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The glucocorticoid receptor (GR) is a ligand-activated transcription factor. In this study, we used the yeast two-hybrid system to isolate cDNAs encoding proteins that interact with the human GR ligand-binding domain (LBD) in a ligand-dependent manner. One isolated cDNA from a HeLa cell library encoded the COOH-terminal portion of the -isoform of the 14-3-3 protein (residues 187-246). Glucocorticoid agonists, triamcinolone acetonide and dexamethasone, induced the GR LBD/14-3-3 protein fragment interaction, but an antagonist, RU486, did not. Glutathione S-transferase pull-down experiments in vitro showed that full-length 14-3-3 protein also interacted with the activated GR. Transient transfection studies using COS-7 cells revealed a stimulatory effect of 14-3-3 protein on transcriptional activation by the GR. The 14-3-3 family members have recently been found to associate with a number of important signaling proteins, such as protein kinase C and Raf-1, as functional modulators. Our findings suggest a novel regulatory role of 14-3-3 protein in GRmediated signaling pathways and also point to a mechanism whereby GR may cross-talk with other signal transduction systems.

In this project, the evidence for the non-monotonic dose-response (NMDR) hypothesis was evaluated by critically reviewing the scientific peer-reviewed literature in the last decade (from 2002 onwards) for substances in the area of food safety. The project was performed according to the systematic review methodology. After a detailed analysis of previous reports published on the issue of NMDRs, a literature search was performed to identify in vivo, in vitro and epidemiological/human studies containing evidence for potential NMDRs. Inclusion and reliability criteria were defined and used to select relevant and reliable studies. Of studies with at least 5 dose groups, dose-response datasets were extracted and analysed by PROAST software package. The resulting dose-response results were evaluated for possible evidence of NMDR by applying six checkpoints. These checkpoints addressed both random and non-random errors. The plausibility of NMDRs was assessed based on the number of fulfilled checkpoints. In total, 202 in vivo datasets (from 49 studies), 311 in vitro datasets (from 91 studies) and 9 epidemiological/human datasets (from 2 studies) were identified. Among them, 179 in vivo and 13 in vitro dose-response datasets were analysed and evaluated using the checkpoints. For 23 in vivo datasets there were data limitations, which made the data unsuitable for dose-response analysis. For the in vitro studies, only continuous dose-response datasets presented in tables were analysed. None of the datasets from epidemiological/human studies could be analysed (one of the epidemiological studies was not analysed due to quantal data and the other due to data limitations).In most of the in vivo datasets, the apparent NMDR might have been caused by a single outlying dose group. In total, only 10 out of the 179 in vivo datasets fulfilled all six checkpoints. The latter datasets included studies on the substances quercetin, resveratrol, alpha-benzene hexachloride, and methylmercury. Review of NMDR of substances for HRA ContributorsAGES: Boehm T., Coja T., Hrdina-Zoedl B., Pacher-Zavisin M., Steinparzer R.ANSES: Charles S., Gouze M.E., Guillou P., Lehegarat L., Manière I., Ormsby J.N., Papadopoulos A., Printemps N., Roudot A.C.RIVM: Smeet E. Acknowledgements:RIVM: Ossendorp B., Vermeire T.IMM: Gornitzki C., Moberg K., Karolinska Institutet University Library Reproduction is authorised provided the source is acknowledged.Permission to reproduce the images must be sought directly from the copyright holder SummaryThe overall objective of this project was to evaluate the evidence for the non-monotonic doseresponse (NMDR) hypothesis by critically reviewing the scientific peer-reviewed literature in the last decade (from 2002 onwards) for substances (other than essential nutrients) in the area of food safety. MethodologyThe project was based on a systematic review including the steps of Preparing the review (review protocol, review question and criteria for study eligibility), Searching for research studies, Selecting the stu...

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Proteins belonging to the 14--3-3 family interact with various regulatory proteins involved in cellular signaling, cell cycle regulation, or apoptosis. 14--3-3 proteins have been suggested to act by regulating the cytoplasmic/nuclear localization of their target proteins or by acting as molecular scaffolds or chaperones. We have previously shown that overexpression of 14--3-3 enhances the transcriptional activity of the glucocorticoid receptor (GR), which is a member of the nuclear receptor family. In this study, we show that 14--3-3 interacts with the nuclear receptor corepressor RIP140. In transfection assays, RIP140 antagonizes 14--3-3- enhanced GR transactivation. Using colocalization studies we demonstrate that 14--3-3 can export RIP140 out of the nucleus and, interestingly, can also change its intranuclear localization. Moreover, we also observed that 14--3-3 can bind various other nuclear receptors and cofactors. In summary, our findings suggest that 14--3-3-mediated intracellular relocalization of the GR corepressor RIP140 might be a novel mechanism to enhance glucocorticoid responsiveness of target genes. They furthermore indicate a more general role for 14--3-3 protein by influencing the nuclear availability of nuclear receptor-associated cofactors.

