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Endocrine disrupting chemicals (EDCs) are able to deregulate the hormone system, notably through interactions with nuclear receptors (NRs). The mechanisms of action and biological effects of many EDCs have mainly been tested on human and mouse but other species such as zebrafish and xenopus are increasingly used as a model to study the effects of EDCs. Among NRs, peroxisome proliferator-activated receptor γ (PPARγ) is a main target of EDCs, for which most experimental data have been obtained from human and mouse models. To assess interspecies differences, we tested known human PPARγ ligands on reporter cell lines expressing either human, mouse, zebrafish, or xenopus PPARγ. Using these cell lines, we were able to highlight major interspecies differences. Known hPPARγ pharmaceutical ligands modulated hPPARγ and mPPARγ activities in a similar manner, while xPPARγ was less responsive and zfPPARγ was not modulated at all by these compounds. On the contrary, human liver X receptor (hLXR) ligands GW 3965 and WAY-252623 were only active on zfPPARγ. Among environmental compounds, several molecules activated the PPARγ of the four species similarly, e.g., phthalates (MEHP), perfluorinated compounds (PFOA, PFOS), and halogenated derivatives of BPA (TBBPA, TCBPA), but some of them like diclofenac and the organophosphorus compounds tri

Simple Summary Many kinase inhibitors have been tested as potential alternatives for the treatment of castration-resistant prostate cancers. However, none of these clinical trials led to drug approval despite interesting responses. Our study reveals that genes involved in drug metabolism and their master regulator PXR (Pregnane X Receptor) could be responsible, at least in part, for these disappointing results as they can modulate tumor cell response to specific kinase inhibitors. We found that stable expression of PXR sensitized prostate cancer cells to erlotinib, dabrafenib, and afatinib, while it rendered cells resistant to dasatinib and had no effect for other inhibitors tested. We also report for the first time that sensitization to afatinib is due to an alteration in drug transport that involves the SLC16A1 monocarboxylate transporter. Together, our results further indicate that PXR might be considered as a biomarker of response to kinase inhibitors in castration-resistant prostate cancers. Abstract Resistance to castration is a crucial issue in the treatment of metastatic prostate cancer. Kinase inhibitors (KIs) have been tested as potential alternatives, but none of them are approved yet. KIs are subject of extensive metabolism at both the hepatic and the tumor level. Here, we studied the role of PXR (Pregnane X Receptor), a master regulator of metabolism, in the resistance to KIs in a prostate cancer setting. We confirmed that PXR is expressed in prostate tumors and is more frequently detected in advanced forms of the disease. We showed that stable expression of PXR in 22Rv1 prostate cancer cells conferred a resistance to dasatinib and a higher sensitivity to erlotinib, dabrafenib, and afatinib. Higher sensitivity to afatinib was due to a ~ 2-fold increase in its intracellular accumulation and involved the SLC16A1 transporter as its pharmacological inhibition by BAY-8002 suppressed sensitization of 22Rv1 cells to afatinib and was accompanied with reduced intracellular concentration of the drug. We found that PXR could bind to the SLC16A1 promoter and induced its transcription in the presence of PXR agonists. Together, our results suggest that PXR could be a biomarker of response to kinase inhibitors in castration-resistant prostate cancers.

International audience; The estrogen-related receptor γ (ERRγ, NR3B3) is a constitutively active nuclear receptor which has been proposed to act as a mediator of the low-dose effects of a number of environmental endocrine-disrupting chemicals (EDCs) such as the xenoestrogen bisphenol-A (BPA). To better characterize the ability of exogenous compounds to bind and activate ERRγ, we used a combination of cell-based, biochemical, structural and computational approaches. A purposely created stable cell line allowed for the determination of the EC50s for over 30 environmental ERRγ ligands, including previously unknown ones. Interestingly, affinity constants (Kds) of the most potent compounds measured by isothermal titration calorimetry were in the 50–500 nM range, in agreement with their receptor activation potencies. Crystallographic analysis of the interaction between the ERRγ ligand-binding domain (LBD) and compounds of the bisphenol, alkylphenol and naphthol families revealed a partially shared binding mode and minimal alterations of the receptor conformation upon ligand binding. Further biophysical characterizations coupled to molecular dynamics simulations suggested a mechanism through which ERRγ ligands would exhibit their agonistic properties by preserving the transcriptionally active form of the receptor while rigidifying some loop regions with associated functions. This unique mechanism contrasts with the classical one involving a ligand-induced repositioning and stabilization of the C-terminal activation helix H12.

