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The sea urchin faces a variety of environmental factors in the ocean, including small molecules. These small molecules can enter the cell and affect development, homeostasis, immunity, and cellular functions. Transcription factors play many roles, including the regulation of how these molecules enter and exit cells by altering expression of membrane proteins. Nuclear receptors (NRs) that aid in responding to small molecules by binding to these ligands, causing a downstream effect of altering the gene expression of target genes. In this thesis, I study a particular group of NRs, the Hepatocyte Nuclear Factors (HNF), which have been shown to play a role in responding to stimuli and to maintain metabolism and regulate development. Sea urchins need to adapt to dynamic environmental conditions by responding to specific stimuli. HNFs have been well studied in mammalian models, but there is a lack of a full description of HNFs within echinoderms. This is particularly useful to further understand these transcription factors in small molecules transport in a genetically enabled species. Here, I have identified and localized the four major types of HNFs - HNF1, HNF3, HNF4, and HNF6 within the sea urchin, Lytechinus pictus during early development.

Phospholipids are ligands for nuclear hormone receptors (NRs) that regulate transcriptional programs relevant to normal physiology and disease. Here, we demonstrate that mimicking phospholipid-NR interactions is a robust strategy to improve agonists of liver receptor homolog-1 (LRH-1), a therapeutic target for colitis. Conventional LRH-1 modulators only partially occupy the binding pocket, leaving vacant a region important for phospholipid binding and allostery. Therefore, we constructed a set of molecules with elements of natural phospholipids appended to a synthetic LRH-1 agonist. We show that the phospholipid-mimicking groups interact with the targeted residues in crystal structures and improve binding affinity, LRH-1 transcriptional activity, and conformational changes at a key allosteric site. The best phospholipid mimetic markedly improves colonic histopathology and disease-related weight loss in a murine T cell transfer model of colitis. This evidence of in vivo efficacy for an LRH-1 modulator in colitis represents a leap forward in agonist development.

Nuclear receptors are a class of ligand-activated transcription factors that affect gene expression. Pharmaceutical drugs that target nuclear receptors make up (13%) of all therapeutics prescribed in the United States. Unlike signaling pathways, nuclear receptors have a wide variety of binding partners that can bind upon activation of a nuclear receptor to upregulate gene transcription. These binding partner proteins, called “coregulators”, are diverse in function. However, the determinants of their binding to nuclear receptors, and how binding to certain coregulators over others occurs, is not known. In this work, we examine how ligands create a preference for nuclear receptors to bind some coregulators over others, a concept previously established in G-protein coupled receptors (GPCRs) known as biased agonism. We define a method to measure bias of ligands, and for the first time to our knowledge, we define structural classes of coregulators that biased ligands can select for. Due to the largely shared coregulator binding surface of nuclear receptors, we expect these findings to be impactful for the field of nuclear receptor research and pharmaceutical drug design. Preclinical works have demonstrated that activation of specific pathways with biased ligands can expand their therapeutic utility and limit side effects. By defining an assay to measure bias and defining clear structural classes of coregulators that ligands can bias binding towards, we define a new method of nuclear receptor ligand design.

Nuclear receptors (NRs) are important biological targets of endocrine-disrupting chemicals (EDCs). Identifying chemicals that can act as EDCs and modulate the function of NRs is difficult because of the time and cost of in vitro and in vivo screening to determine the potential hazards of the 100 000s of chemicals that humans are exposed to. Hence, there is a need for computational approaches to prioritize chemicals for biological testing. Machine learning (ML) techniques are alternative methods that can quickly screen millions of chemicals and identify those that may be an EDC. Computational models of chemical binding to multiple NRs have begun to emerge. Recently, a Nuclear Receptor Activity (NuRA) dataset, describing experimentally derived small-molecule activity against various NRs has been created. We have used the NuRA dataset to develop an ensemble of ML-based models to predict the agonism, antagonism, binding and effector binding of small molecules to nine different human NRs. We defined the applicability domain of the ML models as a measure of Tanimoto similarity to the molecules in the training set, which enhanced the performance of the developed classifiers. We further developed a user-friendly web server named 'NR-ToxPred' to predict the binding of chemicals to the nine NRs using the best-performing models for each receptor. This web server is freely accessible at http://nr-toxpred.cchem.berkeley.edu. Users can upload individual chemicals using Simplified Molecular-Input Line-Entry System, CAS numbers or sketch the molecule in the provided space to predict the compound's activity against the different NRs and predict the binding mode for each.

I recettori nucleari sono fattori di trascrizione coinvolti in processi critici per la salute umana e sono un obiettivo rilevante per la valutazione del rischio tossicologico e il processo di scoperta dei farmaci. I modelli computazionali possono essere uno strumento utile (i) per dare priorità alla sperimentazione di sostanze chimiche che possono imitare gli ormoni naturali e quindi essere interferenti endocrini e (ii) per identificare nuovi possibili candidati farmaci. Pertanto, l'obiettivo principale di questo progetto è quello di studiare le potenziali interazioni tra sostanze chimiche e recettori nucleari, con il duplice scopo di sviluppare strumenti in silico per la ricerca di nuovi modulatori e di identificare possibili sostanze chimiche che alterano il sistema endocrino. Dopo aver creato una collezione esaustiva di modulatori di recettori nucleari, abbiamo applicato metodi di apprendimento automatico per colmare il vuoto di dati e predire nuovi possibili modulatori tramite modelli predittivi. In particolare, le strategie di modellazione hanno incluso algoritmi di apprendimento automatico multi-tasking per indagare le complesse relazioni tra sostanze chimiche e diversi recettori nucleari., Nuclear receptors are transcription factors involved in processes critical to human health and are a relevant target for toxicological risk assessment and the drug discovery process. Computational models can be a useful tool (i) to prioritize chemicals that can mimic natural hormones and thus be endocrine disruptors and (ii) to identify new possible lead for drug discovery. Therefore, the main goal of this project is to study potential interactions between chemicals and nuclear receptors, with the dual purpose of developing in silico tools to search for new modulators and to identify possible endocrine disrupting chemicals. After creating an exhaustive collection of nuclear receptor modulators, we applied machine learning methods to fill the data gap and prioritize modulators by building predictive models. In particular, modeling strategies included multi-tasking machine learning algorithms to investigate the complex relationships between chemicals and multiple nuclear receptors.

