Manipulation of proprotein convertase 1 expression in AtT20 cells changes proopiomelanocortin processing and trafficking: Implications for ACTH regulatory pathways
Tom Day, Anne Warhurst, Robert L Oliver, Lynn E Pritchard & Anne White
We have previously shown that the ACTH precursor, POMC, is present in human plasma and is the predominant secreted POMC peptide. This suggests that POMC processing is incomplete in neuroendocrine cells and regulation of the post-translational cleavage pathway is important in controlling secretion of ACTH. We have therefore analysed the dynamics of POMC processing in both AtT20 pituitary adenoma cells and primary rat pituitary cells. In both cases, large quantities of POMC, as well as ACTH, were detected in media. Acute treatment of cells with either BaCl2 or CRH stimulated the release of ACTH but not POMC, demonstrating that POMC is not released from secretory vesicles. Chronic treatment of AtT20 cells with CRH led to a significant increase in ACTH:POMC ratio in media, suggesting that the extent of POMC processing is increased in pituitary cells in response to physiological cues.
We hypothesised that changes in the expression/activity of proprotein convertase 1 (PC1) may impact on intracellular trafficking and processing of POMC, leading to changes in ACTH:POMC ratio. To test the effect of changes in PC1 expression on POMC processing we transfected AtT20 cells with small interfering RNA interference (siRNAi) probes directed against (a) PC1 and (b) proSAAS, a putative endogenous PC1 inhibitor. PC1 knockdown increased POMC, and reduced ACTH secretion in response to BaCl2. These changes were proportional to percentage PC1 knockdown and indicated that PC1 regulates the extent of POMC to ACTH conversion in secretory granules. Interestingly, PC1 inhibition also increased basal secretion of POMC, suggesting that PC1 plays a role in sorting/retention of POMC in the secretory pathway. In contrast, knockdown of proSAAS decreased secretion of POMC, indicating that changes in proSAAS expression may underlie altered PC1 activity.
These studies demonstrate that changes in PC1 expression regulate steady-state ACTH production by controlling POMC processing and sorting in vitro.