ACTH signalling at the adrenal zona fasciculata induces rapid synthesis of glucocorticoids, accompanied by rapid transcription of genes encoding steroidogenic enzymes, including the labile, rate-limiting protein, StAR protein. ACTH induction of StAR transcription has been demonstrated to be predominantly CREB-dependent, and this mechanism is regulated by both CREB phosphorylation and its binding to the CREB co-activator TORC (transducer of regulated CREB activity, also known as CREB regulated transcription coactivator (CRTC)). Here we show that CREB phosphorylation, and nuclear localisation of the TORC2 and TORC3 isoforms follows ACTH treatment in adrenocortical Y1-BS1 and ATC7-L cells, and this precedes a rapid increase in star transcription (StAR hnRNA). Furthermore, by using chromatin immunoprecipitation we demonstrate that both isoforms bind the endogenous StAR promoter by 30 min after ACTH treatment in ATC7-L cells. In order to further establish the roles of these two isoforms during different types of HPA activity in the adrenal, we used two rat in vivo models; firstly, i.v. injection of a low dose of ACTH was used to mimic an ACTH ultradian pulse; secondly, rats were subjected to an immune stressor through an i.v. LPS challenge. Following both treatments, adrenals were harvested at various time-points and processed for RNA and protein measurements. As expected, we found rapid increases in both CREB phosphorylation and StAR hnRNA in both the i.v. pulse and LPS experiment. Interestingly, we found differential activation of the two TORC isoforms, with only significant TORC3 nuclear localisation induced by the i.v. ACTH ultradian pulse, whilst both TORC2 and TORC3 were induced by the immune stressor. These data demonstrate differing sensitivities of TORC2 and TORC3 to ACTH signalling, suggesting different roles for each in their mediation of ACTH-induced StAR transcription.
02 Nov 2015 - 04 Nov 2015