Endocrine Abstracts

The mechanisms which control the cell cycle and growth of adrenocortical cells remain largely uncharacterized. ACTH binds to its G protein-coupled receptor (GPCR), the melanocortin 2 receptor (MC2R) in the adrenal cortex and mediates the production of cAMP resulting in steroidogenesis. ACTH however also remains a candidate for the control of growth in the adrenal cortex. It is well known to have a growth promoting effect in vivo but has an opposing effect in some cell systems in which it is growth inhibitory. The aim of this study was to characterize the mitogenic signalling mechanisms initiated by ACTH in the human NCI-H295R adrenocortical cell line (H295R). Using Western blotting for phosphorylated Erk1/2 we studied the activation of this growth pathway in response to ACTH. We found a 5-10 fold increase in phosphorylated Erk1/2 in response to ACTH, maximally at 5 min, and maintained for up to 30 mins. Furthermore that this activation is likely to be mediated through the MC2R. We have demonstrated that this response is not mediated through a cAMP/PKA dependent mechanism unlike many other Gs coupled GPCRs, since the response is not inhibited by pre-treatment with H89. Furthermore we have shown that receptor internalization is a necessary pre-requisite for ACTH induced Erk1/2 activation since pre-treatment of cells with inhibitors of internalization, hypertonic sucrose and monodansylcadaverine, which reduce internalization of I125-labelled ACTH, also ameliorate the phosphorylation of Erk1/2 following ACTH treatment. We have further demonstrated that the induction of this pathway by ACTH is dependent on a src family kinase and does not require transactivation of a growth factor receptor.