Glucocorticoids (Gc) are the most potent anti-inflammatory agents known. A major factor limiting their clinical use is the wide variation in responsiveness to treatment between individuals and over time. Generating a reversible model of Gc resistance in vitro that will mimic the cellular microenvironment in vivo is critical in (i) defining biomarkers that predict patient steroid responsiveness; (ii) provide a model to screen new GR modulators capable of potentiating GR function. Various models were tested to induce readily reversible changes in Gc responses. Culture in 50% serum significantly impaired regulation of a simple Gc-responsive reporter (MMTV-luc) in a dose dependent manner. This was overcome by overexpressing the glucocorticoid receptor (GR) coactivator, SRC1, suggesting a reversible model of Gc resistance. Immunoblotting revealed no alteration in GR expression or phosphorylation after dexamethasone treatment that could account for the reduction of GR transactivation in 50% serum. Immunofluorescent illustrated no differences in the subcellular trafficking of GR, suggesting GR compartmentalisation was not affected by serum. Analysis of endogenous gene transactivation identified gene specific effects with FKBP5 and MT1X transactivation specifically impaired by serum shock. The resistant cell phenotype was reversed by 4 hours washout into 10%, or serum-free media, suggesting rapid reversal of the serum effect in target cells. Defining which serum factors induce this reversible model of Gc resistance will not only provide insight into Gc action but represent a tractable model for testing GR co-modulation.
Declaration of interest: There is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding: No specific grant from any funding agency in the public, commercial or not-for-profit sector.