Twilight for the dissociated model of glucocorticoid action

DIEGO M. PRESMAN; MARIA FLORENCIA OGARA; MARTÍN STORTZ; LAUTARO D. ALVAREZ; JOHN R. POOLEY; R. LOUIS SCHILTZ; LARS GRØNTVED; THOMAS A. JOHNSON; PAUL R. MITTELSTADT; JONATHAN D. ASHWELL; SUNDAR GANESAN; GERARDO BURTON; VALERIA LEVI; ADALI PECCI; GORDON L. HAGER

Rockville, Maryland

Conferencia; Center for Cancer Research Fellows & Young Investigators Colloquium; 2015

National Cancer Institute, NIH

Background and HypothesesThe powerful anti-inflammatory and immunosuppressive action of glucocorticoids have made them one of the most prescribed drugs worldwide. Unfortunately, chronic treatment presents severe side-effects. If the desired anti-inflammatory action could somehow be detached from their side effects the clinical benefits would be massive. Glucocorticoids bind to the glucocorticoid receptor (GR), a ligand-dependent transcription factor. GR regulates gene expression directly by binding to DNA (transactivation) or indirectly by modulating the activity of other transcription factors (transrepression). It is currently accepted that the transactivation pathway is mostly responsible for glucocorticoids side-effects and that the oligomerization state of the GR (whether it is a dimer or a monomer) determines which pathway (direct or indirect) will prevail. This ?dissociated model? of GC action owns it genesis to the characterization of a point mutation in the GR, commonly known as the GRdim. Originally characterized as incapable of dimerization and DNA binding, this mutant does not transactivate but still transrepresses. Because the GRdim supports anti-inflammatory responses to GCs but does not mediate GC-induced side effects it was reasonable to believe that ligands that mimic GRdim properties on the wild type receptor would lead to the same dissociated properties of the GRdim mutation. Hence, GR ligands that preferentially induce the transrepression rather than the transactivation pathway should retain the anti-inflammatory effects of GCs, but will lack the undesired adverse reactions. Because the GRdim is allegedly monomeric, ligands that induce the monomeric form of the receptor should therefore have a better chance to retain desire actions and have a detriment in adverse reactions. The search for dissociated glucocorticoids under this paradigm has produced no significant results for the last 20 years. Study Design and MethodsWe re-analyzed some of the key premises of the dissociated model of glucocorticoid action. Different GR mutants were evaluated in terms of its oligomerization state, DNA binding capabilities and transcriptional activity. We performed the novel Number and Brightness analysis in living cells expressing fluorescently tagged GR. This technique, based on moment-analysis, provides the average number of moving fluorescent molecules and their brightness with high spatial resolution. With this information, the relative oligomerization state of a protein can be determined. In combination with ChIP and transcriptional activity assays we tested whether a correlation exists between GR?s oligomerization status, DNA-binding and transcriptional activity.Results and ConclusionsOur results suggest a complete, DNA-independent ligand-induced model for GR dimerization. We demonstrate that the GRdim forms dimers in vivo whereas adding another mutation in the ligand-binding domain (I634A) severely compromises homodimer formation. Contrary to dogma, no correlation between the GR monomeric/dimeric state and transcriptional activity was observed. This implies that the simplified monomer/dimer model equilibrium does not explain GR transactivation vs. transrepression activity. Relevance and importanceIt seems that the prevailing view was established without rigorous verification and new approaches for mitigating the side effects of chronic glucocorticoid treatment should be explored. These results have major implications on future searches for therapeutic glucocorticoids with reduced side effects.