Repression of DNA-binding dependent glucocorticoid receptor-mediated gene expression

The glucocorticoid receptor (GR) affects the transcription of genes involved in diverse processes, including energy metabolism and the immune response, through DNA-binding dependent and independent mechanisms. The DNA-binding dependent mechanism occurs by direct binding of GR to glucocorticoid response elements (GREs) at regulatory regions of target genes. The DNA-binding independent mechanism involves binding of GR to transcription factors and coactivators that, in turn, contact DNA. A small molecule that competes with GR for binding to GREs could be expected to affect the DNA-dependent pathway selectively by interfering with the protein-DNA interface. We show that a DNA-binding polyamide that targets the consensus GRE sequence binds the glucocorticoid-induced zipper (GILZ) GRE, inhibits expression of GILZ and several other known GR target genes, and reduces GR occupancy at the GILZ promoter. Genome-wide expression analysis of the effects of this polyamide on a set of glucocorticoid-induced and -repressed genes could help to elucidate the mechanism of GR regulation for these genes

Differential cytokine profiles upon comparing selective versus classic glucocorticoid receptor modulation in human peripheral blood mononuclear cells and inferior turbinate tissue

BACKGROUND: Glucocorticoid Receptor agonists, particularly classic glucocorticoids, are the mainstay among treatment protocols for various chronic inflammatory disorders, including nasal disease. To steer away from steroid-induced side effects, novel GR modulators exhibiting a more favorable therapeutic profile remain actively sought after. Currently, the impact of 2-(4-acetoxyphenyl)-2-chloro-N-methylethylammonium chloride a plant-derived selective glucocorticoid receptor modulator named compound A, on cytokine production in ex vivo human immune cells and tissue has scarcely been evaluated. METHODS AND RESULTS: The current study aimed to investigate the effect of a classic glucocorticoid versus compound A on cytokine and inflammatory mediator production after stimulation with Staphylococcus aureus-derived enterotoxin B protein in peripheral blood mononuclear cells (PBMCs) as well as in inferior nasal turbinate tissue. To this end, tissue fragments were stimulated with RPMI (negative control) or Staphylococcus aureus-derived enterotoxin B protein for 24 hours, in presence of solvent, or the glucocorticoid methylprednisolone or compound A at various concentrations. Supernatants were measured via multiplex for pro-inflammatory cytokines (IL-1beta, TNFalpha) and T-cell- and subset-related cytokines (IFN-gamma, IL-2, IL-5, IL-6, IL-10, and IL-17). In concordance with the previously described stimulatory role of superantigens in the development of nasal polyposis, a 24h Staphylococcus aureus-derived enterotoxin B protein stimulation induced a significant increase of IL-2, IL-1beta, TNF-alpha, and IL-17 in PBMCs and in inferior turbinates and of IL-5 and IFN-gamma in PBMCs. CONCLUSION: Notwithstanding some differences in amplitude, the overall cytokine responses to methylprednisolone and compound A were relatively similar, pointing to a conserved and common mechanism in cytokine transrepression and anti-inflammatory actions of these GR modulators. Furthermore, these results provide evidence that selective glucocorticoid receptor modulator-mediated manipulation of the glucocorticoid receptor in human tissues, supports its anti-inflammatory potential

The Anticancer Plant Triterpenoid, Avicin D, Regulates Glucocorticoid Receptor Signaling: Implications for Cellular Metabolism

Avicins, a family of apoptotic triterpene electrophiles, are known to regulate cellular metabolism and energy homeostasis, by targeting the mitochondria. Having evolved from “ancient hopanoids,” avicins bear a structural resemblance with glucocorticoids (GCs), which are the endogenous regulators of metabolism and energy balance. These structural and functional similarities prompted us to compare the mode of action of avicin D with dexamethasone (Dex), a prototypical GC. Using cold competition assay, we show that Avicin D competes with Dex for binding to the GC receptor (GR), leading to its nuclear translocation. In contrast to Dex, avicin-induced nuclear translocation of GR does not result in transcriptional activation of GC-dependent genes. Instead we observe a decrease in the expression of GC-dependent metabolic proteins such as PEPCK and FASN. However, like Dex, avicin D treatment does induce a transrepressive effect on the pro-inflammatory transcription factor NF-κB. While avicin's ability to inhibit NF-κB and its downstream targets appear to be GR-dependent, its pro-apoptotic effects were independent of GR expression. Using various deletion mutants of GR, we demonstrate the requirement of both the DNA and ligand binding domains of GR in mediating avicin D's transrepressive effects. Modeling of avicin-GR interaction revealed that avicin molecule binds only to the antagonist confirmation of GR. These findings suggest that avicin D has properties of being a selective GR modulator that separates transactivation from transrepression. Since the gene-activating properties of GR are mainly linked to its metabolic effects, and the negative interference with the activity of transcription factors to its anti-inflammatory and immune suppressive effects, the identification of such a dissociated GR ligand could have great potential for therapeutic use

