The effects of all-trans retinoic acid on estrogen receptor signaling in the estrogen-sensitive MCF/BUS subline.

Estrogen receptor alpha (ERα) and retinoic acid receptors (RARs) play important and opposite roles in breast cancer growth. While exposure to ERα agonists such as 17ß-estradiol (E2) is related to proliferation, RAR agonists such as all-trans retinoic acid (AtRA) induce anti-proliferative effects. Although crosstalk between these pathways has been proposed, the molecular mechanisms underlying this interplay are still not completely unraveled. The aim of this study was to evaluate the effects of AtRA on ERα-mediated signaling in the ERα positive cell lines MCF7/BUS and U2OS-ERα-Luc to investigate some of the possible underlying modes of action. To do so, this study assessed the effects of AtRA on different ERα-related events such as ERα-mediated cell proliferation and gene expression, ERα-coregulator binding and ERα subcellular localization. AtRA-mediated antagonism of E2-induced signaling was observed in the proliferation and gene expression studies. However, AtRA showed no remarkable effects on the E2-driven coregulator binding and subcellular distribution of ERα. Interestingly, in the absence of E2, ERα-mediated gene expression, ERα-coregulator binding and ERα subcellular mobilization were increased upon exposure to micromolar concentrations of AtRA found to inhibit cell proliferation after long-term exposure. Nevertheless, experiments using purified ERα showed that direct binding of AtRA to ERα does not occur. Altogether, our results using MCF7/BUS and U2OS-ERα-Luc cells suggest that AtRA, without being a direct ligand of ERα, can indirectly interfere on basal ERα-coregulator binding and basal ERα subcellular localization in addition to the previously described crosstalk mechanisms such as competition of ERs and RARs for DNA binding sites.

Characterization of the differential coregulator binding signatures of the Retinoic Acid Receptor subtypes upon (ant)agonist action.

Retinoic Acid Receptor alpha (RARα/NR1B1), Retinoic Acid Receptor beta (RARß/NR1B2) and Retinoic Acid Receptor gamma (RARγ/NR1B3) are transcription factors regulating gene expression in response to retinoids. Within the RAR genomic pathways, binding of RARs to coregulators is a key intermediate regulatory phase. However, ligand-dependent interactions between the wide variety of coregulators that may be present in a cell and the different RAR subtypes are largely unknown. The aim of this study is to characterize the coregulator binding profiles of RARs in the presence of the pan-agonist all-trans-Retinoic Acid (AtRA); the subtype-selective agonists Am80 (RARα), CD2314 (RARß) and BMS961 (RARγ); and the antagonist Ro415253. To this end, we used a microarray assay for coregulator-nuclear receptor interactions to assess RAR binding to 154 motifs belonging to >60 coregulators. The results revealed a high number of ligand-dependent RAR-coregulator interactions among all RAR variants, including many binding events not yet described in literature. Next, this work confirmed a greater ligand-independent activity of RARß compared to the other RAR subtypes based on both higher basal and lower ligand-driven coregulator binding. Further, several coregulator motifs showed selective binding to a specific RAR subtype. Next, this work showed that subtype-selective agonists can be successfully discriminated by using coregulator binding assays. Finally this study demonstrated the possible applications of a coregulator binding assay as a tool to discriminate between agonistic/antagonistic actions of ligands. The RAR-coregulator interactions found will be of use to direct further studies to better understand the mechanisms driving the eventual actions of retinoids.

In Vitro Methodologies to Study the Role of Advanced Glycation End Products (AGEs) in Neurodegeneration.

Advanced glycation end products (AGEs) can be present in food or be endogenously produced in biological systems. Their formation has been associated with chronic neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, multiple sclerosis, and amyotrophic lateral sclerosis. The implication of AGEs in neurodegeneration is related to their ability to bind to AGE-specific receptors and the ability of their precursors to induce the so-called "dicarbonyl stress", resulting in cross-linking and protein damage. However, the mode of action underlying their role in neurodegeneration remains unclear. While some research has been carried out in observational clinical studies, further in vitro studies may help elucidate these underlying modes of action. This review presents and discusses in vitro methodologies used in research on the potential role of AGEs in neuroinflammation and neurodegeneration. The overview reveals the main concepts linking AGEs to neurodegeneration, the current findings, and the available and advisable in vitro models to study their role. Moreover, the major questions regarding the role of AGEs in neurodegenerative diseases and the challenges and discrepancies in the research field are discussed.

