Modulating glucocorticoid receptor actions in physiology and pathology: Insights from coregulators.

Glucocorticoids (GCs) are a class of steroid hormones that regulate key physiological processes such as metabolism, immune function, and stress responses. The effects of GCs are mediated by the glucocorticoid receptor (GR), a ligand-dependent transcription factor that activates or represses the expression of hundreds to thousands of genes in a tissue- and physiological state-specific manner. The activity of GR is modulated by numerous coregulator proteins that interact with GR in response to different stimuli assembling into a multitude of DNA-protein complexes and facilitate the integration of these signals, helping GR to communicate with basal transcriptional machinery and chromatin. Here, we provide a brief overview of the physiological and molecular functions of GR, and discuss the roles of GR coregulators in the immune system, key metabolic tissues and the central nervous system. We also present an analysis of the GR interactome in different cells and tissues, which suggests tissue-specific utilization of GR coregulators, despite widespread functions shared by some of them.

Agonist-controlled competition of RAR and VDR nuclear receptors for heterodimerization with RXR is manifested in their DNA binding.

We found previously that nuclear receptors (NRs) compete for heterodimerization with their common partner, retinoid X receptor (RXR), in a ligand-dependent manner. To investigate potential competition in their DNA binding, we monitored the mobility of retinoic acid receptor (RAR) and vitamin D receptor (VDR) in live cells by fluorescence correlation spectroscopy. First, specific agonist treatment and RXR coexpression additively increased RAR DNA binding, while both agonist and RXR were required for increased VDR DNA binding, indicating weaker DNA binding of the VDR/RXR dimer. Second, coexpression of RAR, VDR, and RXR resulted in competition for DNA binding. Without ligand, VDR reduced the DNA-bound fraction of RAR and vice versa, i.e., a fraction of RXR molecules was occupied by the competing partner. The DNA-bound fraction of either RAR or VDR was enhanced by its own and diminished by the competing NR's agonist. When treated with both ligands, the DNA-bound fraction of RAR increased as much as due to its own agonist, whereas that of VDR increased less. RXR agonist also increased DNA binding of RAR at the expense of VDR. In summary, competition between RAR and VDR for RXR is also manifested in their DNA binding in an agonist-dependent manner: RAR dominates over VDR in the absence of agonist or with both agonists present. Thus, side effects of NR-ligand-based (retinoids, thiazolidinediones) therapies may be ameliorated by other NR ligands and be at least partly explained by reduced DNA binding due to competition. Our results also complement the model of NR action by involving competition both for RXR and for DNA sites.

Doxorubicin impacts chromatin binding of HMGB1, Histone H1 and retinoic acid receptor.

Doxorubicin (Dox), a widely used anticancer DNA-binding drug, affects chromatin in multiple ways, and these effects contribute to both its efficacy and its dose-limiting side effects, especially cardiotoxicity. Here, we studied the effects of Dox on the chromatin binding of the architectural proteins high mobility group B1 (HMGB1) and the linker histone H1, and the transcription factor retinoic acid receptor (RARα) by fluorescence recovery after photobleaching (FRAP) and fluorescence correlation spectroscopy (FCS) in live cells. At lower doses, Dox increased the binding of HMGB1 to DNA while decreasing the binding of the linker histone H1. At higher doses that correspond to the peak plasma concentrations achieved during chemotherapy, Dox reduced the binding of HMGB1 as well. This biphasic effect is interpreted in terms of a hierarchy of competition between the ligands involved and Dox-induced local conformational changes of nucleosome-free DNA. Combined, FRAP and FCS mobility data suggest that Dox decreases the overall binding of RARα to DNA, an effect that was only partially overcome by agonist binding. The intertwined interactions described are likely to contribute to both the effects and side effects of Dox.

Agonist binding directs dynamic competition among nuclear receptors for heterodimerization with retinoid X receptor.

