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. The research employs a combination of in vivo and in vitro techniques, including measurement of gene expression, receptor binding and signaling pathways in brain, adrenal and pituitary of rodents subjected to experimental stress models or genetic modifications. Work during the past year focused on the regulation of CRH transcription and the physiological role of the increase in vasopressinergic activity during prolonged stimulation of the HPA axis.? ? Activation of CRH transcription during stress is rapid but transient in spite of persistent stimulation. The decline is independent of the increases in circulating glucocorticoids but it is associated with expression of inducible cAMP early repressor (ICER), an isoform of cAMP responsive element modulator (CREM), in CRH cells of the PVN. An exciting new finding was the fact that blockade of ICER formation by ICER siRNA oligonucleotides prevented the decline of CRH transcription. In these experiments, a reduction in ICER formation following 3h incubation with forskolin in cells transfected with ICER siRNA oligonucleotides was associated with sustained increases of CRH transcription. This was shown in reporter gene assays in 4B cells as enhanced CRH promoter-driven luciferase activity, or by measurement of primary transcript (CRH hnRNA) by qRT-PCR in primary hypothalamic cell cultures. These data confirm the hypothesis that ICER formation is responsible for the inhibitory phase of CRH transcription, and suggest that induction of ICER is part of an intracellular feedback mechanism limiting CRH transcription during stress. ? ? While cAMP appear to be required for positive and negative regulation of CRH transcription, the major regulators of CRH neurons (norepinephrine and glutamate) are not cAMP mediated, suggesting that other messenger systems activate CRH transcription by potentiating the effects of low cAMP levels. This hypothesis was examined in neuronal cell cultures treated with the phorbol ester, PMA, or the cAMP-stimulator, forskolin, or their combination. In the hypothalamic cell line, 4B, transfected with a CRH promoter-driven luciferase reporter, incubation with forskolin markedly increased CRH promoter activity. PMA alone had no effect but it potentiated the stimulatory effect of forskolin. Co-transfection of the dominant negative form of CREB (A-CREB) reduced forskolin-stimulated CRH promoter activity in the absence or presence of PMA, suggesting that pCREB is required for CRH transcriptional activation. However, the lack of effect of PMA on CRH promoter activity, in spite of marked CREB phosphorylation, suggests that pCREB must interact with other transcription factors to induce CRH transcription. PMA also potentiated the stimulatory effect of forskolin on CRH transcription (measured by intronic qRT-PCR) in primary cultures of hypothalamic cells. These data provide a mechanism by which potentiation of cAMP-induced transcription by phospholipid-dependent pathways mediate the marked activation of CRH transcription in response to non-cAMP dependent regulators during stress. ? ? Prolactin (PRL) produced in the brain exerts neuromodulatory effects, attenuating hypothalamo-pituitary-adrenal axis and behavioral stress responses, by acting upon PRL receptors (PRL-R) expressed in hypothalamic and limbic system areas. Studies aimed to understand the actions of PRL in the brain showed marked phosphorylation of ERK1/2 following i.c.v. injection of PRL in vivo or incubation of neuronal cell lines expressing PRL receptors with PRL. Immunohistochemical studies revealed strong co-localization of pERK and AVP in the SON, while in the PVN only a minor population of pERK-expressing cells were AVP-positive. Western blot of protein extracts from 4B cells with a PRL-R antibody showed a 40kDa band corresponding to the short form of the PRL-R. Incubation of the cells with PRL increased pERK from 5 min to 60 min, and potentiated the effect of serum on ERK1/2 phosphorylation. In 4B cells transfected with a CRH promoter-driven luciferase reporter gene, incubation with PRL for 6 h potentiated forskolin-stimulated CRH promoter activity, an effect which was prevented by ERK1/2 inhibitors. PRL induced the expression of early growth response genes 1 and 2 (egr-1 and egr-2) in an ERK1/2 dependent manner. The ability of PRL to induce ERK phosphorylation in the PVN and to increase CRH promoter activity in a hypothalamic cell line, suggests that PRL can directly modulate CRH neuron function. In addition, induction of Egr1/2 may mediate neuronal plasticity associated to high PRL production during lactation. ? ? Vasopressin produced by parvocellular neurons of the PVN potentiates CRH-stimulated pituitary ACTH secretion acting through plasma membrane receptors of the V1b subtype (V1bR). The expression of parvocellular VP and pituitary V1bR but not that of hypothalamic CRH and pituitary CRHR1 increases during chronic stimulation of the hypothalamic pituitary adrenal axis, suggesting that VP becomes the main regulator of ACTH secretion during long term adaptation to stress and that VP mediates the characteristic hyperresponsiveness of the HPA to a novel stress. To test this hypothesis, plasma ACTH responses to ip hypertonic saline injection (ipHS) were measured in rats subjected to daily handling or repeated restraint stress (1h/daily x 14 days) while receiving a minipump infusion of a peptide V1 antagonist or vehicle. The V1 antagonist blunted ACTH responses to exogenous VP without affecting the increases in blood pressure and heart rate induced by acute restraint. In handled rats, exposure to the novel stress of ipHS caused marked increases in plasma ACTH levels, an effect which was significantly reduced in the group receiving V1-antagonist. As expected, ACTH responses to ipHS were higher in repeatedly restrained rats than in handled controls but in contrast to handled rats, ACTH responses to ip HS were unaffected by the V1-antagonist. These data show that VP contributes to the ACTH responses to an acute stressor but that increased vasopressinergic activity is not responsible for the enhanced ACTH responses to the novel stress. ? ? The little impact of vasopressinergic blockade on HPA axis activity during chronic stress, suggests that VP has additional roles, such as controlling the number of pituitary corticotrophs. The role of VP mediating pituitary corticotroph mitogenesis was studied in adrenalectomized rats (ADX) by examining the effect of a peptide VP V1 receptor antagonist (V1-ant) on the number of cells incorporating bromouridine (BrdU). Long term ADX increased the number of both BrdU labeled cells and ACTH stained cells. Infusion of V1-ant for 28 days prevented ADX-induced increases in BrdU incorporation but not changes in the number of ACTH-stained cells. Unexpectedly, co-localization of BrdU uptake in ACTH positive cells was minor and it was unaffected by ADX or the V1-antagonist. No BrdU-stained nuclei were observed in LH, TSH, PRL, GH, folliculo-stellate cells or nestin-labeled progenitor cells. The pituitary corticotroph exclusive transcription factor, Tpit, colocalized in >80% of ACTH containing cells but only in 5% of BrdU labeled nuclei in controls and 10% in ADX rats. The data demonstrates that VP mediates mitogenic activity in the pituitary during long term ADX. The lack of colocalization of ACTH and BrdU, suggest that recruitment of corticotrophs during adrenalectomy occurs from undifferentiated cells.
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