Role of NeuroD1 on the negative regulation of Pomc expression by glucocorticoid

Abstract

The mechanism of the negative regulation of proopiomelanocortin gene (Pomc) by glucocorticoids (Gcs) is still unclear in many points. Here, we demonstrated the involvement of neurogenic differentiation factor 1 (NeuroD1) in the Gc-mediated negative regulation of Pomc. Murine pituitary adrenocorticotropic hormone (ACTH) producing corticotroph tumor-derived AtT20 cells were treated with dexamethasone (DEX) (1–100 nM) and cultured for 24 hrs. Thereafter, Pomc mRNA expression was studied by quantitative real-time PCR and rat Pomc promoter (-703/+58) activity was examined by luciferase assay. Both Pomc mRNA expression and Pomc promoter activity were inhibited by DEX in a dose-dependent manner. Deletion and point mutant analyses of Pomc promoter suggested that the DEX-mediated transcriptional repression was mediated via E-box that exists at -376/-371 in the promoter. Since NeuroD1 is known to bind to and activate E-box of the Pomc promoter, we next examined the effect of DEX on NeuroD1 expression. Interestingly, DEX dose-dependently inhibited NeuroD1 mRNA expression, mouse NeuroD1 promoter (-2.2-kb) activity, and NeuroD1 protein expression in AtT20 cells. In addition, we confirmed the inhibitory effect of DEX on the interaction of NeuroD1 and E-box on Pomc promoter by chromatin immunoprecipitation (ChIP) assay. Finally, overexpression of mouse NeuroD1 could rescue the DEX-mediated inhibition of Pomc mRNA expression and Pomc promoter activity. Taken together, it is suggested that the suppression of NeuroD1 expression and the inhibition of NeuroD1/E-box interaction may play an important role in the Gc-mediated negative regulation of Pomc.

Role of glucocorticoid negative feedback in the regulation of HPA axis pulsatility

“…In addition, GR can inhibit Nur77-induced POMC transcription through a protein–protein interaction mechanism (Martens et al., 2005 ; Philips et al., 1997 ). A recent study suggests an involvement of NeuroD1 in regulating POMC transcription (Parvin et al., 2017 ). In the absence of CORT, NeuroD1 interacts with the E-box on the POMC promoter and activates transcription (Poulin et al., 1997 ).…”

“…In the absence of CORT, NeuroD1 interacts with the E-box on the POMC promoter and activates transcription (Poulin et al., 1997 ). Increased CORT concentration causes a repression of NeuroD1 expression, and hence less activation of POMC transcription (Parvin et al., 2017 ).…”

Inhibitory Effects of a Novel PPAR-γAgonist MEKT1 onPomcExpression/ACTH Secretion in AtT20 Cells

“…RNA isolation, cDNA synthesis, and quantitative real-time polymerase chain reaction (qPCR) were conducted as previously described [ 28 , 29 ]. To confirm the amplification specificity, the PCR products from each primer pair with SYBR green were subjected to a melting curve analysis.…”

“…Data were normalized by β -galactosidase activity. We followed our previously published protocol [ 29 ].…”

“…ChIP assay was performed using anti-Nur77/Nurr1 antibody (SC-990, Santa Cruz Biotechnology); anti TBX19 antibody (GTX77878, GeneTex); normal rabbit IgG (SC-2027, Santa Cruz Biotechnology); and NurRE (Nur response element), TpitRE (Tpit response element), and NBRE (Nur77 response element) region containing primers of mouse Pomc promoter. ChIP assay was conducted as described previously [ 29 ]. DNA fragments were treated with Proteinase K (Wako, Osaka, Japan) and Qiagen DNA Extraction kit was used for purification of DNA.…”