Analysis of the dissociated steroid RU24858 does not exclude a role for inducible genes in the anti-inflammatory actions of glucocorticoids

Although repression of inflammatory gene expression makes glucocorticoids powerful anti-inflammatory agents, side effects limit usage and drive the search for improved glucocorticoid receptor (GR) ligands. In A549 pulmonary cells, dexamethasone and the prototypical dissociated ligand RU24858 (Mol Endocrinol 11:1245-1255, 1997) repress interleukin (IL)-1 beta-induced expression of cyclooxygenase (COX)-2 and IL-8. Although RU24858 is a weaker GR ligand, both glucocorticoids showed similar efficacies on transrepression of nuclear factor KB (NF-KB)-dependent transcription, whereas RU24858 yielded less than 12% of the response to dexamethasone on a classic glucocorticoid response element (GRE) reporter (transactivation). Modest NF-KB-dependent transrepression (similar to 40%), along with analysis of IL-8 transcription rate and the accumulation of unspliced nuclear RNA, indicates that transrepression does not fully account for the repression of genes such as IL-8. This was confirmed by the finding that mRNA degradation is increased by both dexamethasone and RU24858. Analysis of IL-1 beta-induced steady-state mRNA levels for IL-8 and COX-2 show that dexamethasone- and RU24858-dependent repression of these genes is attenuated by inhibitors of transcription and protein synthesis. Because similar effects were observed with respect to COX-2 and IL-8 protein expression, we conclude that glucocorticoid-dependent gene expression is necessary for repression by both glucocorticoids. Despite RU24858 being defective at classic GRE-dependent transactivation, both dexamethasone and RU24858 induced the expression of potentially anti-inflammatory genes and metabolic genes, suggesting the importance of nontraditional glucocorticoid-dependent gene expression. Thus, classic transactivation- and transrepression-based screens for anti-inflammatory dissociated GR ligands may be flawed because they may not reflect the effects on real glucocorticoid-inducible genes.