptor gene are ongoing.
Previous studies conducted in this laboratory indicated a lack of correlation between V1b receptor mRNA and V1b receptor binding, suggesting that regulation at post-transcriptional sites plays an important role in determining the number of VP receptors in the pituitary. In addition, studies during the past year showed that the 5' untranslated region (5'UTR) of the V1b receptor mRNA plays a significant role in controlling translation of the mRNA to receptor protein. 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. These data provide mechanisms by which the 5'UTR of the V1b mRNA can determine negative and positive regulation for translation of V1b receptor mRNA according to the physiological requirements.