Nuclear receptors (NRs), are a wide family of ligand-regulated transcription factors sharing a common modular structure composed by an N-terminal domain and a ligand-binding domain connected by a short hinge linker to a DNA-binding domain. NRs are involved in many physiological processes, including metabolism, reproduction and development. Most of them respond to small lipophilic ligands, such as steroids, retinoids, and phospholipids, which act as conformational switches. Some NRs are still “orphan” and the search for their ligands is still ongoing. Upon DNA binding, NRs can act both as transcriptional activators or repressors of their target genes. Theoretically, the possibility to modulate NRs activity with small molecules makes them ideal therapeutic targets, although the complexity of their signaling makes drug design challenging. In this review, we discuss the role of NRs in erythropoiesis, in both homeostatic and stress conditions. This knowledge is important in view of modulating red blood cells production in disease conditions, such as anemias, and for the expansion of erythroid cells in culture for research purposes and for reaching the long-term goal of cultured blood for transfusion.

The interest in multitask and deep learning strategies has been increasing in the last few years, in application to large and complex dataset for quantitative structure-activity relationship (QSAR) analysis. Multitask approaches allow the simultaneous prediction of molecular properties that are related, through information sharing, whereas deep learning strategies increase the potential of capturing nonlinear relationships. In this work, we compare the binary classification capability of multitask deep and shallow neural networks to single-task strategies used as benchmark (i.e., as k-nearest neighbours, N-nearest neighbours, random forest and Naïve Bayes), as well as multitask supervised self-organizing maps. Comparison was carried out with an extended QSAR dataset containing annotations of molecular binding, agonism and antagonism activity on 11 nuclear receptors, for a total of 14,963 molecules, divided into training and test sets and labelled for their bioactivity on at least one of 30 binary tasks. Additional 304 chemicals were used as external evaluation set to further validate models. Although no approach systematically overperformed the others, task-specific differences were found, suggesting the benefit of multitask learning for tasks that are less represented. On average, some of the single-task approaches and multitask deep learning strategies had similar performances. However, the latter can have advantages, such as a simpler management of predictions and applicability domain assessment for future samples. On the other hand, the parameter tuning required by neural networks are generally time expensive suggesting that the modelling strategy should be evaluated case by case.

Chemical language models enable de novo drug design without the requirement for explicit molecular construction rules. While such models have been applied to generate novel compounds with desired bioactivity, the actual prioritization and selection of the most promising computational designs remains challenging. Herein, we leveraged the probabilities learnt by chemical language models with the beam search algorithm as a model-intrinsic technique for automated molecule design and scoring. Prospective application of this method yielded novel inverse agonists of retinoic acid receptor-related orphan receptors (RORs). Each design was synthesizable in three reaction steps and presented low-micromolar to nanomolar potency towards RORγ. This model-intrinsic sampling technique eliminates the strict need for external compound scoring functions, thereby further extending the applicability of generative artificial intelligence to data-driven drug discovery.

Colorectal cancer (CRC) is the third leading cause of cancer death in the western world. In women, menopausal hormone therapy has been shown to reduce CRC incidence by 20%. Studies demonstrate that estrogen activating estrogen receptor beta (ERβ) protects against CRC. ERβ is a nuclear receptor that regulates gene expression through interactions with the chromatin. This molecular mechanism is, however, not well characterized in colon. Here, we present for the first time, the cistrome of ERβ in different colon cancer cell lines. We use cell lines engineered to express ERβ, optimize and validate an ERβ antibody for chromatin-immunoprecipitation (ChIP), and perform ChIP-Seq. We identify key binding motifs, including ERE, AP-1, and TCF sites, and we determine enrichment of binding to cis-regulatory chromatin sites of genes involved in tumor development, cell migration, cell adhesion, apoptosis, and Wnt signaling pathways. We compare the corresponding cistromes of colon and breast cancer and find that they are conserved for about a third of genes, including GREB1, but that ERβ tethering to TCF and KLF family motifs is characteristic for colon. We exemplify upregulation of putative CRC tumor suppressor gene CST5 where ERβ in colon cells binds to cis-regulatory regions nearby (−351 bp) the transcriptional start site. Our work provides a foundation for understanding the mechanism of action of ERβ in CRC prevention.

Colorectal cancer (CRC) is the third leading cause of cancer death in the western world. In women, menopausal hormone therapy has been shown to reduce CRC incidence by 20%. Studies demonstrate that estrogen activating estrogen receptor beta (ERβ) protects against CRC. ERβ is a nuclear receptor that regulates gene expression through interactions with the chromatin. This molecular mechanism is, however, not well characterized in colon. Here, we present for the first time, the cistrome of ERβ in different colon cancer cell lines. We use cell lines engineered to express ERβ, optimize and validate an ERβ antibody for chromatin-immunoprecipitation (ChIP), and perform ChIP-Seq. We identify key binding motifs, including ERE, AP-1, and TCF sites, and we determine enrichment of binding to cis-regulatory chromatin sites of genes involved in tumor development, cell migration, cell adhesion, apoptosis, and Wnt signaling pathways. We compare the corresponding cistromes of colon and breast cancer and find that they are conserved for about a third of genes, including GREB1, but that ERβ tethering to TCF and KLF family motifs is characteristic for colon. We exemplify upregulation of putative CRC tumor suppressor gene CST5 where ERβ in colon cells binds to cis-regulatory regions nearby (−351 bp) the transcriptional start site. Our work provides a foundation for understanding the mechanism of action of ERβ in CRC prevention.

