Biosynthesis of corticotrophin-releasing hormone (CRH) in mouse corticotrophic tumour cells expressing the human proCRH gene: intracellular storage and regulated secretion

Abstract

The mouse corticotrophic tumour cell line AtT-20 naturally synthesizes pro-opiomelanocortin (POMC) which is proteolytically processed to N-POMC(1-76), ACTH, beta-lipotrophin and beta-endorphin. The processed products are stored in secretory vesicles and released upon stimulation with specific secretagogues. AtT-20 cells which have been stably transfected with the human corticotrophin-releasing hormone (CRH) gene store and secrete immunoreactive CRH. The present results demonstrate that the CRH precursor is proteolytically processed in the transfected cells to yield the 41 amino acid neuropeptide CRH(1-41). On stimulation with the secretagogue noradrenaline, CRH(1-41) was released into the medium, while the precursor was not. Whilst treatment of wild-type AtT-20 cells with exogenous CRH(1-41) (1 nM) caused a fourfold stimulation of ACTH release above basal levels, the peptide had no effect on ACTH release from the stably transfected cells R1 and R4. These results suggest that the endogenous CRH produced by the transfected R1 and R4 cells may cause down-regulation of their CRH receptors, and thus exogenous CRH cannot cause further stimulation of ACTH release in these cells. We propose that the CRH precursor is correctly processed in the transfected AtT-20 cells (R1 and R4) and that the foreign prohormone is sorted into the secretory pathway.

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“…This indicates that both hormones possess distinguishable signals whereby the sorting machinery of a particular cell type is able to differentially segregate these peptides to different vesicle populations. Our results using AtT20 cells, after neuronal differentiation, suggest that prohormones and proneuropeptides expressed in eukaryotic neurons could also be packaged into different subsets of secretory vesicles and secreted differentially in response to an extracellular stimulus (Castro, Brooke, Bullman, Hannah, Glynn & Lowry, 1991; M. J. Perone & M. G. Castro, unpublished observations). We used transfected AtT20 cells expressing human proCRH and endogenous POMC; these cells must be able to direct ACTH and proCRH-derived peptides into different secretory granule populations since the mature peptides could be differentially released in response to an extracellular stimulus.…”

Intracellular trafficking of prohormones and proneuropeptides: cell type‐specific sorting and targeting

“…This indicates that both hormones possess distinguishable signals whereby the sorting machinery of a particular cell type is able to differentially segregate these peptides to different vesicle populations. Our results using AtT20 cells, after neuronal differentiation, suggest that prohormones and proneuropeptides expressed in eukaryotic neurons could also be packaged into different subsets of secretory vesicles and secreted differentially in response to an extracellular stimulus (Castro, Brooke, Bullman, Hannah, Glynn & Lowry, 1991; Castro, unpublished observations). We used transfected AtT20 cells expressing human proCRH and endogenous POMC; these cells must be able to direct ACTH and proCRH-derived peptides into different secretory granule populations since the mature peptides could be differentially released in response to an extracellular stimulus.…”

The Nutrient and Energy Sensor Sirt1 Regulates the Hypothalamic-Pituitary-Adrenal (HPA) Axis by Altering the Production of the Prohormone Convertase 2 (PC2) Essential in the Maturation of Corticotropin-releasing Hormone (CRH) from Its Prohormone in Male Rats

“…Although these results demonstrate that Sirt1 regulates PC1 and PC2 expression in a positive manner, we did not investigate the independent contributions of each convertase on the Sirt1-mediated processing of pro-CRH. However, it is worthy of noting that although AtT20 cells have been used to characterize the processing of pro-CRH by PC1, these cells are adrenocorticotropic in nature and can be influenced by CRH signaling (51). Thus, Sirt1's effect on PC1 may be a secondary consequence to changes in secreted CRH in the in vitro system employed.…”

“…We then investigated whether Sirt1 manipulation affected CRH production and release. As N43-5 cells do not produce functional PC2, we utilized the AtT20 cell line in which pro-CRH processing had been previously characterized (20,22,51). AtT20 cells transfected with cDNAs encoding for preproCRH and preproPC2 were also transfected with Sirt1 or control cDNAs.…”