Neuromedin B Receptor Antagonist Suppresses Pomc Expression in AtT-20 Cells and Human Corticotroph Adenoma Cells

Abstract

Objective: We previously reported that Neuromedin B (NMB) is expressed in murine pituitary corticotrophs under adrenal insufficiency (1). Because NMB is also expressed in several cancer cells and stimulates ACTH secretion, we hypothesized that NMB is related to corticotroph adenoma cell proliferation and hormone secretion. To examine this hypothesis, we investigated the expression of NMB and its receptor NMBR in human corticotroph adenoma and the effects of a NMBR antagonist on AtT-20 cells, a mouse corticotroph adenoma cell line, and patient-derived corticotroph adenoma cells. Methods: 1. NMB and NMBR expression in human pituitary adenoma: We performed real-time qPCR and immunostaining on human pathological specimens of corticotrophs, somatotrophs, and non-functioning pituitary adenoma to investigate NMB and NMBR expression. 2. Experiments in AtT-20 cells: We extracted mRNAs and proteins from AtT-20 cells after incubation with 100nM NMBR antagonist PD168368, and performed real-time qPCR and western blotting analyses to investigate Pomc expression. 3. Experiments in patient-derived corticotroph adenoma cells: We isolated surgically resected human corticotroph adenoma cells from patients who underwent trans-sphenoidal surgery and investigated POMC mRNA expression by real-time qPCR after incubation with PD168368. Statistical analysis: One-way ANOVA was employed to compare values among multiple groups. If the ANOVA revealed significant differences, the Tukey-Kramer post-hoc test was employed to compare values between two specific groups. Dunnett’s post-hoc test was employed to compare values with the control group. Statistical significance was defined as p < 0.05. Results: 1. NMB and NMBR expression levels were significantly higher in human corticotroph adenoma (13 and 33 times higher than non-functioning adenoma, respectively) than in somatotroph adenoma (2 and 3 times higher than non-functioning adenoma, respectively) and non-functioning adenoma in the qPCR analyses. Immunostaining confirmed higher expression of NMB and NMBR in corticotroph adenoma than in somatotroph and non-functioning adenoma. 2. Treatment with 100 nM PD168368 significantly suppressed Pomc mRNA and protein expression in AtT-20 cells by 22%±3% and 25%±10%, respectively. 3. Treatment with 1 µM PD168368 significantly suppressed POMC mRNA expression in human corticotroph adenoma cells by 18%±1%. Conclusions: NMB and NMBR were both expressed in human corticotroph adenoma, suggesting that NMB may stimulate adenoma cell proliferation and hormone secretion in autocrine or paracrine manners. Because the NMBR antagonist suppressed Pomc expression in both AtT-20 cells and human corticotroph adenoma cells, it may represent a potential treatment for Cushing disease. Reference: (1) Kameda H et al., Endocrinology 2014;155(7):2492-9.