Retinoid X receptor (RXR) plays a pivotal role as a transcriptional regulator and serves as an obligatory heterodimerization partner for at least 20 other nuclear receptors (NRs). Given a potentially limiting/sequestered pool of RXR and simultaneous expression of several RXR partners, we hypothesized that NRs compete for binding to RXR and that this competition is directed by specific agonist treatment. Here, we tested this hypothesis on three NRs: peroxisome proliferator-activated receptor gamma (PPARγ), vitamin D receptor (VDR), and retinoic acid receptor alpha (RARα). The evaluation of competition relied on a nuclear translocation assay applied in a three-color imaging model system by detecting changes in heterodimerization between RXRα and one of its partners (NR1) in the presence of another competing partner (NR2). Our results indicated dynamic competition between the NRs governed by two mechanisms. First, in the absence of agonist treatment, there is a hierarchy of affinities between RXRα and its partners in the following order: RARα > PPARγ > VDR. Second, upon agonist treatment, RXRα favors the liganded partner. We conclude that recruiting RXRα by the liganded NR not only facilitates a stimulus-specific cellular response but also might impede other NR pathways involving RXRα.

Simultaneous Mapping of Molecular Proximity and Comobility Reveals Agonist-Enhanced Dimerization and DNA Binding of Nuclear Receptors.

Single Plane Illumination Microscopy (SPIM) revolutionized time lapse imaging of live cells and organisms due to its high speed and reduced photodamage. Quantitative mapping of molecular (co)mobility by fluorescence (cross-)correlation spectroscopy (F(C)CS) in a SPIM has been introduced to reveal molecular diffusion and binding. A complementary aspect of interactions is proximity, which can be studied by Förster resonance energy transfer (FRET). Here, we extend SPIM-FCCS by alternating laser excitation, which reduces false positive cross-correlation and facilitates comapping of FRET. Thus, different aspects of interacting systems can be studied simultaneously, and molecular subpopulations can be discriminated by multiparameter analysis. After demonstrating the benefits of the method on the AP-1 transcription factor, the dimerization and DNA binding behavior of retinoic acid receptor (RAR) and retinoid X receptor (RXR) is revealed, and an extension of the molecular switch model of the nuclear receptor action is proposed. Our data imply that RAR agonist enhances RAR-RXR heterodimerization, and chromatin binding/dimerization are positively correlated. We also propose a ligand induced conformational change bringing the N-termini of RAR and RXR closer together. The RXR agonist increased homodimerization of RXR suggesting that RXR may act as an autonomous transcription factor.

Mental health in Syrian children with a focus on post-traumatic stress: a cross-sectional study from Syrian schools.

PURPOSE: Studies show that conflict can negatively affect psychological health. The Syrian crisis is 8 years old and yet little is known about the impact of the conflict on the well-being of Syrians who remain. This gap was addressed by conducting an empirical study on the mental health burden of Syrian children in two areas of the country. METHODS: 492 children between 8 and 15 years were randomly selected from schools in Damascus and Latakia. The incidence of psychological disorder symptoms was measured using self-report screening instruments, the Children's Revised Impact of Event Scale (CRIES-8) and the Revised Children's Anxiety and Depression Scale (RCADS-25). Simultaneously, sociodemographic and traumatic event information was collected. Binary logistic regression was used to identify factors that influence the development of post-traumatic stress disorder (PTSD) symptoms. RESULTS: In our sample, 50.2% of students were internally displaced and 32.1% reported a negative experience. 60.5% of those tested had at least one probable psychological disorder with PTSD the most common (35.1%), followed by depression (32.0%), and anxiety (29.5%). Binary logistic regression indicated that PTSD symptoms were predicted by: living in Damascus [odds ratio (OR) 2.36, 95% confidence interval (CI) 1.51-3.69], being female (1.54, 1.02-2.34), having depression and anxiety (2.55, 1.48-4.40), and the negative experiences; displacement and daily warzone exposure (1.84, 1.02-3.30 and 2.67, 1.08-6.60). CONCLUSIONS: Syrian children are experiencing traumatic events and war-associated daily stresses that are hugely impacting psychological well-being. Our data offer guidance for mental health providers regarding risk factors and highlights the use of the school system to reach suffering children.