International audience; Bcl-2-associated athanogen-6 (BAG6) is a nucleocytoplasmic shuttling protein involved in protein quality control. We previously demonstrated that BAG6 is essential for autophagy by regulating the intracellular localization of the acetyltransferase EP300, and thus, modifying accessibility to its substrates (TP53 in the nucleus and autophagy-related proteins in the cytoplasm). Here, we investigated BAG6 localization and function in the cytoplasm. First, we demonstrated that BAG6 is localized in the mitochondria. Specifically, BAG6 is expressed in the mitochondrial matrix under basal conditions, and translocates to the outer mitochondrial membrane after mitochondrial depolarization with carbonyl cyanide m-chlorophenyl hydrazine, a mitochondrial uncoupler that induces mitophagy. In SW480 cells, the deletion of BAG6 expression abrogates its ability to induce mitophagy and PINK1 accumulation. On the reverse, its ectopic expression in LoVo colon cancer cells, which do not express endogenous BAG6, reduces the size of the mitochondria, induces mitophagy, leads to the activation of the PINK1/PARKIN pathway and to the phospho-ubiquitination of mitochondrial proteins. Finally, BAG6 contains two LIR (LC3-interacting Region) domains specifically found in receptors for selective autophagy and responsible for the interaction with LC3 and for autophagosome selectivity. Site-directed mutagenesis showed that BAG6 requires wild-type LIRs domains for its ability to stimulate mitophagy. In conclusion, we propose that BAG6 is a novel mitophagy receptor or adaptor that induces PINK1/PARKIN signaling and mitophagy in a LIR-dependent manner.

The environmental risk of natural and synthetic ligands of the nuclear progesterone receptor (nPR) has been pointed out, however there is still a lack of mechanistic information regarding their ability to interact with nuclear PR in aquatic species. To identify possible interspecies differences, we assessed in vitro the ability of manifold progestins to transactivate zebrafish (zf) and human (h) PRs, using two established reporter cell lines, U2OS-zfPR and HELN-hPR, respectively. Reference ligands highlighted some differences between the two receptors. The reference human agonist ligands promegestone and progesterone induced luciferase activity in both cell lines in a concentration-dependent manner, whereas the natural zebrafish progestin 17α,20β-dihydroxy-4-pregnen-3-one activated zfPR but not hPR. The potent human PR antagonist mifepristone (RU486) blocked PR-induced luciferase in both cell models but with different potencies. In addition, a set of 22 synthetic progestins were screened on the two cell lines. Interestingly, all of the tested compounds activated hPR in the HELN-hPR cell line, whereas the majority of them acted as zfPR antagonists in U2OS-zfPR. Such zfPR-specific response was further confirmed in zebrafish liver cells. This study provides novel information regarding the activity of a large set of progestins on human and zebrafish PR and highlights major interspecies differences in their activity, which may result in differential effects of progestins between fish and humans.

To characterize human nuclear receptor (NR) specificity of synthetic pharmaceutical chemicals we established stable cell lines expressing the ligand binding domains (LBDs) of human FXR, LXRα, LXRβ, CAR, and RORγ fused to the yeast GAL4 DNA binding domain (DBD). As we have already done for human PXR, a two-step transfection procedure was used. HeLa cells stably expressing a Gal4 responsive gene (HG5LN cell line) were transfected by Gal4-NRs expressing plasmids. At first, using these cell lines as well as the HG5LN PXR cells, we demonstrated that the basal activities varied from weak (FXR and LXRs), intermediate (PXR), to strong (CAR and RORγ), reflecting the recruitment of HeLa co-regulators in absence of ligand. Secondly, we finely characterized the activities of commercially available FXR, LXRα, LXRβ, CAR, RORγ, and PXR agonists/antagonists GW4064, feraxamine, DY268, T0901317, GW3965, WAY252623, SR9238, SR9243, GSK2033, CITCO, CINPA1, PK11195, S07662, SR1078, SR0987, SR1001, SR2211, XY018, clotrimazole, dabrafenib, SR12813, and SPA70, respectively. Among these compounds we revealed both, receptor specific agonists/antagonists, as well as less selective ligands, activating or inhibiting several nuclear receptors. FXR ligands manifested high receptor selectivity. Vice versa, LXR ligands behaved in non-selective manner, all activating at least PXR. CAR was selectively influenced by their ligands, while it also responded to several LXR ligands. Finally, although PXR was quite selectively activated or antagonized by its own ligands, it responded to several NRs ligands as well. Thus, using these reporter cell lines enabled us to precisely characterize the selectivity of pharmaceutical ligands for different nuclear receptors.