Regulation of gene transcription by nuclear receptors involves association with numerous coregulators.Receptor-interacting protein 140 (RIP140) is a corepressor that negatively regulates the ligand-induced activity of several nuclear receptors, including the glucocorticoid receptor (GR). In the present study, we have characterized the role of the intranuclear localization of RIP140 in its corepressor activity. In the absence of ligand-activated GR, RIP140 is localized in small nuclear foci targeted by a 40-amino-acid-long sequence. Although the focus-targeting domain overlaps with a binding sequence for the corepressor CtBP (C-terminal binding protein), interaction with CtBP is not involved in the localization. RIP140 foci do not correspond to PML bodies but partly colocalize with domains harboring the corepressor SMRT. Upon ligand binding, GR and RIP140 are redistributed to large nuclear domains distinct from the RIP140 foci. The redistribution requires regions of RIP140 with corepressor activity, as well as the DNA-binding domain of GR. Furthermore, we show that full RIP140 corepressor activity is contributed both by C-terminal receptor-binding LXXLL motifs and interaction with the CtBP corepressor. In conclusion, our results suggest that the corepressor function of RIP140 is multifaceted and involves binding to nuclear receptors, as well as additional functions mediated by the formation and intranuclear relocalization of a repressive protein complex.Nuclear hormone receptors are ligand-regulated transcription factors that may either activate or repress gene transcription. Transcriptional activation by nuclear receptors is dependent on the recruitment of numerous coregulator proteins to the receptors (for a review, see reference 18). These encompass ATP-dependent chromatin-remodeling proteins, proteins with histone acetyltransferase activity, and protein complexes involved in recruitment of the basal transcriptional machinery, including RNA polymerase II. Ligand-induced gene activation by nuclear receptors has been suggested to constitute a switch from a repressed state that is maintained by receptor interaction with corepressor proteins such as SMRT and NCoR, recruiting histone deacetylases (HDACs), to an activated state induced by coactivator proteins such as p160/SRC-1 proteins that have histone acetylase activity or recruit proteins that do. The C-terminal ligand-binding domain (LBD) of the receptor is the main docking site for the coregulators (1). Many coregulators have specific LXXLL motifs (NR boxes) that are involved in the interaction with the receptor LBDs (7, 13, 24).Receptor-interacting protein 140 (RIP140), also called nuclear receptor-interacting protein 1 (Nrip1), is a unique coregulator of nuclear receptors (3). It acts mainly as a corepressor reducing gene transcription but, in contrast to other corepressors that interact with unliganded or antagonist-activated receptors, RIP140 interacts with ligand-activated receptors. RIP140 has a restricted number of target proteins that only includes som...

The glucocorticoid receptor (GR) functions as a liganddependent transcription factor. In the present study we describe a specific immunoaffinity chromatography purification of GR from liver cytosol from adrenalectomized rats that may be used to identify hitherto unknown cytosolic GR interacting proteins. We have identified the ubiquitously expressed 14-3-3 as well as Raf-1, a downstream effector of Ras, as GR co-purifying proteins. In our semi-quantitative analysis liganded/activated GR showed the strongest interaction with 14-3-3 and Raf-1, but 14-3-3 was also found to co-purify with GR in a nonliganded/nonactivated state. By extensive salt washes we were also able to demonstrate that the glucocorticoid induced interaction between GR, 14-3-3, and Raf-1, respectively, is remarkably stable and withstood 2.4 M salt. The interaction between GR and 14-3-3 was also verified by 14-3-3 co-immunoprecipitation studies. Our observations that GR and Raf-1 are found within the same protein complex ("receptosome") in the cytoplasm of rat liver cells could provide a mechanistic explanation for glucocorticoid effects on the Raf-1-Ras signaling pathway.

This article reviews the current legislative requirements for risk assessment of combined exposure to multiple chemicals via multiple exposure routes, focusing on human health and particularly on foodrelated chemicals. The aim is to identify regulatory needs and current approaches for this type of risk assessment as well as challenges of the implementation of appropriate and harmonized guidance at international level. It provides an overview of the current legal requirements in the European Union (EU), the United States and Canada. Substantial differences were identified in the legal requirements for risk assessment of combined exposure to multiple chemicals and its implementation between EU and non-EU countries and across several regulatory sectors. Frameworks currently proposed and in use for assessing risks from combined exposure to multiple chemicals via multiple routes and different durations of exposure are summarized. In order to avoid significant discrepancies between regulatory sectors or countries, the approach for assessing risks of combined exposure should be based on similar principles for all types of chemicals. OECD and EFSA identified the development of harmonized methodologies for combined exposure to multiple chemicals as a key priority area. The Horizon 2020 project "EuroMix" aims to contribute to the further development of internationally harmonized approaches for such risk assessments by the development of an integrated test strategy using in vitro and in silico tests verified for chemical mixtures based on more appropriate data on potential combined effects. These approaches and testing strategies should be integrated in a scientifically based weight of evidence approach to account for complexity and uncertainty, to improve risk assessment.