The Gut Microbial Metabolite Pyrogallol Is a More Potent Inducer of Nrf2-Associated Gene Expression Than Its Parent Compound Green Tea (-)-Epigallocatechin Gallate.

(-)-Epigallocatechin gallate (EGCG) has been associated with multiple beneficial effects. However, EGCG is known to be degraded by the gut microbiota. The present study investigated the hypothesis that microbial metabolism would create major catechol-moiety-containing microbial metabolites with different ability from EGCG to induce nuclear factor-erythroid 2-related factor 2 (Nrf2)-mediated gene expression. A reporter gene bioassay, label-free quantitative proteomics and reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) were combined to investigate the regulation of Nrf2-related gene expression after exposure of U2OS reporter gene or Hepa1c1c7 cells in vitro to EGCG or to its major microbial catechol-moiety-containing metabolites: (-)-epigallocatechin (EGC), gallic acid (GA) and pyrogallol (PG). Results show that PG was a more potent inducer of Nrf2-mediated gene expression than EGCG, with a 5% benchmark dose (BMD5) of 0.35 µM as compared to 2.45 µM for EGCG in the reporter gene assay. EGC and GA were unable to induce Nrf2-mediated gene expression up to the highest concentration tested (75 µM). Bioinformatical analysis of the proteomics data indicated that Nrf2 induction by PG relates to glutathione metabolism, drug and/or xenobiotics metabolism and the pentose phosphate pathway. Taken together, our findings demonstrate that the microbial metabolite PG is a more potent inducer of Nrf2-associated gene expression than its parent compound EGCG.

The Influence of Intracellular Glutathione Levels on the Induction of Nrf2-Mediated Gene Expression by α-Dicarbonyl Precursors of Advanced Glycation End Products.

α-Dicarbonyl compounds, particularly methylglyoxal (MGO), glyoxal (GO), and 3-deoxyglucosone (3-DG), are highly reactive precursors for the formation of advanced glycation end products (AGEs). They are formed in vivo and during food processing. This study aimed to investigate the role of intracellular glutathione (GSH) levels in the induction of Nrf2-mediated gene expression by α-dicarbonyl compounds. The reactions between α-dicarbonyl compounds (MGO, GO, and 3-DG) and GSH were studied by LC-MS in a cell-free system. It was shown that these three α-dicarbonyl compounds react instantaneously with GSH, with the GSH-mediated scavenging decreasing in the order MGO > GO > 3DG. Furthermore, in a cell-based reporter gene assay MGO, GO, and 3-DG were able to induce Nrf2-mediated gene expression in a dose-dependent manner. Modulation of intracellular GSH levels showed that the cytotoxicity and induction of the Nrf2-mediated pathway by MGO, GO and 3-DG was significantly enhanced by depletion of GSH, while a decrease in Nrf2-activation by MGO and GO but not 3-DG was observed upon an increase of the cellular GSH levels. Our results reveal subtle differences in the role of GSH in protection against the three typical α-dicarbonyl compounds and in their induction of Nrf2-mediated gene expression, and point at a dual biological effect of the α-dicarbonyl compounds, being reactive toxic electrophiles and -as a consequence- able to induce Nrf2-mediated protective gene expression, with MGO being most reactive.

Differences in kinetics and dynamics of endogenous versus exogenous advanced glycation end products (AGEs) and their precursors.

Advanced glycation end products (AGEs) and their precursors, referred to as glycation products, are a heterogenous group of compounds being associated with adverse health effects. They are formed endogenously and in exogenous sources including food. This review investigates the roles of endogenously versus exogenously formed glycation products in the potential induction of adverse health effects, focusing on differences in toxicokinetics and toxicodynamics, which appeared to differ depending on the molecular mass of the glycation product. Based on the available data, exogenous low molecular mass (LMM) glycation products seem to be bioavailable and to contribute to dicarbonyl stress and protein cross-linking resulting in formation of endogenous AGEs. Bioavailability of exogenous high molecular mass (HMM) glycation products appears limited, while these bind to the AGE receptor (RAGE), initiating adverse health effects. Together, this suggests that RAGE-binding in relevant tissues will more likely result from endogenously formed glycation products. Effects on gut microbiota induced by glycation products is proposed as a third mode of action. Overall, studies which better discriminate between LMM and HMM glycation products and between endogenous and exogenous formation are needed to further elucidate the contributions of these different types and sources of glycation products to the ultimate biological effects.

Pre-validation of a reporter gene assay for oxidative stress for the rapid screening of nanobiomaterials.

