Corticotropin-Releasing Hormone Stimulates Proopiomelanocortin Transcription via the CaMKK/CaMKIV Pathway in the AtT20 Cell Line

Abstract

Proopiomelanocortin (POMC) is a critical precursor protein in the pituitary gland that regulates adrenal steroid hormone secretion by producing the adrenocorticotropic hormone (ACTH). Corticotropin-releasing hormone (CRH) modulates ACTH release via calcium influx through the voltage-operated Ca²⁺ channels and activation of Ca²⁺/calmodulin-dependent protein kinase II (CaMKII). In this study, we aimed to investigate the role of the calcium/calmodulin-dependent protein kinase kinase/ calcium/calmodulin-dependent protein kinase IV (CaMKK/CaMKIV) signaling cascade in CRH-induced POMC expression using ACTH-producing AtT20 cells, a cell line isolated from the pituitary gland of a mouse with tumor. Protein expression levels of CaMKK and CaMKIV were determined via western blotting. POMC transcription was analyzed via real-time polymerase chain reaction and reporter gene assays, and ACTH secretion was measured via enzyme-linked immunosorbent assay. In addition, effects of constitutively active CaMKK (CaMKK-CA) and CaMKIV (CaMKIV-CA) and their dominant-negative mutants on POMC promoter activity were assessed. CRH-induced CaMKIV phosphorylation was examined via western blotting. Both CaMKK and CaMKIV were expressed in the rat pituitary tissues; three random rats were used. Moreover, 10 nM CRH significantly increased POMC transcription and ACTH secretion in AtT20 cells. Inhibition of CaMKK and protein kinase A by STO-609 and H89, respectively, suppressed CRH-induced POMC transcription. Furthermore, CaMKK-CA and CaMKIV-CA independently activated the POMC promoter. CRH rapidly induced CaMKIV phosphorylation and nuclear localization, but these effects were blocked by STO-609. Overall, these findings suggest that the CaMKK/CaMKIV signaling pathway plays a crucial role in CRH-mediated POMC transcription and ACTH secretion in AtT20 cells.

Keywords: adrenocorticotropic hormone; att20; camkk/camkiv pathway; corticotropin-releasing hormone; proopiomelanocortin.

Figure 1. Expression levels of calmodulin-dependent protein kinase (CaMK)-IV and CaMKKα in rat pituitary tissues.

(a) CaMKIV levels. (b) CaMKKα levels. Protein levels in INS-1 cells were used as positive controls.

Figure 2. Roles of the protein kinase A (PKA)/CaMKK cascade in corticotropin-releasing hormone (CRH)-induced proopiomelanocortin (POMC) transcriptional activity and adrenocorticotropic hormone (ACTH) secretion in AtT20 cells.

Effects of CaMKK inhibitor STO-609, PKA inhibitor H89, phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002, or no treatment (control) on CRH (10 nM)-induced (a) POMC mRNA levels, (b) POMC promoter activity, and (c) ACTH secretion. Data were normalized to the control group data. Data are represented as the mean ± standard error of the mean (SEM). ^*P < 0.05 vs. control; ^#P < 0.05 vs. CRH derived from ordinary one-way ANOVA, followed by Tukey’s multiple-comparison test and Student’s t-test.

Figure 3. Effect of the CaMKK/CaMKIV cascade on the POMC promoter activity.

AtT20 cells were co-transfected with the rat POMC promoter-luciferase construct (r-pomc-Luc) or empty vector plasmid cloning DNA (pcDNA) and expression plasmids of (a) CaMKK-constitutively active (CA), CaMKK-dominant-negative (DN), (b) CaMKIV-CA, or CaMKIV-DN for 48 hours. Then, pcDNA- and CaMKK-DN/CaMKIV-DN-transfected cells were exposed to 10 nM CRH for 24 hours. The total luciferase activity was measured. Data were normalized to the control group data. Data are represented as the mean ± SEM. ^*P < 0.05 vs. control; ^#P < 0.05 vs. pcDNA + CRH derived from ordinary one-way ANOVA, followed by Tukey’s multiple-comparison test.

Figure 4. Time course of CaMKIV phosphorylation by CRH in AtT20 cells.

(a) CRH stimulates CaMKIV phosphorylation. The cells were exposed to 10 nM CRH for 0, 5, 10, 15, and 30 minutes. High phosphorylated CaMKIV levels were determined via western blotting analysis of the total cell proteins using the phospho-Thr196-CaMKIV antibody. The p-CaMKIV/CaMKIV ratio is shown as a percentage of the basal ratio. Data were normalized to the control group data. (b) CRH stimulates CaMKIV phosphorylation via the CaMKK signaling pathway. The cells were treated with the CAMKK inhibitor STO-609 and exposed to 10 nM CRH for 0, 5, 10, 15, and 30 minutes. Cells treated with CRH alone for 10 minutes were used as positive controls. The p-CaMKIV/CaMKIV ratio is shown as a percentage of the basal ratio. Data were normalized to the control group data. Data are represented as the mean ± SEM. ^*P < 0.05 derived from ordinary one-way ANOVA, followed by Dunnett’s multiple-comparison test.