Colorectal cancer (CRC) is the third leading cause of cancer death in the western world. In women, menopausal hormone therapy has been shown to reduce CRC incidence by 20%. Studies demonstrate that estrogen activating estrogen receptor beta (ERβ) protects against CRC. ERβ is a nuclear receptor that regulates gene expression through interactions with the chromatin. This molecular mechanism is, however, not well characterized in colon. Here, we present for the first time, the cistrome of ERβ in different colon cancer cell lines. We use cell lines engineered to express ERβ, optimize and validate an ERβ antibody for chromatin-immunoprecipitation (ChIP), and perform ChIP-Seq. We identify key binding motifs, including ERE, AP-1, and TCF sites, and we determine enrichment of binding to cis-regulatory chromatin sites of genes involved in tumor development, cell migration, cell adhesion, apoptosis, and Wnt signaling pathways. We compare the corresponding cistromes of colon and breast cancer and find that they are conserved for about a third of genes, including GREB1, but that ERβ tethering to TCF and KLF family motifs is characteristic for colon. We exemplify upregulation of putative CRC tumor suppressor gene CST5 where ERβ in colon cells binds to cis-regulatory regions nearby (−351 bp) the transcriptional start site. Our work provides a foundation for understanding the mechanism of action of ERβ in CRC prevention.

Colorectal cancer (CRC) is the third leading cause of cancer death in the western world. In women, menopausal hormone therapy has been shown to reduce CRC incidence by 20%. Studies demonstrate that estrogen activating estrogen receptor beta (ERβ) protects against CRC. ERβ is a nuclear receptor that regulates gene expression through interactions with the chromatin. This molecular mechanism is, however, not well characterized in colon. Here, we present for the first time, the cistrome of ERβ in different colon cancer cell lines. We use cell lines engineered to express ERβ, optimize and validate an ERβ antibody for chromatin-immunoprecipitation (ChIP), and perform ChIP-Seq. We identify key binding motifs, including ERE, AP-1, and TCF sites, and we determine enrichment of binding to cis-regulatory chromatin sites of genes involved in tumor development, cell migration, cell adhesion, apoptosis, and Wnt signaling pathways. We compare the corresponding cistromes of colon and breast cancer and find that they are conserved for about a third of genes, including GREB1, but that ERβ tethering to TCF and KLF family motifs is characteristic for colon. We exemplify upregulation of putative CRC tumor suppressor gene CST5 where ERβ in colon cells binds to cis-regulatory regions nearby (−351 bp) the transcriptional start site. Our work provides a foundation for understanding the mechanism of action of ERβ in CRC prevention.

The nuclear receptor ligand-binding domain (LBD) is a highly dynamic entity. The FXR LBD shows multiple low-energy conformational states of the activation function-2 (AF-2) coregulator binding surface upon ligand binding, indicating the complexity of FXR activation. However, it is unknown how ligand binding leads to different conformational states within the AF-2 region centered on helix 12 (H-12) of the LBD. Here we observe the conformation of the coregulator binding surface (H-12 specifically) of FXR upon ligand binding in solution using fluorine-19 (19F) nuclear magnetic resonance (NMR) and simulations of this surface using molecular dynamics. Fluorescence anisotropy of fluorescein-labeled coregulator peptides reveals a correlation between structural conformations of the coregulator binding surface and the function of FXR. While the coregulator surface of apo FXR and partial-agonist bound FXR exchanges between multiple low energy conformations, full-agonist bound FXR is restricted to few conformations, which favor coactivator binding. Furthermore, we find that two ligands that induce similar affinities for a coactivator peptide have distinct coregulator binding surface structures.

© The Author(s), 2021. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Goksoyr, S. O., Goldstone, J., Lille-Langoy, R., Lock, E.-J., Olsvik, P. A., Goksoyr, A., & Karlsen, O. A. Polycyclic aromatic hydrocarbons modulate the activity of Atlantic cod (Gadus morhua) vitamin D receptor paralogs in vitro. Aquatic Toxicology, 238, (2021): 105914, https://doi.org/10.1016/j.aquatox.2021.105914., Vitamin D receptor (VDR) mediates the biological function of the steroid hormone calcitriol, which is the metabolically active version of vitamin D. Calcitriol is important for a wide array of physiological functions, including calcium and phosphate homeostasis. In contrast to mammals, which harbor one VDR encoding gene, teleosts possess two orthologous vdr genes encoding Vdr alpha (Vdra) and Vdr beta (Vdrb). Genome mining identified the vdra and vdrb paralogs in the Atlantic cod (Gadus morhua) genome, which were further characterized regarding their phylogeny, tissue-specific expression, and transactivational properties induced by calcitriol. In addition, a selected set of polycyclic aromatic hydrocarbons (PAHs), including naphthalene, phenanthrene, fluorene, pyrene, chrysene, benzo[a]pyrene (BaP), and 7-methylbenzo[a]pyrene, were assessed for their ability to modulate the transcriptional activity of gmVdra and gmVdrb in vitro. Both gmVdra and gmVdrb were activated by calcitriol with similar potencies, but gmVdra produced significantly higher maximal fold activation. Notably, none of the tested PAHs showed agonistic properties towards the Atlantic cod Vdrs. However, binary exposures of calcitriol together with phenanthrene, fluorene, or pyrene, antagonized the activation of gmVdra, while chrysene and BaP significantly potentiated the calcitriol-mediated activity of both receptors. Homology modeling, solvent mapping, and docking analyses complemented the experimental data, and revealed a putative secondary binding site in addition to the canonical ligand-binding pocket (LBP). Calcitriol was predicted to interact with both binding sites, whereas PAHs docked primarily to the LBP. Importantly, our in vitro data suggest that PAHs can interact with the paralogous gmVdrs and interfere with their transcriptional activities, and thus potentially modulate the vitamin D signaling pathway and contribute to adverse effects of crude oil and PAH exposures on cardiac development a, This study was funded by the Research Council of Norway through the ”iCod 2.0: Integrative environmental genomics of Atlantic cod” project (project no. 244564) and the ”dCod 1.0: decoding systems toxicology of Atlantic cod” project (Center for Digital Life Norway project no. 248840).

