Endocrine Abstracts

JOINT2764

In a physiological state, activation of hypothalamic-pituitary-adrenal (HPA) axis induces the release of ACTH by pituitary and, in turn, of cortisol from adrenal glands according to a circadian rhythm. HPA axis undergoes negative feedback regulation due to the elevated circulating cortisol that induces negative feedback on the hypothalamus and pituitary. In corticotroph cells, the control is provided by the coordinated action of the co-chaperones FKBP4 and FKBP5, which regulate the cytoplasmic and nuclear localization of glucocorticoid receptor (GR) and, consequently, the action of glucocorticoids (GCs). Cortisol excess in Cushing’s syndrome (CS) is associated with a complete loss of clock gene rhythmicity and reduced clock gene BMAL1 acrophase; therefore, the current study aimed at investigating the BMAL1 potential impacton GCs negative feedback on pituitary corticotroph cells. A wild-type murine corticotroph cell line, AtT20^WT, and AtT20 BMAL1 knockout (AtT20^KO), by CRISPR-Cas9, were used. GR, BMAL1, FKBP5, FKBP4 and POMC protein expression were evaluated by western blot (WB) in both cell models treated with or without dexamethasone 10^-7M (DEX) for 6 h and 24 h. Bioinformatic analysis predicted miRNAs targeting BMAL1, and concomitantly, the expression of these potential miRNAs was analyzed by RT-qPCR in both cell models treated with or without DEX and in 17 CS patients and 17 healthy subjects. AtT20^KO exhibited FKBP4 (91.2%, P=0.04 and FKBP5 (346%, P=0.002) higher expression compared to AtT20^WT. Interestingly, in AtT20^WT, DEX induced GR lower expression (52.2%, P=0.01) at 6 h, and FKBP5 higher expression (134%, P=0.03) and POMC lower expression (65.5%, P=0.01) at 24 h compared to untreated cells, while in AtT20^KO DEX effect on FKBP5 and POMC was lost and only lower expression of GR was observed at 6 h (40.7%, P=0.005) and 24 h (29.4%, P=0.03). Bioinformatic analysis revealed that miR-27a-3p and miR-27b-3p might target BMAL1. miR-27a-3p and miR-27b-3p circulating levels resulted upregulated in CS patients compared to healthy subjects (P<0.0001 respectively). AtT20^KO expressed miR-27b-3p lower levels (P=0.03) compared to AtT20^WT. Notably, 24 h DEX increased miR-27b-3p (P=0.02) in AtT20^WT but not in AtT20^KO. Even POMC could be a target of miR-27. Accordingly, AtT20^WT treated with 24 h DEX almost completely reduced POMC protein expression, but this effect was reverted when miR-27a-3p and miR-27b-3p inhibitors were added to DEX. In conclusion, these results added a small piece to the physiological knowledge by unveiling a new mechanism of negative feedback regulation of HPA at the pituitary level where BMAL1 expression allows DEX to increase miR-27b-3p levels, which in turn reduces POMC expression.