Studies of this laboratory 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 hypothalamic pituitary adrenal (HPA) axis. Both peptides co-expressed in the same parvocellular neuron of the paraventricular nucleus (PVN) are differentially regulated during stress or exposure to glucocorticoids. CRH coordinates behavioral, autonomic and hormonal responses to stress and is the main regulator of ACTH secretion in acute and chronic conditions. Following CRH release, activation of CRH transcription is required to restore mRNA and peptide levels, but termination of the response is essential to prevent pathology associated with chronic elevation of CRH and glucocorticoid production. This laboratory has made important contributions on the understanding of the mechanisms controlling negative and positive transcriptional regulation of CRH. CRH transcription is under positive control by cAMP/phospho-CREB signaling and negatively regulated by glucocorticoid feedback. Concerning the positive regulation, this laboratory has reported solid evidence that cAMP/phospho-CREB signaling, believed to mediate activation of the CRH promoter, is essential but not sufficient to activate CRH transcription. This finding strongly suggested that transcriptional activation requires a co-activator of CREB. In a number of systems it has been shown that CREB mediated transcription potentially involved in the regulation of CRH transcription is Transducer Of Regulated CREB activity (TORC) is required for CREB mediated transcription. The ability of TORC to regulate CRH transcription was examined in the hypothalamic cell line 4B transfected with a CRH promoter driven luciferase reporter gene. Studies were performed in the hypothalamic cell line, 4B, to examine the hypothesis that the CREB co-activator, TORC, is required for activation of CRH transcription. This cell line does not express endogenous CRH but it has been proven to provide a good system for studying CRH transcription using reporter gene assays. Quantitative real time PCR indicated that all 3 TORC subtypes are present in 4B cells with TORC 2 being the most abundant expressed (TORC 2, 5-fold>TORC 1, 15x>TORC 3). Western blot analysis of cytoplasmic and nuclear proteins revealed rapid and transient nuclear translocation of TORC 2 and 3, and to a minor extent TORC 1, by forskolin in a dose dependent manner. In contrast, the phorbol ester, PMA, had no effect on nuclear TORC levels and caused a delay in migration in the cytoplasm suggesting hyper-phosphorylation. In reporter gene assays, co-transfection of expression vectors for TORC 1 or 2 increased basal CRH promoter activity and potentiated the stimulatory effect of forskolin. The phorbol ester PMA had no significant effect of CRH promoter activity, with or without TORC over-expression. Silencing RNA knock out of each endogenous TORC subtype partially inhibited forskolin-stimulated CRH promoter activity, while simultaneous knockout of TORC 2 and 3 was sufficient to prevent it. Co-immunoprecipitation and chromatin immunoprecipitation experiments revealed association of CREB and TORC in the nucleus, and recruitment of TORC 2 by the CRH promoter, following 30min incubation with forskolin. The data demonstrates that TORC 2 is required for transcriptional activation of the CRH promoter in the hypothalamic cell line 4B, by acting as a CREB co-activator. In addition, cytoplasmic retention of TORC during PMA treatment is likely to explain the failure of phorbolesters to activate CRH transcription in spite of efficiently phosphorylating CREB. The physiological relevance of these findings was studied in primary cultures of hypothalamic neurons in vitro and hypothalamic tissue from control and stressed rats. Western blot analysis of hypothalamic proteins and TORC 1, 2 and 3 antibodies revealed the presence of TORC 1 and 2 in cytoplasmic and nuclear fractions. Thirty min restraint stress caused a slight increase of TORC 2 in the nuclear fractions followed by a decrease to basal by 2h. There were no significant changes in TORC1 levels at either 30min or 2h of the stress in cytoplasmic of nuclear proteins. Similar results were observed in hypothalamic neuronal cultures following 20 min incubation with forskolin, with only TORC 2 translocating to the nucleus. Immunohistochemical studies using TORC2 antibody in rat hypothalamic tissue, revealed specific staining in the parvocellular and magnocellular areas of the paraventricular nucleus (PVN). Staining was mostly cytoplasmic in controls and 4h after restraint, at 30 min restraint, there was a significant increase in nuclear staining in the parvocellular but not magnocellular region. Double fluorescent immunostaining and confocal microscopy showed that 61 3.5% of CRH immunoreactive cells also stained for TORC2. The demonstration of the presence of TORC 2 in CRH neurons and its transient translocation to the nucleus during stress supports the involvement of this TORC subtype in CRH regulation. Current research is focused on the interactions of TORC with the CRH promoter and on the importance of the co-activator TORC during physiological regulation of CRH transcription in vivo. During the past year considerable effort was placed in studying the long-term consequences of early life stress on the function of the HPA axis. It is well recognized that stress exposure during early development causes long-lasting alterations in behavior and HPA axis activity, including increased levels of CRH mRNA in the PVN.
The aim of this study was to test the hypothesis that early life stress causes epigenetic changes in the CRH promoter leading to increased CRH transcription. Groups of 8-week old female and male rats, which had been subjected to maternal deprivation between days 2and 10 post-birth, were killed either in basal conditions, or following 30 or 60 min restraint stress for evaluation of ACTH and corticosterone, and CRH primary transcript or hnRNA levels (as an index of CRH transcription). Additional groups of control and MD were used for methylation analysis of the CRH promoter in the PVN and the amygdala. Adrenal weight, basal levels of plasma corticosterone and hypothalamic CRH hnRNA were higher in MD females but not in males. However, plasma corticosterone and CRH hnRNA responses to acute restraint stress were higher in MD of both sexes. DNA methylation analysis of the CRH promoter in the PVN and amygdala revealed a lower percent of methylation specifically in 2 CpGs located immediately preceding (1) and inside (2) the cAMP-responsive element (CRE) at -230 in the CRH promoter, in both sexes. This CRE has been shown to be an absolute requirement for activation of the CRH promoter. In contrast to the PVN, the percentage of methylation of CpGs 1 and 2 in the amygdala were identical in control in rats subjected to maternal deprivation. These findings demonstrate that HPA axis hypersensitivity caused by neonatal stress causes long-lasting enhanced CRH transcriptional activity in the PVN of both sexes. Hypomethylation of the -230 CRE in the CRH promoter is likely to serve as a mechanism for the increased transcriptional responses to stress observed in maternal deprivation in rats. Current efforts are directed to elucidate the functional consequences of hypomethylation of CpGs 1 and 2. Specifically studies will be performed to examine on the capacity of the CRH promoter CRE to bind the transcription factor, phospho-CREB, and the co-activator, TORC 2, as well as the association of acetylated histones and methyl DNA binding protein to the CRH promoter.
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