© The Author(s), 2021. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Goksoyr, S. O., Goldstone, J., Lille-Langoy, R., Lock, E.-J., Olsvik, P. A., Goksoyr, A., & Karlsen, O. A. Polycyclic aromatic hydrocarbons modulate the activity of Atlantic cod (Gadus morhua) vitamin D receptor paralogs in vitro. Aquatic Toxicology, 238, (2021): 105914, https://doi.org/10.1016/j.aquatox.2021.105914., Vitamin D receptor (VDR) mediates the biological function of the steroid hormone calcitriol, which is the metabolically active version of vitamin D. Calcitriol is important for a wide array of physiological functions, including calcium and phosphate homeostasis. In contrast to mammals, which harbor one VDR encoding gene, teleosts possess two orthologous vdr genes encoding Vdr alpha (Vdra) and Vdr beta (Vdrb). Genome mining identified the vdra and vdrb paralogs in the Atlantic cod (Gadus morhua) genome, which were further characterized regarding their phylogeny, tissue-specific expression, and transactivational properties induced by calcitriol. In addition, a selected set of polycyclic aromatic hydrocarbons (PAHs), including naphthalene, phenanthrene, fluorene, pyrene, chrysene, benzo[a]pyrene (BaP), and 7-methylbenzo[a]pyrene, were assessed for their ability to modulate the transcriptional activity of gmVdra and gmVdrb in vitro. Both gmVdra and gmVdrb were activated by calcitriol with similar potencies, but gmVdra produced significantly higher maximal fold activation. Notably, none of the tested PAHs showed agonistic properties towards the Atlantic cod Vdrs. However, binary exposures of calcitriol together with phenanthrene, fluorene, or pyrene, antagonized the activation of gmVdra, while chrysene and BaP significantly potentiated the calcitriol-mediated activity of both receptors. Homology modeling, solvent mapping, and docking analyses complemented the experimental data, and revealed a putative secondary binding site in addition to the canonical ligand-binding pocket (LBP). Calcitriol was predicted to interact with both binding sites, whereas PAHs docked primarily to the LBP. Importantly, our in vitro data suggest that PAHs can interact with the paralogous gmVdrs and interfere with their transcriptional activities, and thus potentially modulate the vitamin D signaling pathway and contribute to adverse effects of crude oil and PAH exposures on cardiac development a, This study was funded by the Research Council of Norway through the ”iCod 2.0: Integrative environmental genomics of Atlantic cod” project (project no. 244564) and the ”dCod 1.0: decoding systems toxicology of Atlantic cod” project (Center for Digital Life Norway project no. 248840).

Psoriasis is a chronic inflammatory skin disease characterized by epidermal hyperplasia and cellular infiltration. Studies have shown that disease development depends on proinflammatory cytokines, such as interleukin (IL)-23 and IL-17. It has been suggested that IL-23 produced by innate immune cells, such as macrophages, stimulates a subset of helper T cells to release IL-17, promoting neutrophil recruitment and keratinocyte proliferation. However, recent studies have revealed the crucial role of gamma delta T cells in psoriasis pathogenesis as the primary source of dermal IL-17. The nuclear receptors REV-ERBs are ligand-dependent transcription factors recognized as circadian rhythm regulators. REV-ERBs negatively regulate IL-17-producing helper T cells, whereas the involvement of REV-ERBs in regulating IL-17-producing gamma delta T (gamma delta T17) cells remains unclear. Here we revealed the regulatory mechanism involving gamma delta T17 cells through REV-ERBs. gamma delta T17 cell levels were remarkably elevated in the secondary lymphoid organs of mice that lacked an isoform of REV-ERBs. A synthetic REV-ERB agonist, 5R9009, suppressed gamma delta T17 cells in vitro and in vivo. Topical application of SR9009 to the skin reduced the inflammatory symptoms of psoriasiform dermatitis in mice. The results of this study provide a novel therapeutic approach for psoriasis targeting REV-ERBs in gamma delta T17 cells.

Steroid hormones control development and homeostasis in a wide variety of animals by interacting with intracellular nuclear receptors. Recent discoveries in the fruit fly Drosophila melanogaster revealed that insect steroid hormones or ecdysteroids are incorporated into cells through a membrane transporter named Ecdysone Importer (EcI), which may become a novel target for manipulating steroid hormone signaling in insects. In this study, we established an assay system that can rapidly assess EcI-mediated ecdysteroid entry into cultured cells. Using NanoLuc Binary Technology (NanoBiT), we first developed an assay to detect ligand-dependent heterodimerization of the ecdysone receptor (EcR) and retinoid X receptor (RXR) in human embryonic kidney (HEK) 293T cells. We also developed HEK293 cells that stably express EcI. By combining these tools, we can monitor ecdysteroid entry into the cells in real time, making it a reliable system to assess EcI-mediated steroid hormone incorporation into animal cells.

