New insights into the anti-inflammatory mechanisms of glucocorticoids : an emerging role for glucocorticoid-receptor-mediated transactivation

Glucocorticoids are anti-inflammatory drugs that are widely used for the treatment of numerous (autoimmune) inflammatory diseases. They exert their actions by binding to the glucocorticoid receptor (GR), a member of the nuclear receptor family of transcription factors. Upon ligand binding, the GR translocates to the nucleus, where it acts either as a homodimeric transcription factor that binds glucocorticoid response elements (GREs) in promoter regions of glucocorticoid (GC)-inducible genes, or as a monomeric protein that cooperates with other transcription factors to affect transcription. For decades, it has generally been believed that the undesirable side effects of GC therapy are induced by dimer-mediated transactivation, whereas its beneficial anti-inflammatory effects are mainly due to the monomer-mediated transrepressive actions of GR. Therefore, current research is focused on the development of dissociated compounds that exert only the GR monomer-dependent actions. However, many recent reports undermine this dogma by clearly showing that GR dimer-dependent transactivation is essential in the anti-inflammatory activities of GR. Many of these studies used GR(dim/dim) mutant mice, which show reduced GR dimerization and hence cannot control inflammation in several disease models. Here, we review the importance of GR dimers in the anti-inflammatory actions of GCs/GR, and hence we question the central dogma. We summarize the contribution of various GR dimer-inducible anti-inflammatory genes and question the use of selective GR agonists as therapeutic agents

Glucocorticoid-induced microRNA-511 protects against TNF by down-regulating TNFR1

TNF is a central actor during inflammation and a well-recognized drug target for inflammatory diseases. We found that the mouse strain SPRET/Ei, known for extreme and dominant resistance against TNF-induced shock, displays weak expression of TNF receptor 1 protein (TNFR1) but normal mRNA expression, a trait genetically linked to the major TNFR1 coding gene Tnfrsf1a and to a locus harbouring the predicted TNFR1-regulating miR-511. This miRNA is a genuine TNFR1 regulator in cells. In mice, overexpression of miR-511 down-regulates TNFR1 and protects against TNF, while anti-miR-511 up-regulates TNFR1 and sensitizes for TNF, breaking the resistance of SPRET/Ei. We found that miR-511 inhibits endotoxemia and experimental hepatitis and that this miR is strongly induced by glucocorticoids and is a true TNFR1 modulator and thus an anti-inflammatory miR. Since minimal reductions of TNFR1 have considerable effects on TNF sensitivity, we believe that at least part of the anti-inflammatory effects of glucocorti-coids are mediated by induction of this miR, resulting in reduced TNFR1 expression

An acute phase protein ready to go therapeutic for sepsis

While APP are well-known inflammation biomarkers, A2MG found in sepsis patients' sera within lipid microparticles is an essential player in the host response to sepsis and has diagnostic as well as therapeutic potentials.image

LPS resistance of SPRET/Ei mice is mediated by Gilz, encoded by the Tsc22d3 gene on the X chromosome

Natural variation for LPS-induced lethal inflammation in mice is useful for identifying new genes that regulate sepsis, which could form the basis for novel therapies for systemic inflammation in humans. Here we report that LPS resistance of the inbred mouse strain SPRET/Ei, previously reported to depend on the glucocorticoid receptor (GR), maps to the distal region of the X-chromosome. The GR-inducible gene Tsc22d3, encoding the protein Gilz and located in the critical region on the X-chromosome, showed a higher expressed SPRET/Ei allele, regulated in cis. Higher Gilz levels were causally related to reduced inflammation, as shown with knockdown and overexpression studies in macrophages. Transient overexpression of Gilz by hydrodynamic plasmid injection confirmed that Gilz protects mice against endotoxemia Our data strongly suggest that Gilz is responsible for the LPS resistance of SPRET/Ei mice and that it could become a treatment option for sepsis

Glucocorticoid receptor-mediated transactivation is hampered by Striatin-3, a novel interaction partner of the receptor

The transcriptional activity of the glucocorticoid receptor (GR) is co-determined by its ability to recruit a vast and varying number of cofactors. We here identify Striatin-3 ( STRN3) as a novel interaction partner of GR that interferes with GR's ligand-dependent transactivation capacity. Remarkably, STRN3 selectively affects only GR-dependent transactivation and leaves GR-dependent transrepression mechanisms unhampered. We found that STRN3 down-regulates GR transactivation by an additional recruitment of the catalytic subunit of protein phosphatase 2A (PPP2CA) to GR. We hypothesize the existence of a functional trimeric complex in the nucleus, able to dephosphorylate GR at serine 211, a known marker for GR transactivation in a target gene-dependent manner. The presence of STRN3 appears an absolute prerequisite for PPP2CA to engage in a complex with GR. Herein, the C-terminal domain of GR is essential, reflecting ligand-dependency, yet other receptor parts are also needed to create additional contacts with STRN3

Glucocorticoid receptor dimers control intestinal STAT1 and TNF-induced inflammation in mice

TNF is an important mediator in numerous inflammatory diseases, e.g., in inflammatory bowel diseases (IBDs). In IBD, acute increases in TNF production can lead to disease flares. Glucocorticoids (GCs), which are steroids that bind and activate the glucocorticoid receptor (GR), are able to protect animals and humans against acute TNF-induced inflammatory symptoms. Mice with a poor transcriptional response of GR dimer-dependent target genes were studied in a model of TNF-induced lethal inflammation. In contrast to the GRWT/WT mice, these GRdim/dim mice displayed a substantial increase in TNF sensitivity and a lack of protection by the GC dexamethasone (DEX). Unchallenged GRdim/dim mice had a strong IFN-stimulated gene (ISG) signature, along with STAT1 upregulation and phosphorylation. This ISG signature was gut specific and, based on our studies with antibiotics, depended on the gut microbiota. GR dimers directly bound to short DNA sequences in the STAT1 promoter known as inverted repeat negative GRE (IR-nGRE) elements. Poor control of STAT1 in GRdim/dim mice led to failure to repress ISG genes, resulting in excessive necroptosis induction by TNF. Our findings support a critical interplay among gut microbiota, IFNs, necroptosis, and GR in both the basal response to acute inflammatory challenges and pharmacological intervention by GCs

