Exploring rapid non-genomic glucocorticoid signaling in hepatic cells through FRET-based biosensors

Glucocorticoids (GCs) are steroid hormones produced from cholesterol under the control of the hypothalamic–pituitary–adrenal axis. They play essential roles in metabolism, immune regulation, and stress adaptation, and synthetic analogs are widely used therapeutically. While the classical genomic actions of GCs are mediated by the glucocorticoid receptor (GR) acting as a transcription factor, increasing evidence indicates that GCs can also trigger rapid, non- genomic effects independent of direct DNA binding. These non-genomic pathways often involve membrane-associated or cytoplasmic GR pools that activate kinase signaling cascades such as cAMP/PKA. This study investigated compartment-specific, non-genomic GC signaling in hepatocyte- derived cells using FRET-based A-kinase activity reporter (AKAR) biosensors targeted to the plasma membrane, mitochondria, and nucleus. Treatment with hydrocortisone, dexamethasone, and the membrane-impermeable BSA-conjugated cortisol induced dose- dependent activation of PKA, with distinct spatial patterns. Notably, BSA-cortisol produced a rapid increase in PKA activity at the plasma membrane, demonstrating the presence of a membrane-initiated, GR-dependent signaling route. Pharmacological inhibition with mifepristone and the selective GR modulator relacorilant confirmed GR involvement. Mitochondrial and nuclear PKA activation were also detected at higher hormone concentrations, suggesting signal propagation across subcellular compartments. Nuclear PKA likely phosphorylates CREB and histone H3, linking acute non-genomic signaling to chromatin remodeling and transcriptional regulation. Immunofluorescence confirmed a membrane- associated GR population consistent with functional data. Overall, these findings demonstrate that glucocorticoids can activate PKA in distinct intracellular microdomains via GR-dependent, non-genomic mechanisms. This spatially i organized signaling integrates rapid metabolic regulation with long-term genomic effects and highlights new opportunities to pharmacologically target specific GR pools to reduce adverse outcomes of glucocorticoid therapy.