Prostate cancer is the most commonly diagnosed malignancy in men. Its growth mainly relies on the activity of the androgen receptor (AR), justifying the use of androgen deprivation therapy as a gold standard treatment for the metastatic disease. Inhibition of the androgen axis using second generation antagonists has improved patients&rsquo, survival, but is systematically confronted to resistance mechanisms, leading to a median survival that does not exceed 5 years. Counteracting this resistance has been the object of a large number of investigations, with a particular emphasis towards the identification of new AR inhibitors, whether they antagonize the receptor by a competitive or a non-competitive binding. To this end, many high content screens have been performed, to identify new non-steroidal AR antagonists, using a variety of approaches, but reported somewhat controversial results, depending on the approach and on the cell model that was used for screening. In our study, we used the U2OS osteosarcoma cells stably transfected with AR or ARv7 and a luciferase reporter as a previously validated model to screen the Prestwick Phytochemical library. The results of our screen identified ellipticine, harmol, and harmine hydrochloride as confirmed hits. Surprisingly, we could demonstrate that harmol hydrochloride, previously identified as a non-competitive inhibitor of AR or a weak inhibitor of androgen signaling, was actually a competitive antagonist of AR, which inhibits the growth of VCaP prostate cancer line, at concentrations for which it did not affect the growth of the AR negative DU145 and PC3 cells. Interestingly, we also report for the first time that harmol hydrochloride was selective for AR, as it could not alter the activity of other nuclear receptors, such as the glucocorticoid receptor (GR), the progesterone receptor (PR), or the mineralocorticoid receptor (MR). Additionally, we demonstrate that, conversely to enzalutamide, harmol hydrochloride did not show any agonistic activity towards the pregnane X receptor (PXR), a master regulator of drug metabolism. Together, our results shed light on the importance of the cellular context for the screening of new AR antagonists. They further indicate that some of the potential hits that were previously identified may have been overlooked.

The human pregnane X receptor (hPXR) is activated by a large set of endogenous and exogenous compounds and plays a critical role in the control of detoxifying enzymes and transporters regulating liver and gastrointestinal drug metabolism and clearance. hPXR is also involved in both the development of multidrug resistance and enhanced cancer cells aggressiveness. Moreover, its unintentional activation by pharmaceutical drugs can mediate drug&ndash, drug interactions and cause severe adverse events. In that context, the potential of the anticancer BRAF inhibitor dabrafenib suspected to activate hPXR and the human constitutive androstane receptor (hCAR) has not been thoroughly investigated yet. Using different reporter cellular assays, we demonstrate that dabrafenib can activate hPXR as efficiently as its reference agonist SR12813, whereas it does not activate mouse or zebrafish PXR nor hCAR. We also showed that dabrafenib binds to recombinant hPXR, induces the expression of hPXR responsive genes in colon LS174T-hPXR cancer cells and human hepatocytes and finally increases the proliferation in LS174T-hPXR cells. Our study reveals that by using a panel of different cellular techniques it is possible to improve the assessment of hPXR agonist activity for new developed drugs.

International audience; Bisphenol-A (BPA) is one of the most abundant chemicals produced worldwide. Exposure to BPA has been associated with various physiological dysregulations, involving reproduction, development, metabolism, as well as genesis and progression of hormone-dependent cancers. It has been well published that BPA along with its analogs bind and activate estrogen receptors (ER) α and β, estrogen related receptor (ERR) γ and pregnan X receptor (PXR). BPA has been also characterized as an inhibitor of the androgen (AR) and progesterone (PR) receptor. Thus, the need for safer alternatives to BPA among bisphenols is rising. In this regard, we used reporter cell lines to analyze the effects of 24 bisphenols on the selected nuclear receptors (NRs), known and potential targets of BPA. We showed that bisphenols differently modulated the activities of NRs. ERs, ERRγ and PXR were generally activated by bisphenols, whereas many compounds of this family acted as AR, PR, GR and MR antagonists. On the other hand, some bisphenols such as BPA, BPC and BPE modulated the activity of several NRs, but others lacked the activity of other NRs. Altogether, these data provide the guidelines for development of safer BPA substitutes with reduced hormonal activity.

