Endocrine Abstracts

Glucocorticoids act through the glucocorticoid receptor (GR) to regulate cell function. GR gamma (GRγ) is a constitutive exon3/4 splice variant, contributing 4–8% of the total GR transcripts and is highly conserved through mammalian evolution, but its biological function remains unknown. Using microarray analysis we identified the transcriptome response for GRγ in stable HEK-Flp cells. This revealed that although GRγ effectively modulated gene expression in response to the synthetic glucocorticoid dexamethasone, a significant proportion of the GRγ specific genes were regulated in the absence of ligand (when compared to control transfected or GRα expressing cells). Immunofluorescent microscopy suggested that GRγ was predominantly cytoplasmic at rest, showing distinctly slower ligand-induced nuclear translocation kinetics, compared to the more heterogeneously distributed GRα. The apparent paradox of GRγ regulation of transcription, from a cytoplasmic location suggested a novel mechanism of action, which may be through altered trafficking kinetics, or cytoplasmic signalling. A novel in-silico strategy, utilizing forward engineering (a conservative strategy) to construct the connection topology, and reverse engineering (a probabilistic inference model) to identify genomic regulatory responses with transcription factor networks, was used to investigate this further. Using this approach we identified a panel of transcription factors with inferred activity, specifically in the GRγ expressing cells in the absence of ligand. These included the SP1 and AP1 families of transcription factors, known GR partner proteins, and downstream targets of JNK and p38 pathways. Phosphoimmunoblot analysis revealed that GRγ activates the JNK signalling pathway independent of ligand, compared to GRα, providing a link between localisation of GRγ in the cytoplasm and ligand-independent regulation of target genes. GRγ therefore subserves a variety of biological effects that appear to be less reliant on ligand activation than GRα. The evolutionary conservation of GRγ also supports the importance of this mechanism of action.