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 in the hypothalamic cell line, 4B, demonstrated that the CREB co-activator, TORC, is required for activation of CRH transcription. 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, overexpression of TORC 1 or 2 increased basal CRH promoter activity and potentiated the stimulatory effect of forskolin but had no effect during incubationwith the phorbol ester PMA. 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 can 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. 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. Immunohistochemistry revealed TORC 2 immunoreactivity (irTORC 2) in the dorsolateral (magnocellular) and dorsomedial (parvocellular) regions of the hypothalamic paraventricular nucleus (PVN). While staining was mostly cytosolic in basal conditions, there was a marked increase in nuclear irTORC 2 in the dorsomedial region at 30 min restraint, concomitant with increases in CRH hnRNA levels. Levels of nuclear irTORC 2 and CRH hnRNA had returned to basal 3h after stress. Double staining immunohistochemistry showed TORC 2 co-staining in 100% of CRH neurons, and nuclear translocation following 30min restraint in 61%. Cellular distribution of TORC 2 in the dorsolateral PVN was unaffected by restraint. Chromatin immunoprecipitation (ChIP) experiments revealed recruitment of TORC 2 and phospho-CREB by the CRH promoter following 30min restraint, but 3h after stress only phospho-CREB was associated with the CRH promoter. The demonstration that TORC 2 translocates to the nucleus of hypothalamic CRH neurons and interacts with the CRH promoter in conjunction with the activation of CRH transcription during restraint stress, provides strong evidence for the involvement of TORC 2 in the physiological regulation of CRH transcription. Current research is focused on importance of the co-activator TORC during physiological regulation of CRH transcription in vivo and the mechanism regulating TORC activition and nuclear tarnslocation. 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. To examine potential epigenetic changes in the CRH promoter induced by early life stress, rats were subjected to maternal deprivation (MD) between 2 and 10 days post-birth. At 8-weeks of age, control and MD animals were sacrificed in basal conditions or following restraint stress for evaluation of HPA axis activity, including plasma corticosterone and CRH heteronuclear (hn) RNA. Additional groups were used for methylation analysis of the CRH promoter in the hypothalamic paraventricular nucleus (PVN). Corticosterone and CRH hnRNA responses to restraint stress were higher in MD rats. These results were observed in both sexes, and demonstrate that HPA axis hypersensitivity caused by neonatal stress involves enhanced CRH transcription in the PVN. DNA methylation analysis of the CRH promoter revealed decreased methylation of two CpG sites immediately preceding (Met-C1) and within (Met-C2) the cAMP-response element (CRE) in MD rats. This suggests that hypomethylation of the CRH promoter CRE is a mechanism for increased transcriptional responses to stress by affecting binding of the transcription factor cAMP response element binding protein, CREB. The molecular consequences of CRE methylation on CREB binding were examined by gel-shift assays, using CRH promoter CRE oligonucleotides methylated either at Met-C1 or Met-C2, both Met-C1 and 2, or without methylation. When labeled oligos were incubated with nuclear protein extracts from hypothalamic 4B cells and resolved on an acrylimide gel, a strong band corresponding to bound phosphorylated CREB (p-CREB) was observed for both the unmethylated and Met-C1 oligos. The identity of this shifted band was verified by supershift following incubation with a p-CREB antibody. Methylation of the intra CRE CpG, Met-C2, significantly decreased pCREB binding by 50%, and a similar decrease in binding was observed for the Met-C1 and 2 oligo. These results demonstrate that the methylation state of the intra CRE CpG of the CRH promoter significantly affects transcription factor binding, providing a molecular mechanism for the long-term effects of maternal deprivation on CRH transcription.
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