The high volume production compound bisphenol A (BPA) is of environmental concern largely because of its estrogenic activity. Consequently, BPA analogues have been synthesized to be considered as replacement molecules for BPA. These analogues need to be thoroughly evaluated for their estrogenic activity. Here, we combined mechanism zebrafish-based assays to examine estrogenic and anti-estrogenic activities of BPA and two of its analogues, bisphenol AF (BPAF) and bisphenol C (BPC) in vitro and in vivo. In vitro reporter cell lines were used to investigate agonistic and antagonistic effects of the three bisphenols on the three zebrafish estrogen receptors. The transgenic Tg(5 × ERE:GFP) and Cyp19a1b-GFP zebrafish lines were then used to analyze estrogenic and anti-estrogenic responses of the three bisphenols in vivo. BPA, BPAF and BPC were agonists with different potencies for the three zebrafish estrogen receptors in vitro. The potent zfERα-mediated activity of BPA and BPAF in vitro resulted in vivo by activation of GFP expression in zebrafish larvae in the heart (zfERα-dependent) at lower concentrations, and in the liver (zfERβ-dependent) at higher concentrations. BPC induced zfERβ-mediated luciferase expression in vitro, and the zfERβ agonism led to activation of GFP expression in the liver and the brain in vivo. In addition, BPC acted as a full antagonist on zfERα, and completely inhibited estrogen-induced GFP expression in the heart of the zebrafish larvae. To summarize, applying a combination of zebrafish-based in vitro and in vivo methods to evaluate bisphenol analogues for estrogenic activity will facilitate the prioritization of these chemicals for further analysis in higher vertebrates as well as the risk assessment in humans.

International audience; Histone lysine acetylation is an epigenetic mark regulated by histone acetyltransferases and histone deacetylases (HDAC) which plays an important role in tumorigenesis. In this study, we observed a strong overexpression of class IIa HDAC9, at the mRNA and protein levels, in the most aggressive human breast cancer cell lines (i.e. in basal breast cancer cells vs luminal ones or in malignant vs begnin MCF10A breast epithelial cell lines). HDAC9 overexpression was associated with higher rates of gene transcription and increased epigenetic marks on the HDAC9 promoter. Ectopic expression of HDAC9 in MCF7 luminal breast cancer cells led to an increase in cell proliferation and to a decrease in apoptosis. These effects were associated with a deregulated expression of several genes controlled by HDAC inhibitors such as CDKN1A, BAX and TNFRSF10A. Inversely, knock-down of HDAC9 expression in MDA-MB436 basal breast cancer cells reduced cell proliferation. Moreover, high HDAC9 expression decreased the efficacy of HDAC inhibitors to reduce cell proliferation and to regulate CDKN1A gene expression. Interestingly, the gene encoding the transcription factor SOX9 was identified by a global transcriptomic approach as an HDAC9 target gene. In stably transfected MCF7 cells, SOX9 silencing significantly decreased HDAC9 mitogenic activity. Finally, in a large panel of breast cancer biopsies, HDAC9 expression was significantly increased in tumors of the basal subtype, correlated with SOX9 expression and associated with poor prognosis. Altogether, these results indicate that HDAC9 is a key factor involved in mammary carcinogenesis and in the response to HDAC inhibitors.

In vitro bioassays are increasingly applied to detect endocrine disrupting chemicals (EDCs) in environmental waters. Most studies use human nuclear receptor assays, but this raises questions about their relevance for evaluating ecosystem health. The current study aimed to assess species-specific differences in the activation or inhibition of a range of human and zebrafish nuclear receptors by different water extracts. Wastewater and surface water extracts were run in transactivation assays indicative of the estrogen receptor (ER), androgen receptor (AR), glucocorticoid receptor (GR), progesterone receptor (PR), mineralocorticoid receptor (MR), pregnane X receptor (PXR) and peroxisome proliferator-activated receptor gamma (PPARγ). The transactivation assays were complemented with competitive binding assays for human AR, GR, PR and MR. In most cases, both human and zebrafish nuclear receptor activity were detected in the water extracts. Only some species-specific differences in potency and activity were observed. Water extracts were more active in zebrafish PXR compared to human PXR whereas the opposite was observed for PPARγ. Further, all water extracts inhibited zebrafish PR, while only one extract showed weak anti-progestagenic activity for human PR. Due to these observed differences, zebrafish nuclear receptor assays may be preferable over human nuclear receptor assays to assess the potential risks of EDCs to aquatic organisms. However, recognizing issues with availability of zebrafish nuclear receptor assays and the relatively small differences in responsiveness for many of the human and zebrafish nuclear receptors, including the widely studied ER, the current study supports the continued use of human nuclear receptor assays for water quality monitoring.

