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