Glucocorticoid receptor-JNK interaction mediates inhibition of the JNK pathway by glucocorticoids

Inhibition of the c-Jun N-terminal kinase (JNK) pathway by glucocorticoids (GCs) results in AP-1 repression. GC antagonism of AP-1 relies mainly on the transrepression function of the GC receptor (GR) and mediates essential physiological and pharmacological actions. Here we show that GCs induce the disassembly of JNK from mitogen-activated protein kinase kinase 7 (MKK7) by promoting its association with GR. Moreover, we have characterized a hormone-regulated JNK docking site in the GR ligand-binding domain that mediates GR-JNK interaction. The binding of GR to JNK is required for inhibition of JNK activation and induction of inactive JNK nuclear transfer by GCs. The dissociation of these two hormone actions shows that JNK nuclear transfer is dispensable for the downregulation of JNK activation by GCs. Nonetheless, nuclear accumulation of inactive JNK may still be relevant for enhancing the repression of AP-1 activity by GCs. In this regard, chromatin immunoprecipitation assays show that GC-induced GR-JNK association correlates with an increase in the loading of inactive JNK on the AP-1-bound response elements of the c-jun gene.     

Glucocorticoid ligands specify different interactions with NF-kappaB by allosteric effects on the glucocorticoid receptor DNA binding domain

Glucocorticoids inhibit inflammation by acting through the glucocorticoid receptor (GR) and powerfully repressing NF-kappaB function. Ligand binding to the C-terminal of GR promotes the nuclear translocation of the receptor and binding to NF-kappaB through the GR DNA binding domain. We sought how ligand recognition influences the interaction between NF-kappaB and GR. Both dexamethasone (agonist) and RU486 (antagonist) promote efficient nuclear translocation, and we show occupancy of the same intranuclear compartment as NF-kappaB with both ligands. However, unlike dexamethasone, RU486 had negligible activity to inhibit NF-kappaB transactivation. This failure may stem from altered co-factor recruitment or altered interaction with NF-kappaB. Using both glutathione S-transferase pull-down and bioluminescence resonance energy transfer approaches, we identified a major glucocorticoid ligand effect on interaction between the GR and the p65 component of NF-kappaB, with RU486 inhibiting recruitment compared with dexamethasone. Using the bioluminescence resonance energy transfer assay, we found that RU486 efficiently recruited NCoR to the GR, unlike dexamethasone, which recruited SRC1. Therefore, RU486 promotes differential protein recruitment to both the C-terminal and DNA binding domain of the receptor. Importantly, using chromatin immunoprecipitation, we show that impaired interaction between GR and p65 with RU486 leads to reduced recruitment of the GR to the NF-kappaB-responsive region of the interleukin-8 promoter, again in contrast to dexamethasone that significantly increased GR binding. We demonstrate that ligand-induced conformation of the GR C-terminal has profound effects on the functional surface generated by the DNA binding domain of the GR. This has implications for understanding ligand-dependent interdomain communication.     

Identification and characterization of glucocorticoid receptor-binding sites in the human genome

Glucocorticoids regulate gene expression via binding of the ligand-activated glucocorticoid receptor (GR) to glucocorticoid-responsive elements (GRE). To identify GR-binding sites, we developed a modified yeast one-hybrid system which enables rapid and efficient identification of genomic targets for DNA-binding proteins. The human GR expression vector was transformed into yeast cells containing a library of human genomic fragments cloned upstream of the reporter gene URA3. The genomic fragments with GR-binding sites were identified by growth of yeast clones in media lacking uracil but containing dexamethasone. DNA fragments were recovered by colony-direct PCR and GRE sequences were predicted by in silico analysis. Using electrophoretic mobility shift assay and fluorescence correlation spectroscopy, we demonstrated that 314 predicted GREs could directly interact with recombinant human GR proteins. In addition, when the genomic fragments were inserted in front of the heterologous SV40 promoter, at least 150 fragments could function as GREs in HEK293 cells. Furthermore, we identified four functional regulatory polymorphisms which may influence individual variation in sensitivity to glucocorticoids. These results provide insights into the molecular mechanisms underlying the physiological and pathological actions of glucocorticoid.