Psoriasis is a chronic inflammatory skin disease characterized by epidermal hyperplasia and cellular infiltration. Studies have shown that disease development depends on proinflammatory cytokines, such as interleukin (IL)-23 and IL-17. It has been suggested that IL-23 produced by innate immune cells, such as macrophages, stimulates a subset of helper T cells to release IL-17, promoting neutrophil recruitment and keratinocyte proliferation. However, recent studies have revealed the crucial role of gamma delta T cells in psoriasis pathogenesis as the primary source of dermal IL-17. The nuclear receptors REV-ERBs are ligand-dependent transcription factors recognized as circadian rhythm regulators. REV-ERBs negatively regulate IL-17-producing helper T cells, whereas the involvement of REV-ERBs in regulating IL-17-producing gamma delta T (gamma delta T17) cells remains unclear. Here we revealed the regulatory mechanism involving gamma delta T17 cells through REV-ERBs. gamma delta T17 cell levels were remarkably elevated in the secondary lymphoid organs of mice that lacked an isoform of REV-ERBs. A synthetic REV-ERB agonist, 5R9009, suppressed gamma delta T17 cells in vitro and in vivo. Topical application of SR9009 to the skin reduced the inflammatory symptoms of psoriasiform dermatitis in mice. The results of this study provide a novel therapeutic approach for psoriasis targeting REV-ERBs in gamma delta T17 cells.

The immune system plays an essential role in protecting the host from infectious diseases and cancer. Notably, B and T lymphocytes from the adaptive arm of the immune system can co-operate to form long-lived antibody responses and are therefore the main target in vaccination approaches. Nevertheless, protective immune responses must be tightly regulated to avoid hyper-responsiveness and responses against self that can result in autoimmunity. Nuclear receptors (NRs) are perfectly adapted to rapidly alter transcriptional cellular responses to altered environmental settings. Their functional role is associated with both immune deficiencies and autoimmunity. Despite extensive linking of nuclear receptor function with specific CD4 T helper subsets, research on the functional roles and mechanisms of specific NRs in CD4 follicular T helper cells (Tfh) and germinal center (GC) B cells during the germinal center reaction is just emerging. We review recent advances in our understanding of NR regulation in specific cell types of the GC response and discuss their implications for autoimmune diseases such as systemic lupus erythematosus (SLE).

Variants of NR5A1 are often found in individuals with 46,XY disorders of sex development (DSD) and manifest with a very broad spectrum of clinical characteristics and variable sex hormone levels. Such complex phenotypic expression can be due to the inheritance of additional genetic hits in DSD-associated genes that modify sex determination, differentiation and organ function in patients with heterozygous NR5A1 variants. Here we describe the clinical, biochemical and genetic features of a series of seven patients harboring monoallelic variants in the NR5A1 gene. We tested the transactivation activity of novel NR5A1 variants. We additionally included six of these patients in a targeted diagnostic gene panel for DSD and identified a second genetic hit in known DSD-causing genes STAR, AMH and ZFPM2/FOG2 in three individuals. Our study increases the number of NR5A1 variants related to 46,XY DSD and supports the hypothesis that a digenic mode of inheritance may contribute towards the broad spectrum of phenotypes observed in individuals with a heterozygous NR5A1 variation.

Hazard assessment, based on new approach methods (NAM), requires the use of batteries of assays, where individual tests may be contributed by different laboratories. A unified strategy for such collaborative testing is presented. It details all procedures required to allow test information to be usable for integrated hazard assessment, strategic project decisions and/or for regulatory purposes. The EU-ToxRisk project developed a strategy to provide regulatorily valid data, and exemplified this using a panel of > 20 assays (with > 50 individual endpoints), each exposed to 19 well-known test compounds (e.g. rotenone, colchicine, mercury, paracetamol, rifampicine, paraquat, taxol). Examples of strategy implementation are provided for all aspects required to ensure data validity: (i) documentation of test methods in a publicly accessible database; (ii) deposition of standard operating procedures (SOP) at the European Union DB-ALM repository; (iii) test readiness scoring accoding to defined criteria; (iv) disclosure of the pipeline for data processing; (v) link of uncertainty measures and metadata to the data; (vi) definition of test chemicals, their handling and their behavior in test media; (vii) specification of the test purpose and overall evaluation plans. Moreover, data generation was exemplified by providing results from 25 reporter assays. A complete evaluation of the entire test battery will be described elsewhere. A major learning from the retrospective analysis of this large testing project was the need for thorough definitions of the above strategy aspects, ideally in form of a study pre-registration, to allow adequate interpretation of the data and to ensure overall scientific/toxicological validity.

Variants of NR5A1 are often found in individuals with 46,XY disorders of sex development (DSD) and manifest with a very broad spectrum of clinical characteristics and variable sex hormone levels. Such complex phenotypic expression can be due to the inheritance of additional genetic hits in DSD-associated genes that modify sex determination, differentiation and organ function in patients with heterozygous NR5A1 variants. Here we describe the clinical, biochemical and genetic features of a series of seven patients harboring monoallelic variants in the NR5A1 gene. We tested the transactivation activity of novel NR5A1 variants. We additionally included six of these patients in a targeted diagnostic gene panel for DSD and identified a second genetic hit in known DSD-causing genes STAR, AMH and ZFPM2/FOG2 in three individuals. Our study increases the number of NR5A1 variants related to 46,XY DSD and supports the hypothesis that a digenic mode of inheritance may contribute towards the broad spectrum of phenotypes observed in individuals with a heterozygous NR5A1 variation.

The hypothalamic-pituitary-adrenal (HPA) axis plays a critical role in adaptive stress responses and maintaining organism homeostasis. The pituitary corticotroph is the central player in the HPA axis and is regulated by a plethora of hormonal and stress related factors that synergistically interact to activate and temper pro-opiomelanocortin (POMC) transcription, to either increase or decrease adrenocorticotropic hormone (ACTH) production and secretion as needed. Nuclear receptors are a family of highly conserved transcription factors that can also be induced by various physiologic signals, and they mediate their responses via multiple targets to regulate metabolism and homeostasis. In this review, we summarize the modulatory roles of nuclear receptors on pituitary corticotroph cell POMC transcription, describe the unique and complex role these factors play in hypothalamic-pituitary-adrenal axis (HPA) regulation and discuss potential therapeutic targets in disease states.