Compound A, a Dissociated Glucocorticoid Receptor Modulator, Inhibits T-bet (Th1) and Induces GATA-3 (Th2) Activity in Immune Cells

Background: Compound A (CpdA) is a dissociating non-steroidal glucocorticoid receptor (GR) ligand which has antiinflammatory properties exerted by down-modulating proinflammatory gene expression. By favouring GR monomer formation, CpdA does not enhance glucocorticoid (GC) response element-driven gene expression, resulting in a reduced side effect profile as compared to GCs. Considering the importance of Th1/Th2 balance in the final outcome of immune and inflammatory responses, we analyzed how selective GR modulation differentially regulates the activity of T-bet and GATA-3, master drivers of Th1 and Th2 differentiation, respectively. Results: Using Western analysis and reporter gene assays, we show in murine T cells that, similar to GCs, CpdA inhibits T-bet activity via a transrepressive mechanism. Different from GCs, CpdA induces GATA-3 activity by p38 MAPK-induction of GATA-3 phosphorylation and nuclear translocation. CpdA effects are reversed by the GR antagonist RU38486, proving the involvement of GR in these actions. ELISA assays demonstrate that modulation of T-bet and GATA-3 impacts on cytokine production shown by a decrease in IFN-c and an increase in IL-5 production, respectively. Conclusions: Taken together, through their effect favoring Th2 over Th1 responses, particular dissociated GR ligands, fo

Keratinocyte-Targeted Overexpression of the Glucocorticoid Receptor Delays Cutaneous Wound Healing

Delayed wound healing is one of the most common secondary adverse effects associated to the therapeutic use of glucocorticoid (GC) analogs, which act through the ligand-dependent transcription factor GC-receptor (GR). GR function is exerted through DNA-binding-dependent and –independent mechanisms, classically referred to as transactivation (TA) and transrepression (TR). Currently both TA and TR are thought to contribute to the therapeutical effects mediated by GR; however their relative contribution to unwanted side effects such as delayed wound healing is unknown. We evaluated skin wound healing in transgenic mice with keratinocyte-restricted expression of either wild type GR or a mutant GR that is TA-defective but efficient in TR (K5-GR and K5-GR-TR mice, respectively). Our data show that at days (d) 4 and 8 following wounding, healing in K5-GR mice was delayed relative to WT, with reduced recruitment of granulocytes and macrophages and diminished TNF-α and IL-1β expression. TGF-β1 and Kgf expression was repressed in K5-GR skin whereas TGF-β3 was up-regulated. The re-epithelialization rate was reduced in K5-GR relative to WT, as was formation of granulation tissue. In contrast, K5-GR-TR mice showed delays in healing at d4 but re-established the skin breach at d8 concomitant with decreased repression of pro-inflammatory cytokines and growth factors relative to K5-GR mice. Keratinocytes from both transgenic mice closed in vitro wounds slower relative to WT, consistent with the in vivo defects in cell migration. Overall, the delay in the early stages of wound healing in both transgenic models is similar to that elicited by systemic treatment with dexamethasone. Wound responses in the transgenic keratinocytes correlated with reduced ERK activity both in vivo and in vitro. We conclude that the TR function of GR is sufficient for negatively regulating early stages of wound closure, while TA by GR is required for delaying later stages of healing

The Calcitonin and Glucocorticoids Combination: Mechanistic Insights into Their Class-Effect Synergy in Experimental Arthritis

PMCID: PMC3564948This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Pharmacological levels of withaferin A (Withania somnifera) trigger clinically relevant anticancer effects specific to triple negative breast cancer cells

Withaferin A (WA) isolated from Withania somnifera (Ashwagandha) has recently become an attractive phytochemical under investigation in various preclinical studies for treatment of different cancer types. In the present study, a comparative pathway-based transcriptome analysis was applied in epithelial-like MCF-7 and triple negative mesenchymal MDA-MB-231 breast cancer cells exposed to different concentrations of WA which can be detected systemically in in vivo experiments. Whereas WA treatment demonstrated attenuation of multiple cancer hallmarks, the withanolide analogue Withanone (WN) did not exert any of the described effects at comparable concentrations. Pathway enrichment analysis revealed that WA targets specific cancer processes related to cell death, cell cycle and proliferation, which could be functionally validated by flow cytometry and real-time cell proliferation assays. WA also strongly decreased MDA-MB-231 invasion as determined by single-cell collagen invasion assay. This was further supported by decreased gene expression of extracellular matrix-degrading proteases (uPA, PLAT, ADAM8), cell adhesion molecules (integrins, laminins), pro-inflammatory mediators of the metastasis-promoting tumor microenvironment (TNFSF12, IL6, ANGPTL2, CSF1R) and concomitant increased expression of the validated breast cancer metastasis suppressor gene (BRMS1). In line with the transcriptional changes, nanomolar concentrations of WA significantly decreased protein levels and corresponding activity of uPA in MDA-MB-231 cell supernatant, further supporting its anti-metastatic properties. Finally, hierarchical clustering analysis of 84 chromatin writer-reader-eraser enzymes revealed that WA treatment of invasive mesenchymal MDA-MB-231 cells reprogrammed their transcription levels more similarly towards the pattern observed in non-invasive MCF-7 cells. In conclusion, taking into account that sub-cytotoxic concentrations of WA target multiple metastatic effectors in therapy-resistant triple negative breast cancer, WA-based therapeutic strategies targeting the uPA pathway hold promise for further (pre)clinical development to defeat aggressive metastatic breast cancer

Is there a role for glucocorticoid receptor beta in asthma?