International audience; BACKGROUND:Thyroid hormone receptors (TRs) are tightly regulated by the corepressors nuclear receptor corepressor (NCoR) and silencing mediator of retinoic acid and thyroid hormone receptors. Three conserved corepressor/NR signature box motifs (CoRNR1-3) forming the nuclear receptor interaction domain have been identified in these corepressors. Whereas TRs regulate multiple normal physiological and developmental pathways, mutations in TRs can result in endocrine diseases and be associated with cancers due to impairment of corepressor release. Three mutants that are located in helix H11 of TRs are of special interest: TRα-M388I, a mutant associated with the development of renal clear cell carcinomas (RCCCs), and TRβ-Δ430 and TRβ-Δ432, two deletion mutants causing resistance to thyroid hormone syndrome.METHODS:Several cell-based and biophysical methods were used to measure the affinity between wild-type and mutant TRα and TRβ and all the CoRNR motifs from corepressors to quantify the effects of different thyroid hormone analogues on these interactions. This study was coupled with the measurement of interactions between wild-type and mutant TRs in the context of a heterodimer with RXR to a NCoR fragment in the presence of the same ligands. Structural insights into the binding mode of corepressors to TRs were assessed in parallel by nuclear magnetic resonance spectroscopy.RESULTS:The study shows that TRs interact more avidly with the silencing mediator of retinoic acid and thyroid hormone receptors than with NCoR peptides, and that TRα binds most avidly to S-CoRNR3, whereas TRβ binds preferentially to S-CoRNR2. In the studied TR mutants, a transfer of the CoRNR-specificity toward CoRNR1 was observed, coupled with a significant increase in the binding strength. In contrast to 3,5,3'-triiodothyronine (T3), the agonist TRIAC and the antagonist NH-3 were very efficient at dissociating the abnormally strong interactions between mutant TRβs and corepressors. A strong impairment of T3-binding for TRβ mutants was shown compared to TRIAC and NH-3 and could explain the different efficiencies of the different ligands in releasing corepressors from the studied TRβ mutants. Consequently, TRIAC was found to be more effective than T3 in facilitating coactivator recruitment and decreasing the dominant activity of TRβ-Δ430.CONCLUSION:This study helps to clarify the specific interaction surfaces involved in the pathologic phenotype of TR mutants and demonstrates that TRIAC is a potential therapeutic agent for patients suffering from resistance to thyroid hormone syndromes.

Mutations of the androgen receptor (AR) gene are the most frequent cause of 46,XY disorders of sex development. They are associated with a variety of phenotypes, ranging from phenotypic women (complete androgen insensitivity syndrome, CAIS) to milder degrees of undervirilization (partial forms) or men with only infertility (mild form). We identified a new W752G AR mutation responsible for a familial case of CAIS and performed an in vitro study and structural analysis of this mutation and the only other reported substitution affecting the same amino acid (W752R). Although sex assignment is not discussed in cases of CAIS, we show how the phenotype-genotype correlation can be refined by in vitro and structural studies according to the nature of the amino acid substitution, which in turn may have interesting impacts on the follow-up of these patients.

Reactive oxygen species (ROS) are key players in the apoptotic effects induced by short-term tamoxifen treatment of breast cancer cells, but also in acquired resistance following long-term treatment. Whereas the use of the selective estrogen receptor down-regulator fulvestrant is promising, especially in patients who develop tamoxifen resistance, only few studies addressed its implication in the modulation of cellular redox status.The regulation of (pro/anti)-oxidant players were first investigated at the mRNA level in a MCF-7-derived cell line after short-term (24 h) estradiol treatment. Long-term anti-estrogen treated MCF-7 derived cell lines were also developed: 3 months of 4-hydroxytamoxifen alone (MCF7L-OHTLT) or followed by 3 months of fulvestrant (MCF7L-ICILT). Growth properties, hormone sensitivity, receptor content, ROS production and relative mRNA expression of pro or antioxidant enzymes were evaluated in these long-term treated cell lines.Short-term estradiol treatment showed a hormone sensitivity of Nox2, GPx1, GPx2 and SOD1 mRNA levels. The long-term fulvestrant treatment (3 months) of MCF7L-OHTLT led to a reduced level of ROS production accompanied with a drastic drop of the accessory protein p22(phox) mRNA. This ROS reduction, although not clearly related to antioxidant enzymes level, seems to be involved in fulvestrant sensitivity of long-term anti-estrogen treated cells, as suggested by the effects of antiradical tempol treatment.When compared to long-term 4-hydroxytamoxifen-treated breast cancer cells, addition of fulvestrant treatment was able to diminish ROS production and p22(phox) mRNA level, and made cells more sensitive to growth inhibition induced by tempol. These effects may be a valuable asset of the fulvestrant treatment.

