Insights on Glucocorticoid Receptor Activity Modulation through the Binding of Rigid Steroids

Abstract

BackgroundThe glucocorticoid receptor (GR) is a transcription factor that regulates gene expression in a ligand-dependent fashion. This modular protein is one of the major pharmacological targets due to its involvement in both cause and treatment of many human diseases. Intense efforts have been made to get information about the molecular basis of GR activity.Methodology/Principal FindingsHere, the behavior of four GR-ligand complexes with different glucocorticoid and antiglucocorticoid properties were evaluated. The ability of GR-ligand complexes to oligomerize in vivo was analyzed by performing the novel Number and Brightness assay. Results showed that most of GR molecules form homodimers inside the nucleus upon ligand binding. Additionally, in vitro GR-DNA binding analyses suggest that ligand structure modulates GR-DNA interaction dynamics rather than the receptor's ability to bind DNA. On the other hand, by coimmunoprecipitation studies we evaluated the in vivo interaction between the transcriptional intermediary factor 2 (TIF2) coactivator and different GR-ligand complexes. No correlation was found between GR intranuclear distribution, cofactor recruitment and the homodimerization process. Finally, Molecular determinants that support the observed experimental GR LBD-ligand/TIF2 interaction were found by Molecular Dynamics simulation.Conclusions/SignificanceThe data presented here sustain the idea that in vivo GR homodimerization inside the nucleus can be achieved in a DNA-independent fashion, without ruling out a dependent pathway as well. Moreover, since at least one GR-ligand complex is able to induce homodimer formation while preventing TIF2 coactivator interaction, results suggest that these two events might be independent from each other. Finally, 21-hydroxy-6,19-epoxyprogesterone arises as a selective glucocorticoid with potential pharmacological interest. Taking into account that GR homodimerization and cofactor recruitment are considered essential steps in the receptor activation pathway, results presented here contribute to understand how specific ligands influence GR behavior.

The rigid steroid 21-hydroxy-6,19-epoxyprogesterone (21OH-6,19OP) is a dissociated glucocorticoid receptor modulator potentially useful as a novel coadjuvant in breast cancer chemotherapy

“…by direct tethering of NF-κB and AP-1 transcription factors [35,47], by down-regulating the p38 activity through the induction of MKP-1 phosphatase [48] and by inducing the expression of factors involved in controlling mRNA stability [49], 21-OH-6,19OP was unable to repress pRelA-and pcJun-dependent COX-2 induction but it did increase MKP-1 protein levels and consequently led to a decrease in COX-2 levels by p38 inactivation in A549 cells. This suggests a certain tissue selectivity of this compound towards the up-regulation of the MKP-1 mediated pathway over the direct transrepression mechanism, in accordance with the fact that the rigid steroid was shown to inhibit NF-κB and AP-1 activities in other cell types [15][16].…”

“…Upon GR binding, 19OP activates translocation of the receptor to the nucleus and its dimerization [15]. This rigid steroid has the potential function of a dissociated GC since it inhibits Rel A and activator protein 1 (AP-1) dependent gene expression in BHK and Cos-1 cells [15][16] but it is unable to induce tyrosine aminotransferase or to increase glycogen deposits in rat liver [14] and MMTV-LUC reporter gene expression in L929 fibroblasts [17] and in Cos-1 cells transfected with the GR receptor [16].…”

“…Nevertheless, the ability of the GRdim/DEX mutant to dimerize is actually under revision. Taking into account that the GR/21OH-6,19OP was able to dimerize but unable to induce the expression of genes driven by GREs [15], it seems possible that the increased levels of MKP-1 could be a consequence of the indirect transactivation pathway triggered by the rigid compound.…”

DNA binding triggers tetramerization of the glucocorticoid receptor in live cells

“…However, this paradigm has been established exclusively from in vitro studies, working mostly with the DBD fragment (8)(9)(10), only using the whole GR protein in rare cases (11,12). The small number of experiments performed in live cells only addresses the entire nuclear population, lacking specific information regarding the GR fraction bound to chromatin (13)(14)(15)(16). Furthermore, these studies were unable to discriminate between dimers or higher oligomeric states.…”

Roles of GILZ in protein metabolism of L6 muscle cells exposed to serum from septic rats

“…It is induced under stress and has an extensive and strong anti-inflammatory effect. It also promotes protein degradation and inhibits protein synthesis (Sen et al, 1997;Seene et al, 2003;Presman et al, 2010;Smith et al, 2010). GC functions in combination with the cytoplasmic glucocorticoid receptor (GR) and the biological effects of GC directly depend on the expression levels of the GR protein (McNally et al, 2000).…”