Glucocorticoids (GCs) are routinely used as anti-inflammatory drugs in the treatment of asthma. They act through binding to glucocorticoid receptor α (GRα), which represses numerous genes encoding pro-inflammatory mediators. A hormone binding deficient GR isoform named GRβ has been isolated in humans. When overexpressed by transfection, GRβ may function as a dominant negative modulator of GRα. However, to act as such, GRβ has to be more abundant than GRα, and conflicting data have been obtained concerning the relative levels of the two isoforms in cell lines and freshly isolated cells. Moreover, the dominant negative effect was not confirmed by independent laboratories. In GC-resistant asthmatics, GRβ was expressed by an increased number of peripheral blood mononuclear cells (PBMCs), airway T cells, and cells found in skin biopsies of tuberculin responses. However, the relative amounts of GRα and GRβ in these cells were not determined. In GC-dependent asthmatics, PBMCs expressed GRα predominantly. No cells containing higher levels of GRβ than GRα have yet been reported in asthmatics. Even if the existence of such cells is demonstrated, the role of GRβ in asthma will remain a matter of controversy because functional studies have given discrepant data

The SMILE study: a study of medical information and lifestyles in Eindhoven, the rationale and contents of a large prospective dynamic cohort study

<p>Abstract</p> <p>Background</p> <p>Health problems, health behavior, and the consequences of bad health are often intertwined. There is a growing need among physicians, researchers and policy makers to obtain a comprehensive insight into the mutual influences of different health related, institutional and environmental concepts and their collective developmental processes over time.</p> <p>Methods/Design</p> <p>SMILE is a large prospective cohort study, focusing on a broad range of aspects of disease, health and lifestyles of people living in Eindhoven, the Netherlands. This study is unique in its kind, because two data collection strategies are combined: first data on morbidity, mortality, medication prescriptions, and use of care facilities are continuously registered using electronic medical records in nine primary health care centers. Data are extracted regularly on an anonymous basis. Secondly, information about lifestyles and the determinants of (ill) health, sociodemographic, psychological and sociological characteristics and consequences of chronic disease are gathered on a regular basis by means of extensive patient questionnaires. The target population consisted of over 30,000 patients aged 12 years and older enrolled in the participating primary health care centers.</p> <p>Discussion</p> <p>Despite our relatively low response rates, we trust that, because of the longitudinal character of the study and the high absolute number of participants, our database contains a valuable set of information.</p> <p>SMILE is a longitudinal cohort with a long follow-up period (15 years). The long follow-up and the unique combination of the two data collection strategies will enable us to disentangle causal relationships. Furthermore, patient-reported characteristics can be related to self-reported health, as well as to more validated physician registered morbidity. Finally, this population can be used as a sampling frame for intervention studies. Sampling can either be based on the presence of certain diseases, or on specific lifestyles or other patient characteristics.</p

Principles of Modular Tumor Therapy

Nature is interwoven with communication and is represented and reproduced through communication acts. The central question is how may multimodal modularly acting and less toxic therapy approaches, defined as modular therapies, induce an objective response or even a continuous complete remission, although single stimulatory or inhibitingly acting drugs neither exert mono-activity in the respective metastatic tumor type nor are they directed to potentially ‘tumor-specific’ targets. Modularity in the present context is a formal pragmatic communicative systems concept, describing the degree to which systems objects (cells, pathways etc.) may be communicatively separated in a virtual continuum, and recombined and rededicated to alter validity and denotation of communication processes in the tumor. Intentional knowledge, discharging in reductionist therapies, disregards the risk-absorbing background knowledge of the tumor’s living world including the holistic communication processes, which we rely on in every therapy. At first, this knowledge constitutes the validity of informative intercellular processes, which is the prerequisite for therapeutic success. All communication-relevant steps, such as intentions, understandings, and the appreciation of messages, may be modulated simultaneously, even with a high grade of specificity. Thus, modular therapy approaches including risk-absorbing and validity-modifying background knowledge may overcome reductionist idealizations. Modular therapies show modular events assembled by the tumor’s living world as an additional evolution-constituting dimension. This way, modular knowledge may be acquired from the environment, either incidentally or constitutionally. The new communicatively defined modular coherency of environment, i.e. the tumor-associated microenvironment, and tumor cells open novel ways for the scientific community in ‘translational medicine’