SAT-117 - NR3C1 Signaling Is Required for Transcriptional Control of Somatotrope Differentiation and Function

Glucocorticoids control cellular proliferation, inflammation and metabolism through their binding to the glucocorticoid receptor, also known as nuclear receptor subfamily 3 group member 1 (NR3C1). They are essential modulators of pituitary development and endocrine function, yet their direct role in establishing somatotrope differentiation and growth hormone (GH) regulatory programs remains unclear. We hypothesized that NR3C1 signaling is required for differentiation of Pou1f1-positive progenitors to mature into functional somatotropes by directly regulating transcriptional networks controlling GH synthesis. To test this hypothesis, we generated Nr3c1 loss-of-function mutations in somatotrope-like GH3 cells using CRISPR–Cas9–mediated knockout with gRNAs targeting exon 2, which encodes the ligand independent NR3C1 transactivation domain. Nr3c1 knockout in GH3 cells was confirmed by its reduced protein expression on western blots. We treated the knockout clones and WT controls with 100 nM dexamethasone for 96 hours to compare glucocorticoid responsiveness and analyzed the effect of Nr3c1 knockout by accessing key somatotrope-associated genes, including Gh1, Ghrhr and Foxo1. Lack of Nr3c1 leads to a significant reduction in Gh1 and Foxo1 expression in untreated clones, and a failure in glucocorticoid induction of all three genes. FOXO1 binding to the Gh1 gene in WT cells is significantly increased with dexamethasone treatment, indicating glucocorticoids enhance FOXO1 binding to this target gene in somatotropes. Differential gene expression analysis of bulk RNA-Seq data comparing dexamethasone treated WT and Nr3c1 knockout cells shows significant downregulation of somatotrope identity and function genes, including Gh1, Ghr, Ghrhr and Foxo1. Furthermore, the zinc finger and BTB domain containing 16 (Zbtb16) gene, an established NR3C1 target gene which functions either as a transcriptional activator or repressor, was also significantly downregulated in the knockout clones. These findings support a model in which NR3C1 activity contributes to the transcriptional control of somatotrope differentiation and function. To define the direct genomic targets underlying these effects, ChIP sequencing of NR3C1 is being performed in wild-type cells. In conclusion, this work demonstrates the importance of NR3C1-regulated gene networks that govern somatotrope differentiation and function, providing mechanistic understanding of glucocorticoid control of pituitary endocrine development.