The immune system plays an essential role in protecting the host from infectious diseases and cancer. Notably, B and T lymphocytes from the adaptive arm of the immune system can co-operate to form long-lived antibody responses and are therefore the main target in vaccination approaches. Nevertheless, protective immune responses must be tightly regulated to avoid hyper-responsiveness and responses against self that can result in autoimmunity. Nuclear receptors (NRs) are perfectly adapted to rapidly alter transcriptional cellular responses to altered environmental settings. Their functional role is associated with both immune deficiencies and autoimmunity. Despite extensive linking of nuclear receptor function with specific CD4 T helper subsets, research on the functional roles and mechanisms of specific NRs in CD4 follicular T helper cells (Tfh) and germinal center (GC) B cells during the germinal center reaction is just emerging. We review recent advances in our understanding of NR regulation in specific cell types of the GC response and discuss their implications for autoimmune diseases such as systemic lupus erythematosus (SLE).

The immune system plays an essential role in protecting the host from infectious diseases and cancer. Notably, B and T lymphocytes from the adaptive arm of the immune system can co-operate to form long-lived antibody responses and are therefore the main target in vaccination approaches. Nevertheless, protective immune responses must be tightly regulated to avoid hyper-responsiveness and responses against self that can result in autoimmunity. Nuclear receptors (NRs) are perfectly adapted to rapidly alter transcriptional cellular responses to altered environmental settings. Their functional role is associated with both immune deficiencies and autoimmunity. Despite extensive linking of nuclear receptor function with specific CD4 T helper subsets, research on the functional roles and mechanisms of specific NRs in CD4 follicular T helper cells (Tfh) and germinal center (GC) B cells during the germinal center reaction is just emerging. We review recent advances in our understanding of NR regulation in specific cell types of the GC response and discuss their implications for autoimmune diseases such as systemic lupus erythematosus (SLE).

Variants of NR5A1 are often found in individuals with 46,XY disorders of sex development (DSD) and manifest with a very broad spectrum of clinical characteristics and variable sex hormone levels. Such complex phenotypic expression can be due to the inheritance of additional genetic hits in DSD-associated genes that modify sex determination, differentiation and organ function in patients with heterozygous NR5A1 variants. Here we describe the clinical, biochemical and genetic features of a series of seven patients harboring monoallelic variants in the NR5A1 gene. We tested the transactivation activity of novel NR5A1 variants. We additionally included six of these patients in a targeted diagnostic gene panel for DSD and identified a second genetic hit in known DSD-causing genes STAR, AMH and ZFPM2/FOG2 in three individuals. Our study increases the number of NR5A1 variants related to 46,XY DSD and supports the hypothesis that a digenic mode of inheritance may contribute towards the broad spectrum of phenotypes observed in individuals with a heterozygous NR5A1 variation.

The immune system plays an essential role in protecting the host from infectious diseases and cancer. Notably, B and T lymphocytes from the adaptive arm of the immune system can co-operate to form long-lived antibody responses and are therefore the main target in vaccination approaches. Nevertheless, protective immune responses must be tightly regulated to avoid hyper-responsiveness and responses against self that can result in autoimmunity. Nuclear receptors (NRs) are perfectly adapted to rapidly alter transcriptional cellular responses to altered environmental settings. Their functional role is associated with both immune deficiencies and autoimmunity. Despite extensive linking of nuclear receptor function with specific CD4 T helper subsets, research on the functional roles and mechanisms of specific NRs in CD4 follicular T helper cells (Tfh) and germinal center (GC) B cells during the germinal center reaction is just emerging. We review recent advances in our understanding of NR regulation in specific cell types of the GC response and discuss their implications for autoimmune diseases such as systemic lupus erythematosus (SLE).

Nuclear receptors (NRs) are key regulators of human health and constitute a relevant target for medicinal chemistry applications as well as for toxicological risk assessment. Several open databases dedicated to small molecules that modulate NRs exist; however, depending on their final aim (i.e., adverse effect assessment or drug design), these databases contain a different amount and type of annotated molecules, along with a different distribution of experimental bioactivity values. Stemming from these considerations, in this work we aim to provide a unified dataset, NURA (NUclear Receptor Activity) dataset, collecting curated information on small molecules that modulate NRs, to be intended for both pharmacological and toxicological applications. NURA contains bioactivity annotations for 15,247 molecules and 11 selected NRs, and it was obtained by integrating and curating data from toxicological and pharmacological databases (i.e., Tox21, ChEMBL, NR-DBIND and BindingDB). Our results show that NURA dataset is a useful tool to bridge the gap between toxicology- and medicinal-chemistry-related databases, as it is enriched in terms of number of molecules, structural diversity and covered atomic scaffolds compared to the single sources. To the best of our knowledge, NURA dataset is the most exhaustive collection of small molecules annotated for their modulation of the chosen nuclear receptors. NURA dataset is intended to support decision-making in pharmacology and toxicology, as well as to contribute to data-driven applications, such as machine learning. The dataset and the data curation pipeline can be downloaded free of charge on Zenodo at the following DOI: https://doi.org/10.5281/zenodo.3991561.

The immune system plays an essential role in protecting the host from infectious diseases and cancer. Notably, B and T lymphocytes from the adaptive arm of the immune system can co-operate to form long-lived antibody responses and are therefore the main target in vaccination approaches. Nevertheless, protective immune responses must be tightly regulated to avoid hyper-responsiveness and responses against self that can result in autoimmunity. Nuclear receptors (NRs) are perfectly adapted to rapidly alter transcriptional cellular responses to altered environmental settings. Their functional role is associated with both immune deficiencies and autoimmunity. Despite extensive linking of nuclear receptor function with specific CD4 T helper subsets, research on the functional roles and mechanisms of specific NRs in CD4 follicular T helper cells (Tfh) and germinal center (GC) B cells during the germinal center reaction is just emerging. We review recent advances in our understanding of NR regulation in specific cell types of the GC response and discuss their implications for autoimmune diseases such as systemic lupus erythematosus (SLE).

