Abstract
The activation and nuclear translocation of cAMP‐response element binding protein (CREB)‐regulated transcription coactivator (CRTC)2 occurs in the rat adrenal gland, in response to adrenocorticotrophic hormone (ACTH) and stressors, and has been implicated in the transcriptional regulation of steroidogenic acute regulatory protein (StAR). We have recently demonstrated the activation of CRTC isoforms, CRTC1 and CRTC3, in adrenocortical cell lines. In the present study, we aimed to determine the activation and expression of the three CRTC isoforms in vivo in relation to Star transcription, under basal conditions and following a robust endotoxic stress challenge. Rat adrenal glands and blood plasma were collected following i.v. administration of either an ultradian‐sized pulse of ACTH or administration of lipopolysaccharide, as well as under unstressed conditions across the 24‐hour period. Plasma ACTH and corticosterone (CORT) were measured and the adrenal glands were processed for measurement of protein by western immunoblotting, RNA by a quantitative reverse transcriptase‐polymerase chain reaction and association of CRTC2 and CRTC3 with the Star promoter by chromatin immunoprecipitation. An increase in nuclear localisation of CRTC2 and CRTC3 followed increases in both ultradian and endotoxic stress‐induced plasma ACTH, and this was associated with increased CREB phosphorylation and corresponding increases in Star transcription. Both CRTC2 and CRTC3 were shown to associate with the Star promoter, with the dynamics of CRTC3 binding corresponding to that of nuclear changes in protein levels. CRTC isoforms show little variation in ultradian expression or variation across 24 hours, although evidence of long‐term down‐regulation following endotoxic stress was found. We conclude that co‐transcription factors CRTC2 and, more clearly, CRTC3 appear to act alongside phosphorylated CREB in the generation of ultradian pulses of Star transcription, essential for the maintenance of basal StAR expression. Similarly, our findings suggest CRTC2 and CRTC3 mediate Star transcriptional initiation following an endotoxic stressor; however, other transcription factors are likely to be responsible for the long‐term up‐regulation of adrenal Star transcription.
Highlights
The release of glucocorticoid hormones is tightly regulated by the hypothalamic-pituitary-adrenal (HPA) axis, enabling the body to adapt in response to stress alongside the autonomic nervous system.[1]
We demonstrate that nuclear translocation of endogenous CRTC2 and CRTC3 in the rat adrenal gland occurs in response to both basal, ultradian HPA axis activity, and following endotoxic stress, complimenting the findings from previous in vitro and in vivo studies in which activation of CRTC2 and CRCT3 was shown in response to adrenocorticotrophic hormone (ACTH) and restraint stress.[13,14,16,17,18]
Using a chromatin immunoprecipitation (ChIP) assay, we confirm that association of CRTC2 and CRTC3 with the Star promoter occurs in vivo, and demonstrate clear stimulation of CRTC3 binding at the Star promoter in response to ultradian and endotoxic stress stimuli during periods of increased Star transcription
Summary
The release of glucocorticoid hormones (corticosterone in rodents and as cortisol in humans, here referred as CORT) is tightly regulated by the hypothalamic-pituitary-adrenal (HPA) axis, enabling the body to adapt in response to stress alongside the autonomic nervous system.[1]. CRTC is a co-transcription factor that enhances binding of CREB to the gene promoter through its binding to the CREB bZIP domain, working independently of CBP.[12] CRTC is sequestered in the cytoplasm until activated by dephosphorylation, following increased intracellular cAMP levels.[13,14] This allows CRTC to translocate into the nucleus and bind CREB at the transcription site.[15] Three isoforms (CRTC1, 2 and 3) have been identified, with CRTC2 and CRTC3 shown to be the most highly expressed in the adrenal gland.[12,16] CRTC2 has long been implicated in the regulation of Star transcription in vitro[13,14] and in vivo, where nuclear levels of phosphorylated CREB (pCREB) and CRTC2 in the rat adrenal gland have been shown to increase in response to both high and low dose ACTH and restraint stress.[17,18] More recently, using murine adrenocortical cell lines, we have demonstrated that, in addition to CRTC2, ACTH stimulates rapid nuclear translocation of CRTC1 and CRTC3, and that both CRTC2 and CRTC3 bind at the Star promoter in response to ACTH, suggesting a role for these isoforms in mediating the initiation of Star transcription.[16]. Because several key steroidogenic regulators exhibit an ultradian and circadian pattern of expression in basal condition, as well as in response to stress, we investigated whether CRTC 1-3 exhibit strong endogenous regulation of transcription and expression
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