UMR AGAP équipe DAAV Diversité, adaptation et amélioration de la vigne; International audience; Mesoporous silica nanoparticles (MSNs) were covalently coated with antioxidant molecules, namely, caffeic acid (MSN-CAF) or rutin (MSN-RUT), in order to diminish the impact of oxidative stress induced after transfection into cells, thus generating safer carriers used for either drug delivery or other applications. Two cellular models involved in the entry of NPs in the body were used for this purpose: the intestinal Caco-2 and the epidermal HaCaT cell lines. Rutin gave the best results in terms of antioxidant capacities preservation during coupling procedures, cellular toxicity alleviation, and decrease of ROS level after 24 h incubation of cells with grafted nanoparticles. These protective effects of rutin were found more pronounced in HaCaT than in Caco-2 cells, indicating some cellular specificity toward defense against oxidative stress. In order to gain more insight about the Nrf2 response, a stable transfected HaCaT cell line bearing repeats of the antioxidant response element (ARE) in front of a luciferase reporter gene was generated. In this cell line, both tBHQ and quercetin (Nrf2 agonists), but not rutin, were able to induce, in a dose-dependent fashion, the luciferase response. Interestingly, at high concentration, MSN-RUT was able to induce a strong Nrf2 protective response in HaCaT cells, accompanied by a comparable induction of HO-1 mRNA. The level of these responses was again less important in Caco-2 cells. To conclude, in keratinocyte cell line, the coupling of rutin to silica nanoparticles was beneficial in term of ROS reduction, cellular viability, and protective effects mediated through the activation of the Nrf2 antioxidant pathway.

Vitamin B6 is well-known for its role as a cofactor in many enzymatic reactions and recently, several epidemiological studies have highlighted the importance of this vitamin as a protective agent against various cancers: elevated vitamin B6 plasma levels were associated with a lower risk of colorectal cancer development, for example. In vivo studies have shown that vitamin B6 decreased cell proliferation and enhanced the immune response. At the cellular level, antioxidant, pro-apoptotic and anti-angiogenic effects have been identified. At the molecular level, vitamin B6 is able to inhibit the transactivation potential of various nuclear receptors. Interestingly, a recent paper has described the conjugation of vitamin B6 to RIP140 (receptor interacting protein of 140 kDa), a protein that acts as a transcriptional corepressor of nuclear receptors. This post-translational modification increases the transcriptional repression of RIP140 and regulates its subcellular localization and its ability to interact with different protein partners. Finally, vitamin B6 is involved in the methyl donor cycle ant thus, some of the antitumor properties of vitamin B6 may involve an indirect effect on the level of DNA or histone methylation. All of these mechanistic and clinical data justify further studies to decipher the mechanism of action of vitamin B6 and its clinical interest in combination with molecules typically used in chemotherapy or hormonal therapy.

International audience; Background: The occurrence of halogenated analogs of the xenoestrogen bisphenol A (BPA) has been recently demonstrated both in environmental and human samples. These analogs include brominated [e.g., tetrabromobisphenol A (TBBPA)] and chlorinated [e.g., tetrachlorobisphenol A (TCBPA)] bisphenols, which are both flame retardants. Because of their structural homology with BPA, such chemicals are candidate endocrine disruptors. However, their possible target(s) within the nuclear hormone receptor superfamily has remained unknown.Objectives: We investigated whether BPA and its halogenated analogs could be ligands of estrogen receptors (ERs) and peroxisome proliferator–activated receptors (PPARs) and act as endocrine-disrupting chemicals.Methods: We studied the activity of compounds using reporter cell lines expressing ERs and PPARs. We measured the binding affinities to PPARγ by competitive binding assays with [3H]‑rosiglitazone and investigated the impact of TBBPA and TCBPA on adipocyte differentiation using NIH3T3-L1 cells. Finally, we determined the binding mode of halogenated BPAs to PPARγ by X‑ray crystallography.Results: We observed that TBBPA and TCBPA are human, zebrafish, and Xenopus PPARγ ligands and determined the mechanism by which these chemicals bind to and activate PPARγ. We also found evidence that activation of ERα, ERβ, and PPARγ depends on the degree of halogenation in BPA analogs. We observed that the bulkier brominated BPA analogs, the greater their capability to activate PPARγ and the weaker their estrogenic potential.Conclusions: Our results strongly suggest that polyhalogenated bisphenols could function as obesogens by acting as agonists to disrupt physiological functions regulated by human or animal PPARγ.