総説, 胆汁酸とはステロイド骨格に水酸基とカルボキシル基をもつ両親媒性有機陰イオンである.胆汁酸は肝内でコレステロールの異化により生成されて,胆汁主成分として分泌されるとともに腸管内での脂質の消化吸収に重要な役割を果たす.その大部分は回腸末端部にて再吸収されて肝内に還流する腸肝循環を形成するため,その代謝は極めて効率的であるとともにその破綻を惹起する“黄疸・胆汁うっ滞性疾患"の臨床診断の指標ともなる.近年,その代謝機構において鍵となる様々な分子が発見されて創薬に繋がっている.中でも核内レセプターFarnesoid X receptor(FXR)は大腸炎,大腸発癌と関わることが知られておりそのリガンドによる治療戦略については関心が高い.本稿では去る2019年2月13日開催の第153回東邦大学医学会で講演させて頂いた,“臨床医として知っておきたい胆汁酸の役割"について消化器病診療への関わりを中心に概説する.

This thesis uses a multimodality approach to gain more insight into the complexity of the genomic actions of thyroid hormone receptors.

Nurr1, a nuclear receptor essential for the development, maintenance, and survival of midbrain dopaminergic neurons, is a potential therapeutic target for Parkinson's disease, a neurological disorder characterized by the degeneration of these same neurons. Efforts to identify Nurr1 agonists have been hampered by the recognition that it lacks several classic regulatory elements of nuclear receptor function, including the canonical ligand-binding pocket. Here we report that the dopamine metabolite 5,6-dihydroxyindole (DHI) binds directly to and modulates the activity of Nurr1. Using biophysical assays and X-ray crystallography, we show that DHI binds to the ligand-binding domain within a non-canonical pocket, forming a covalent adduct with Cys566. In cultured cells and zebrafish, DHI stimulates Nurr1 activity, including the transcription of target genes underlying dopamine homeostasis. These findings suggest avenues for developing synthetic Nurr1 ligands to ameliorate the symptoms and progression of Parkinson's disease.

The nuclear receptors are transcription factors involved in the regulation of a variety of physiological processes whose activity can be modulated by binding to relevant small molecule ligands. Their dysfunction has been shown to play a role in disease states such as diabetes, cancer, inflammatory diseases, and hormonal resistance ailments, which makes them interesting targets for drug discovery. The nuclear receptor REV-ERBα is involved in regulating the circadian rhythm and metabolism. Its natural ligand is heme and there is significant interest in identifying novel synthetic modulators to serve as tools to characterize its function and to serve as drugs in treating metabolic disorders. To do so, we established a mammalian cell-based two-hybrid assay system capable of measuring the interaction between REV-ERBα and its co-repressor, nuclear co-repressor 1. This assay was validated to industry standard criteria and was used to screen a subset of the LOPAC®1280 library and 29568 compounds from a diverse compound library. Profiling of the primary hits in a panel of counter and selectivity assays confirmed that REV-ERBα activity can be modulated pharmacologically and chemical scaffolds have been identified for optimization.

The nuclear receptors are transcription factors involved in the regulation of a variety of physiological processes whose activity can be modulated by binding to relevant small molecule ligands. Their dysfunction has been shown to play a role in disease states such as diabetes, cancer, inflammatory diseases, and hormonal resistance ailments, which makes them interesting targets for drug discovery. The nuclear receptor REV-ERBα is involved in regulating the circadian rhythm and metabolism. Its natural ligand is heme and there is significant interest in identifying novel synthetic modulators to serve as tools to characterize its function and to serve as drugs in treating metabolic disorders. To do so, we established a mammalian cell-based two-hybrid assay system capable of measuring the interaction between REV-ERBα and its co-repressor, nuclear co-repressor 1. This assay was validated to industry standard criteria and was used to screen a subset of the LOPAC®1280 library and 29568 compounds from a diverse compound library. Profiling of the primary hits in a panel of counter and selectivity assays confirmed that REV-ERBα activity can be modulated pharmacologically and chemical scaffolds have been identified for optimization.

Generative artificial intelligence offers a fresh view on molecular design. We present the first-time prospective application of a deep learning model for designing new druglike compounds with desired activities. For this purpose, we trained a recurrent neural network to capture the constitution of a large set of known bioactive compounds represented as SMILES strings. By transfer learning, this general model was fine-tuned on recognizing retinoid X and peroxisome proliferator-activated receptor agonists. We synthesized five top-ranking compounds designed by the generative model. Four of the compounds revealed nanomolar to low-micromolar receptor modulatory activity in cell-based assays. Apparently, the computational model intrinsically captured relevant chemical and biological knowledge without the need for explicit rules. The results of this study advocate generative artificial intelligence for prospective de novo molecular design, and demonstrate the potential of these methods for future medicinal chemistry

Author Posting. © The Author(s), 2018. This is the author's version of the work. It is posted here under a nonexclusive, irrevocable, paid-up, worldwide license granted to WHOI. It is made available for personal use, not for redistribution. The definitive version was published in Journal of Steroid Biochemistry and Molecular Biology 184 (2018): 3-10, doi:10.1016/j.jsbmb.2018.02.010., Nuclear receptors are a superfamily of transcription factors restricted to animals. These transcription factors regulate a wide variety of genes with diverse roles in cellular homeostasis, development, and physiology. The origin and specificity of ligand binding within lineages of nuclear receptors (e.g., subfamilies) continues to be a focus of investigation geared toward understanding how the functions of these proteins were shaped over evolutionary history. Among early-diverging animal lineages, the retinoid X receptor (RXR) is first detected in the placozoan, Trichoplax adhaerens. To gain insight into RXR evolution, we characterized ligand- and DNA-binding activity of the RXR from T. adhaerens (TaRXR). Like bilaterian RXRs, TaRXR specifically bound 9-cis-retinoic acid, which is consistent with a recently published result and supports a conclusion that the ancestral RXR bound ligand. DNA binding site specificity of TaRXR was determined through protein binding microarrays (PBMs) and compared with human RXR. The binding sites for these two RXR proteins were broadly conserved (~85% shared high-affinity sequences within a targeted array), suggesting evolutionary constraint for the regulation of downstream genes. We searched for predicted binding motifs of the T. adhaerens genome within 1000 bases of annotated genes to identify potential regulatory targets. We identified 648 unique protein coding regions with predicted TaRXR binding sites that had diverse predicted functions, with enriched processes related to intracellular signal transduction and protein transport. Together, our data support hypotheses that the original RXR protein in animals bound a ligand with structural similarity to 9-cis-retinoic acid; the DNA motif recognized by RXR has changed little in more than 1 billion years of evolution; and the suite of processes regulated by this transcription factor diversified early in animal evolution., Support for AMT was provided by the Tropical Research Initiative and an Internal Research and Development Award from the Woods Hole Oceanographic Institution. AMR was supported by NIH award R15GM114740. JM was supported by NSF award 1536530 to AMR. DM-P, BF and FMS were supported by NIH award R01DK094707 to FMS.

