Pituitary Somatostatin Receptor Subtype 5 Modulates the Hypothalamic-Pituitary-Adrenal Axis Stress Response

Presentation Number: OR02-1
Date of Presentation: April 4th, 2017

Masaaki Yamamoto*, Anat Ben-Shlomo, Hiraku Kameda, Evelyn Ding and Shlomo Melmed
Cedars-Sinai Medical Center, Los Angeles, CA

Abstract

Adaptation to stress is critical for survival. The hypothalamic–pituitary–adrenal (HPA) axis regulates the stress response via hypothalamic corticotrophin releasing hormone (CRH), which stimulates ACTH secretion from pituitary corticotrophs to increase adrenal cortisol production. Under physiologic conditions, activation of pituitary corticotroph somatostatin receptor subtype 5 (SST5) inhibits ACTH secretion. As ACTH deficiency results in central adrenal insufficiency and an inability to appropriately respond to stress, we sought to clarify the role of SST5 in the HPA axis stress response.

We generated transgenic PH5 mice overexpressing SST5 in pituitary cells producing the ACTH precursor proopiomelanocortin (POMC). We compared their phenotype vs control wild type (WT) mice at baseline and under biochemical stress using CRH (40 μg/kg) and physiological stress using 30-minute restraint stress test and lipopolysaccharide (LPS, 10 and 5 mg/kg) administration. To elucidate molecular mechanisms underlying phenotypic stress responses, we created mouse pituitary AtT20 corticotroph cells stably overexpressing SST5 (SST5TG) or CRISPR/Cas9 SST5 knock-out (SST5KO) and compared gene expression and functional properties to WT cells.

PH5 mice showed no abnormalities in growth or fertility. At 10 months of age, both PH5 and WT females had 3-fold higher corticosterone levels vs males (p<0.001), and PH5 females had lower basal corticosterone (p=0.048) but not ACTH or fasting glucose level vs WT. We therefore used females as our model in the experiments. Thirty minutes after CRH injection, PH5 females exhibited reduced ACTH (p=0.03) and corticosterone (p=0.005) vs WT. ACTH and corticosterone were reduced in PH5 mice (p=0.01 and p=0.005, respectively) after 30-minute restraint test, and corticosterone was reduced 1 hour after LPS 5 mg/kg injection (p=0.04) and 3 hours after LPS 10 mg/kg (p=0.001). Mortality was higher in PH5 than WT mice under stressful conditions during restriction test (20% vs 0%), after 5 mg/kg LPS (33.3% vs 14%), and after 10 mg/kg LPS (50% vs 20%). PH5 pituitary corticotroph CRHR1 mRNA expression was reduced compared to WT. In SST5TG, POMC levels were attenuated, and CRH receptor subtype 1 (CRHR1) mRNA expression and cAMP production by CRH stimulation were reduced. In contrast, SST5KO cells exhibited increased CRHR1 mRNA expression and enhanced cAMP response to CRH. We isolated 8 microRNAs positively regulated by SST5 and potentially binding the 3’UTR of CRHR1, and found 12-fold enhanced expression of CRHR1 mRNA expression after treatment with miR-449 inhibitor.

Our findings show that SST5 attenuates the HPA axis stress response via downregulation of CRHR1 transcription, mediated in part by microRNAs, suggesting that corticotroph SST5 may contribute to the pathophysiology of ACTH failure and pituitary-derived adrenal insufficiency.

 

Disclosure: SM: Planning Group Member, Ipsen, Principal Investigator, Pfizer, Inc., Advisory Group Member, chiasma, Ad Hoc Consultant, ionis, Ad Hoc Consultant, Novartis Pharmaceuticals. Nothing to Disclose: MY, AB, HK, ED