Endocrine-disrupting chemicals (EDCs) are exogenous substances interfering with hormone biosynthesis, metabolism, or action, and consequently causing disturbances in the endocrine system. Various pathways are activated by EDCs, including interactions with nuclear receptors (NRs), which are primary targets of numerous environmental contaminants. The main NRs targeted by environmental contaminants are the estrogen (ER α, β) and the androgen (AR) receptors. ERs and AR have pleiotropic regulatory roles in a diverse range of tissues, notably in the mammary gland, the uterus, and the prostate. Thus, dysfunctional ERs and AR signaling due to inappropriate exposure to environmental pollutants may lead to hormonal cancers and infertility. The pregnane X receptor (PXR) is also recognized by many environmental molecules. PXR has a protective role of the body through its ability to regulate proteins involved in the metabolism, the conjugation, and the transport of many exogenous and endogenous compounds. However, the permanent activation of this receptor by xenobiotics may lead to premature drug metabolism, the formation, and accumulation of toxic metabolites and defects in hormones homeostasis. The activity of other NRs can also be affected by environmental molecules. Compounds capable of inhibiting or activating the estrogen related (ERRγ), the thyroid hormone (TRα, β), the retinoid X receptors (RXRα, β, γ), and peroxisome proliferator-activated (PPAR α, γ) receptors have been identified and are highly suspected to promote developmental, reproductive, neurological, or metabolic diseases in humans and wildlife. In this review, we provide an overview of reporter cell lines established to characterize the human NR activities of a large panel of EDCs including natural as well as industrial compounds such as pesticides, plasticizers, surfactants, flame retardants, and cosmetics.

Humans are chronically exposed to multiple exogenous substances, including environmental pollutants, drugs and dietary components. Many of these compounds are suspected to impact human health, and their combination in complex mixtures could exacerbate their harmful effects. Here we demonstrate that a pharmaceutical oestrogen and a persistent organochlorine pesticide, both exhibiting low efficacy when studied separately, cooperatively bind to the pregnane X receptor, leading to synergistic activation. Biophysical analysis shows that each ligand enhances the binding affinity of the other, so the binary mixture induces a substantial biological response at doses at which each chemical individually is inactive. High-resolution crystal structures reveal the structural basis for the observed cooperativity. Our results suggest that the formation of ‘supramolecular ligands' within the ligand-binding pocket of nuclear receptors contributes to the synergistic toxic effect of chemical mixtures, which may have broad implications for the fields of endocrine disruption, toxicology and chemical risk assessment., Endocrine-disrupting chemicals act on nuclear hormone receptors, such as PXR. Here, Delfosse et al. show how two such chemicals interact with each other in the PXR ligand-binding pocket, forming a so-called supramolecular ligand that is a more potent PXR activator than each of the two chemicals alone.

In this study, we have investigated the role of C-terminal binding proteins (CtBPs) and histone deacetylases (HDACs) in the repressive activity of the nuclear receptor cofactor Receptor-Interacting Protein 140 (RIP140). We have defined the interaction of both CtBP1 and CtBP2 with RIP140 and delineated two motifs (PIDLS and PINLS) differentially required for in vitro interaction. Using different approaches (titration of endogenous CtBPs, mutagenesis and transfection in CtBP knock-out cells), we find that recruitment of CtBPs only partially explains the negative regulation exerted by RIP140. We then demonstrate that RIP140 associates in vitro not only with class I HDACs but also with class II enzymes such as HDAC5. This interaction mainly involves the N-terminus of RIP140 (residues 27–199) and two domains of HDAC5. Moreover, the two proteins functionally interfere in transfection experiments, and confocal microscopy indicates that they co-localize in the nucleus. Interestingly, using the specific HDAC inhibitor trichostatin A, we show that HDAC activity is dispensable for active transrepression by RIP140. Finally, we demonstrate that the C-terminal region of RIP140 contains two additional silencing domains and confers strong active transrepression independently of HDAC activity and CtBPs. Altogether, these data indicate that transcriptional inhibition by the cofactor RIP140 involves complex mechanisms relying on multiple domains and partners.

