Neuroendocrine Control Of The Stress Response

Child Health And Human Development

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Abstract

Summary: The purpose of the research is to study the cellular and molecular mechanisms of adaptation to stress with emphasis on the regulation of the various components of the hypothalamic pituitary adrenal (HPA) axis. This includes the expression of hypothalamic corticotropin releasing hormone (CRH) and vasopressin (VP), pituitary CRH and V1b VP receptors, and adrenal steroidogenesis. At the hypothalamic level, in vivo and in vitro studies suggest that cAMP can mediate positive and negative regulation of CRH transcription. Activation of CRH expression by cAMP is markedly dependent on downstream elements in the CRH gene. Stimulation of cAMP production by forskolin increased luciferase activity driven by the CRH-promoter. This was caused by increases in transcription, shown by nuclear run-on-assay, as well as increases in mRNA stability and translation, shown by experiments using tetracyclin inducible constructs. Addition of the 3 prime untranslated region downstream of luciferase in a CRH promoter driven reporter construct markedly potentiated foskolin-stimulated CRH luciferase activity due to a potentiation of cAMP-stimulated mRNA translation. Concerning negative regulation, in vivo studies showed that stress induces expression of the CREM isoform, inducible cAMP early repressor (ICER) in the hypothalamic paraventricular nucleus (PVN). Studies using the recently characterized cell lines (4B and H32) revealed that transfection of ICER markedly inhibit cAMP-induced stimulation of CRH promoter activity after cotransfection with a luciferase reporter gene driven by a CRH promoter fragment. Western blot analysis of nuclear extracts of cells incubated with forskolin revealed time dependent increases in ICER. This paralleled formation of ICER-CRH CRE complexes in the electromobility gel shift assay (EMSA) suggesting that endogenous levels of ICER can interact with the CRH CRE. EMSA using hypothalamic nuclear extracts of control and stressed rats and CRH CRE radiolabeled oligonuclotides showed several shifted bands, of which the two lower bands were supershifted with CREM antibody. Extension of studies on immune-neuroendocrine interactions showed that alterations in fluid homeostasis secondary to endotoxemia are due primarily to renal insensitivity to VP rather than defective hypothalamic VP secretion. These studies revealed marked and sustained decreases in VP V2 receptors and aquaporin 2 in kidney medulla following LPS induced endotoxemia, and that these alterations lead to impaired capacity to concentrate urine. Transcriptional and post-transcriptional mechanisms regulating the number of CRH and VP receptors in the pituitary play an important role in the control of HPA axis activity. An essential element regulating V1b receptor transcription is a GAGA box located in the proximal promoter, which binds a protein complex found in pituitary nuclear extracts. Stress causes transient increases in GAGA binding activity an effect possible mediated by VP. Experiments using the hypothalamic cell line, H32, which expresses endogenous VP receptors showed that VP caused a rapid increase in GAGA binding activity followed by a decline. This effect of VP paralleled changes in ERK phosphorylation and it was mimicked by epidermal growth factor (EGF) and blocked by inhibitors of the EGF receptor and MAPK pathway. While the early increase was blocked by intracellular calcium chelators, the declining phase was prevented by protein kinase C inhibition. Studies using receptor subtype specific analogs or different cell lines transfected with either V1a or V1b receptors showed that the pathways involved in transactivation of the MAPK pathway by VP are cell specific and independent of the V1 receptor subtype. This data suggest that VP released into the pituitary portal circulation during stress activates V1b receptor transcription through transactivation of the EGF receptor. At the translational level, studies showed that the 5 prime untranslated region (5?UTR) of the V1b receptor mRNA contains elements, which can inhibit or stimulate translation. While the presence of upstream open reading frames in the 5 prime untranslated region play a role maintaining low translational activity in basal conditions, an internal ribosome entry site (IRES) can initiate translation independently of cap. Stress conditions which upregulate V1b receptors stimulate IRES activity through protein kinase C and PI3 kinase dependent pathways, providing a mechanism for rapid stimulation of V1b receptor translation to meet physiological requirements.