Endocrine Abstracts

Objective: Cushing’s disease is caused by hypercortisolism due to adrenocorticotropic hormone (ACTH) hypersecretion from functioning pituitary adenomas (FCA). Endoplasmic reticulum (ER) protein processing is important for hormone production and is upregulated in FCA as shown previously. ER protein processing can be disturbed by ER stress. We hypothesized that ACTH production and secretion can be inhibited by agents that cause ER stress by attenuating ER protein processing.

Methods: Cells from the AtT-20 mouse pituitary corticotroph tumor cell line and primary cells isolated from FCA were treated for 6 and 24 hours with 50-µM-Hsp90 inhibitor 17-N-allylamino-17-demethoxygeldanamycin (17-AAG, tanespimycin) and 250 nM of the sarco- and endoplasmatic reticulum Ca-ATPase inhibitor thapsigargin (Tg) to induce ER stress. The stimulation was followed by gene and protein expression analyses, and ACTH measurement in culture media and cell lysate.

Results: In AtT-20 cells, gene expression involved in ER protein processing, including CALR, GRP78, GRP94, UGGT1 were significantly upregulated by Tg and 17-AAG at 24 h compared with controls. Tg increased all unfolded proteins signalling pathway responses (ATF4, IRE1 and ATF6) at both 6 and 24 h, whereas 17-AAG increased only ATF4 at 6 h, and IRE1 at 24 h, and no effect was observed on ATF6. The ratio of s/uXBP1, member of IRE1 pathway, was downregulated by 17-AAG at both time points (P< 0.001 for both), suggesting that the cells are in recovery phase from ER stress. POMC gene expression was downregulated by Tg at both 6 and 24 h (P< 0.0001, both), whereas 17-AAG was upregulated at 6 h (P= 0.0497). At 24 h, Tg decreased secreted ACTH in media by 24% in AtT-20 cells (P< 0.0001) and 32% in primary cells (P< 0.0001) compared with controls. However, 17-AAG increased secreted ACTH by 64% (P= 0.0052) with marked response variation between FCA patients’ tumor cells. No effect of 17-AAG was observed in AtT-20 cells. Intracellular ACTH level in both cell types showed no change by Tg, while 17-AAG increased it in AtT-20 by 17% (P= 0.0075) and by 38% in primary cells (P= 0.0354).

Conclusion: ER stress may disturb ACTH production and secretion from the corticotroph tumour cells. The broad induction of ER stress by Tg effectively reduced ACTH production and secretion. On the contrary, targeted ER stress induced by 17-AAG showed increased ACTH production and secretion by FCA primary cells, possibly by inducing ER compensatory mechanisms.