Comprehensive overview of the structure and regulation of the glucocorticoid receptor

Glucocorticoids are among the most prescribed drugs worldwide for the treatment of numerous immune and inflammatory disorders. They exert their actions by binding to the glucocorticoid receptor (GR), a member of the nuclear receptor superfamily. There are several GR isoforms resulting from alternative RNA splicing and translation initiation of the GR transcript. Additionally, these isoforms are all subject to several transcriptional, post-transcriptional, and post-translational modifications, all of which affect the protein's stability and/or function. In this review, we summarize recent knowledge on the distinct GR isoforms and the processes that generate them. We also review the importance of all known transcriptional, post-transcriptional, and post-translational modifications, including the regulation of GR by microRNAs. Moreover, we discuss the crucial role of the putative GR-bound DNA sequence as an allosteric ligand influencing GR structure and activity. Finally, we describe how the differential composition and distinct regulation at multiple levels of different GR species could account for the wide and diverse effects of glucocorticoids

Study of the molecular mechanisms of glucocorticoid receptor function and resistance in TNF-induced toxicity

TNF is a pleiotropic cytokine described to play a prominent role in several inflammatory disorders, such as rheumatoid arthritis, inflammatory bowel disease (IBD) and psoriasis. For the treatment of many of these pathologies, synthetic glucocorticoids (GCs) are prescribed. GCs exert their function by binding to their receptor, the glucocorticoid receptor (GR), which is one of the strongest anti-inflammatory molecules encoded in mammalian genomes. However, the success of GC therapy is hampered due to two major drawbacks: i) long-term use of GCs is often accompanied with severe side effects and; ii) depending on the disease, a subset of patients do not respond to GC therapy, referred to as GC resistance (GCR). Moreover, there is limited knowledge on the exact molecular mechanisms of GC-mediated anti-inflammatory actions. These effects of GR are classically believed to result from tethering protein-protein interactions between GR and inflammatory transcription factors, such as NF-κB, which culminates in transrepression (TR) of a big cohort of pro-inflammatory mediators. Furthermore, the side-effects which result from long-term GC treatment were thought to be the result of transactivation (TA) of GC-inducible genes. Hence, research has invested in the generation of so-called Selective GR Agonists (SEGRAs), which favor TR and show a reduced side-effect profile. Such an uncoupling of TA and TR is supposed to be achievable since TA is thought to be based on GR dimerization, while TR is a GR monomer activity. In this thesis, we have aimed to elucidate the importance of TA of GR to resolve TNFinduced inflammation. Results of this PhD work provide evidence that GR dimers, and hence TA, are indispensable, and are even dominant over monomers, in the resolution of TNF-induced toxicity. Moreover, we also conclude that GR dimers protect mice against TNF-induced lethality i) by inducing the GR-dimer-inducible gene Dusp1, coding for the anti-inflammatory protein MKP-1, and hence inhibiting activity of JNK-2, intestinal damage and intestinal permeability; and ii) presumably by protecting the intestinal mucus barrier. For the latter aspect, more in-depth investigation is still warranted. We also were aiming to contribute to the clarification of the exact causes of GCR. We found some mechanisms responsible for GCR. Findings of this thesis demonstrate that TNF induces GCR via i) partly decreasing the levels of GR, independent from endogenous GC-mediated homologous down-regulation of GR; and ii) diminishing GR-DNA binding. As a consequence, the TA potential of GR and the subsequent induction of GC-dimerdependent genes coding for anti-inflammatory proteins is reduced. Both the reduced GR levels and the reduced GR-DNA binding were observations we made, but the exact upstream mechanisms behind these observations are still lacking, but are currently under investigation. In conclusion, in the acute TNF-induced lethal inflammation model, we find an important role for GR dimers, and hence gene induction, in the protection of GCs. It seems that precisely this important function of GR is the one that is undermined by TNF, not by inhibition of dimerization of GR, but by diminishing DNA binding. Three main directions of future studies emerge and should address the following questions: i) what is the actual mechanism of reduced DNA binding and is this effect generic (equally important for each GRE gene)?; ii) can the TNF effect be prevented by e.g. inhibitors of TNF downstream signaling molecules?; iii) is it possible to develop dimer-stimulating SEDIGRAs and, if so, do they have a therapeutic future

On the trail of the glucocorticoid receptor: into the nucleus and back

The glucocorticoid receptor (GR) belongs to the superfamily of steroid receptors and is an important regulator of physiological and metabolic processes. In its inactive state, GR is unbound by ligand and resides in the cytoplasm in a chaperone complex. When it binds glucocorticoids, it is activated and translocates to the nucleus, where it functions as a transcription factor. However, the subcellular localization of GR is determined by the balance between its rates of nuclear import and export. The mechanism of GR nuclear transport has been extensively studied. Originally, it was believed that nuclear import of GR is initiated by dissociation of the chaperone complex in the cytoplasm. However, several studies show that the chaperone machinery is required for nuclear transport of GR. In this review, we summarize the contribution of various chaperone components involved in the nuclear transport of GR and propose an updated model of its nuclear import and export. Moreover, we review the importance of ligand-independent nuclear transport and compare the nuclear transport of GR with that of other steroid receptors