Engineered nanomaterials have been found to induce oxidative stress. Cellular oxidative stress, in turn, can result in the induction of antioxidant and detoxification enzymes which are controlled by the nuclear erythroid 2-related factor 2 (NRF2) transcription factor. Here, we present the results of a pre-validation study which was conducted within the frame of BIORIMA ("biomaterial risk management") an EU-funded research and innovation project. For this we used an NRF2 specific chemically activated luciferase expression reporter gene assay derived from the human U2OS osteosarcoma cell line to screen for the induction of the NRF2 mediated gene expression following exposure to biomedically relevant nanobiomaterials. Specifically, we investigated Fe3O4-PEG-PLGA nanomaterials while Ag and TiO2 "benchmark" nanomaterials from the Joint Research Center were used as reference materials. The viability of the cells was determined by using the Alamar blue assay. We performed an interlaboratory study involving seven different laboratories to assess the applicability of the NRF2 reporter gene assay for the screening of nanobiomaterials. The latter work was preceded by online tutorials to ensure that the procedures were harmonized across the different participating laboratories. Fe3O4-PEG-PLGA nanomaterials were found to induce very limited NRF2 mediated gene expression, whereas exposure to Ag nanomaterials induced NRF2 mediated gene expression. TiO2 nanomaterials did not induce NRF2 mediated gene expression. The variability in the results obtained by the participating laboratories was small with mean intra-laboratory standard deviation of 0.16 and mean inter laboratory standard deviation of 0.28 across all NRF2 reporter gene assay results. We conclude that the NRF2 reporter gene assay is a suitable assay for the screening of nanobiomaterial-induced oxidative stress responses.

Comparative study of the transcriptomes of Caco-2 cells cultured under dynamic vs. static conditions following exposure to titanium dioxide and zinc oxide nanomaterials.

Due to the widespread application of food-relevant inorganic nanomaterials, the gastrointestinal tract is potentially exposed to these materials. Gut-on-chip in vitro systems are proposed for the investigation of compound toxicity as they better recapitulate the in vivo human intestinal environment than static models, due to the added shear stresses associated with the flow of the medium. We aimed to compare cellular responses of intestinal epithelial Caco-2 cells at the gene expression level upon TiO2 (E171) and ZnO (NM110) nanomaterial exposure when cultured under dynamic and conventionally applied static conditions. Whole-genome transcriptome analyses upon exposure of the cells to TiO2 and ZnO nanomaterials revealed differentially expressed genes and related biological processes that were culture condition specific. The total number of differentially expressed genes (p < 0.01) and affected pathways (p < 0.05 and FDR < 0.25) after nanomaterial exposure was higher under dynamic culture conditions than under static conditions for both nanomaterials. The observed increase in nanomaterial-induced responses in the gut-on-chip model indicates that shear stress might be a major factor in cell susceptibility. This is the first report on the application of a gut-on-chip system in which gene expression responses upon TiO2 and ZnO nanomaterial exposure are evaluated and compared to a static system. It extends current knowledge on nanomaterial toxicity assessment and the influence of a dynamic environment on cellular responses. Application of the gut-on-chip system resulted in higher sensitivity of the cells and might thus be an attractive system for use in the toxicological hazard characterization of nanomaterials.

Transcriptome comparisons of in vitro intestinal epithelia grown under static and microfluidic gut-on-chip conditions with in vivo human epithelia.

Gut-on-chip devices enable exposure of cells to a continuous flow of culture medium, inducing shear stresses and could thus better recapitulate the in vivo human intestinal environment in an in vitro epithelial model compared to static culture methods. We aimed to study if dynamic culture conditions affect the gene expression of Caco-2 cells cultured statically or dynamically in a gut-on-chip device and how these gene expression patterns compared to that of intestinal segments in vivo. For this we applied whole genome transcriptomics. Dynamic culture conditions led to a total of 5927 differentially expressed genes (3280 upregulated and 2647 downregulated genes) compared to static culture conditions. Gene set enrichment analysis revealed upregulated pathways associated with the immune system, signal transduction and cell growth and death, and downregulated pathways associated with drug metabolism, compound digestion and absorption under dynamic culture conditions. Comparison of the in vitro gene expression data with transcriptome profiles of human in vivo duodenum, jejunum, ileum and colon tissue samples showed similarities in gene expression profiles with intestinal segments. It is concluded that both the static and the dynamic gut-on-chip model are suitable to study human intestinal epithelial responses as an alternative for animal models.