International audience; : Zebrafish, Danio rerio, is increasingly used as an animal model to study the effects of pharmaceuticals and environmental estrogens. As most of these estrogens have only been tested on human estrogen receptors (ERs), it is necessary to measure their effects on zebrafish ERs. In humans there are two distinct nuclear ERs (hERα and hERβ), whereas the zebrafish genome encodes three ERs, zfERα and two zfERβs (zfERβ1 and zfERβ2). In this study, we established HeLa-based reporter cell lines stably expressing each of the three zfERs. We first reported that estrogens more efficiently activate the zfERs at 28°C as compared to 37°C, thus reflecting the physiological temperature of zebrafish in wildlife. We then showed significant differences in the ability of agonist and antagonist estrogens to modulate activation of the three zfER isotypes in comparison to hERs. Environmental compounds (bisphenol A, alkylphenols, mycoestrogens) which are hER panagonists and hERβ selective agonists displayed greater potency for zfERα as compared to zfERβs. Among hERα selective synthetic agonists, PPT did not activate zfERα while 16α-LE2 was the most zfERα selective compound. Altogether, these results confirm that all hER ligands control in a similar manner the transcriptional activity of zfERs although significant differences in selectivity were observed among subtypes. The zfER subtype selective ligands that we identified thus represent new valuable tools to dissect the physiological roles of the different zfERs. Finally, our work also points out that care has to be taken in transposing the results obtained using the zebrafish as a model for human physiopathology.

International audience; Bisphenol A (BPA) is an industrial compound and a well known endocrine-disrupting chemical with estrogenic activity. The widespread exposure of individuals to BPA is suspected to affect a variety of physiological functions, including reproduction, development, and metabolism. Here we report that the mechanisms by which BPA and two congeners, bisphenol AF and bisphenol C (BPC), bind to and activate estrogen receptors (ER) α and β differ from that used by 17β-estradiol. We show that bisphenols act as partial agonists of ERs by activating the N-terminal activation function 1 regardless of their effect on the C-terminal activation function 2, which ranges from weak agonism (with BPA) to antagonism (with BPC). Crystallographic analysis of the interaction between bisphenols and ERs reveals two discrete binding modes, reflecting the different activities of compounds on ERs. BPA and 17β-estradiol bind to ERs in a similar fashion, whereas, with a phenol ring pointing toward the activation helix H12, the orientation of BPC accounts for the marked antagonist character of this compound. Based on structural data, we developed a protocol for in silico evaluation of the interaction between bisphenols and ERs or other members of the nuclear hormone receptor family, such as estrogen-related receptor γ and androgen receptor, which are two known main targets of bisphenols. Overall, this study provides a wealth of tools and information that could be used for the development of BPA substitutes devoid of nuclear hormone receptor-mediated activity and more generally for environmental risk assessment.

Hormonal regulation of gene activity is mediated by nuclear receptors acting as ligand-activated transcription factors. To achieve efficient regulation of gene expression, these receptors must interact with different type of molecules: 1) the steroid hormone, 2) the DNA response element, and 3) various proteins acting as transcriptional cofactors. In the present study, we have investigated how ligand and DNA binding influence the in vitro interaction between estrogen receptors (ERs) and the transcription intermediary factor hTIF1alpha (human transcriptional intermediary factor 1alpha). We first optimized conditions for the coactivator-dependent receptor ligand assay to lower ED50, and we then analyzed the ability of various natural and synthetic estrogens to allow the binding of the two types of proteins. Results were compared with the respective affinities of these ligands for the receptor. We then developed a protein-protein-DNA assay allowing the quantification of cofactor-ER-estrogen response element (ERE) complex formation in the presence of ligand and used measurements of fluorescence anisotropy to define the equilibrium binding parameters of the interaction. We demonstrated that the leucine-charged domain of hTIF1alpha is sufficient to interact with ERE-bound ERalpha in a ligand-dependent manner and showed that binding of ERalpha onto DNA does not significantly affect its hormone-dependent association with TIF1alpha. Finally, we show that, mainly in the absence of hormone, hTIF1alpha interacts better with ERbeta than with ERalpha independently of the presence of ERE.