Stress induces Parallel Changes in Corticotropin Releasing Hormone (CRH) Transcription and Nuclear Translocation of Transducer of Regulated CREB Activity 2 (TORC 2) in Hypothalamic CRH Neurons

摘要

Recent studies in vitro have shown that the CREB co-activator, Transducer of Regulated CREB activity (TORC), is required for transcriptional activation of the corticotropin releasing hormone (CRH) gene. To determine the physiological importance of TORC 2 regulating CRH transcription during stress, we examined the localization of TORC 2 in CRH neurons, and the relationship between changes in CRH hnRNA, nuclear translocation of TORC 2, and binding of TORC 2 to the CRH promoter. 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 4h after stress. Double staining immunohistochemistry showed TORC 2 co-staining in 100% of >detected CRH neurons, and nuclear translocation following 30 min restraint in 61%. Cellular distribution of TORC 2 in the dorsolateral PVN was unaffected by restraint. Chromatin immunoprecipitation (ChIP) experiments showed recruitment of TORC 2 and phospho-CREB by the CRH promoter following 30min restraint, but 4h 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.