Endocrine Abstracts

JOINT2893

Introduction: Glucocorticoids act via the glucocorticoid receptor (GR) to exert pleiotropic effects on all tissues, with heterogeneity in cellular response, aimed at regulating cellular and metabolic homeostasis. Recent advances in our mechanistic understanding of the GR demonstrate a more complex interplay of functions than traditionally considered. Multiple splice variants and translational isoforms of GR have been elucidated which contribute to cellular glucocorticoid sensitivity and may have diverse functions. The aim was to assess the GR isoform expression in PBMCs of individuals with varying states of glucocorticoid exposure.

Methods: Peripheral blood mononuclear cells (PBMC) were isolated from whole blood of healthy volunteers (HV), patients with endogenous Cushing syndrome (CS), adrenal insufficiency (AI) and treated with exogenous glucocorticoids for other conditions (GC). GR protein isoforms were measured via western blot. RNA was extracted from PBMC aliquots and RNA-Seq libraries prepared using Illumina Ribo-Zero plus and sequenced using NovaSeq X.

Results: PBMC were collected from 42 HV (15 male, 12 pre-menopausal and 15 post-menopausal females), 10 CS, 10 AI and 16 GC. The cohort were 50±1.8 years old, with the GC group being older, and had a mean BMI 27.2±0.8 kg/m² (which was significantly greater in the CS group). GR isoforms GRα-A, α-C, α-D1-3, -A and -P were identified in both the cytoplasm and nucleus of PBMCs across all groups. Isoforms GRα-C and D3 were the most frequently expressed (n=69). Cytoplasmic expression of GRα-C (P=0.005), GR-P (P=0.01), and GR-A (P=0.009) were lower in the GC group. After adjusting for age, BMI and sex/menopausal status, the lower cytoplasmic expression in of GR-P (P=0.002) in GC remained significant. No other differences were detected between groups including in nuclear isoform expression. Preliminary data suggest that isoform expression is correlated with both shared and unique gene expression. Genes correlated with GRα-A were enriched in pathways involving functions associated innate and adaptive immune function including T cell-mediated responses; GRα-D2 correlated genes were enriched in pathways associated with ubiquitin-proteosome function and GRα-D2 and GR-A-gene correlations were enriched in pathways associated with ATP production and mitochondrial function.

Conclusion: Previously described GR isoforms are present in PBMC of humans regardless of glucocorticoid exposure state. There appears to be an in vivo difference in gene regulatory function of individual isoforms which may contribute to the variation in clinical sequelae of glucocorticoid perturbations.