Author Posting. © The Author(s), 2018. This is the author's version of the work. It is posted here under a nonexclusive, irrevocable, paid-up, worldwide license granted to WHOI. It is made available for personal use, not for redistribution. The definitive version was published in Journal of Steroid Biochemistry and Molecular Biology 184 (2018): 3-10, doi:10.1016/j.jsbmb.2018.02.010., Nuclear receptors are a superfamily of transcription factors restricted to animals. These transcription factors regulate a wide variety of genes with diverse roles in cellular homeostasis, development, and physiology. The origin and specificity of ligand binding within lineages of nuclear receptors (e.g., subfamilies) continues to be a focus of investigation geared toward understanding how the functions of these proteins were shaped over evolutionary history. Among early-diverging animal lineages, the retinoid X receptor (RXR) is first detected in the placozoan, Trichoplax adhaerens. To gain insight into RXR evolution, we characterized ligand- and DNA-binding activity of the RXR from T. adhaerens (TaRXR). Like bilaterian RXRs, TaRXR specifically bound 9-cis-retinoic acid, which is consistent with a recently published result and supports a conclusion that the ancestral RXR bound ligand. DNA binding site specificity of TaRXR was determined through protein binding microarrays (PBMs) and compared with human RXR. The binding sites for these two RXR proteins were broadly conserved (~85% shared high-affinity sequences within a targeted array), suggesting evolutionary constraint for the regulation of downstream genes. We searched for predicted binding motifs of the T. adhaerens genome within 1000 bases of annotated genes to identify potential regulatory targets. We identified 648 unique protein coding regions with predicted TaRXR binding sites that had diverse predicted functions, with enriched processes related to intracellular signal transduction and protein transport. Together, our data support hypotheses that the original RXR protein in animals bound a ligand with structural similarity to 9-cis-retinoic acid; the DNA motif recognized by RXR has changed little in more than 1 billion years of evolution; and the suite of processes regulated by this transcription factor diversified early in animal evolution., Support for AMT was provided by the Tropical Research Initiative and an Internal Research and Development Award from the Woods Hole Oceanographic Institution. AMR was supported by NIH award R15GM114740. JM was supported by NSF award 1536530 to AMR. DM-P, BF and FMS were supported by NIH award R01DK094707 to FMS.

Type 2 diabetes mellitus is a disorder characterized by insulin resistance and a relative deficit in insulin secretion, both of which result in elevated blood glucose. Understanding the molecular mechanisms underlying the pathophysiology of diabetes could lead to the development of new therapeutic approaches. An ever-growing body of evidence suggests that members of the NR4A family of nuclear receptors could play a pivotal role in glucose homeostasis. This review aims to present and discuss advances so far in the evaluation of the potential role of NR4A in the regulation of glucose homeostasis and the development of type 2 diabetes., Le diabète de type 2 est caractérisé par une résistance à l’insuline et une détérioration progressive de la sécrétion d’insuline, deux perturbations qui entraînent une élévation de la glycémie. La compréhension des mécanismes moléculaires participant au développement du diabète de type 2 permettra le développement de nouvelles approches thérapeutiques pour le traitement de cette maladie. Il a été suggéré que les membres de la famille des récepteurs nucléaires NR4A pourraient jouer un rôle central dans l’homéostasie du glucose. Dans cette minirevue, nous décrirons l’état actuel des connaissances et discuterons du rôle potentiel des NR4A dans l’homéostasie du glucose et le diabète de type 2.

Steroid hormones are a group of lipophilic hormones that are believed to enter cells by simple diffusion to regulate diverse physiological processes through intracellular nuclear receptors. Here, we challenge this model in Drosophila by demonstrating that Ecdysone Importer (EcI), a membrane transporter identified from two independent genetic screens, is involved in cellular uptake of the steroid hormone ecdysone. EcI encodes an organic anion transporting polypeptide of the evolutionarily conserved solute carrier organic anion superfamily. In vivo, EcI loss of function causes phenotypes indistinguishable from ecdysone- or ecdysone receptor (EcR)-deficient animals, and EcI knockdown inhibits cellular uptake of ecdysone. Furthermore, EcI regulates ecdysone signaling in a cell-autonomous manner and is both necessary and sufficient for inducing ecdysone-dependent gene expression in culture cells expressing EcR. Altogether, our results challenge the simple diffusion model for cellular uptake of ecdysone and may have wide implications for basic and medical aspects of steroid hormone studies.

The Aedes aegypti mosquito is the principal vector for many dangerous human viral diseases. Carbohydrate metabolism (CM) is essential for supplying the energy necessary for host seeking, blood digestion and rapid egg development of this vector insect. The steroid hormone 20-hydroxyecdysone (20E) and the ecdysone receptor (EcR) are important regulators of CM, coordinating it with female reproductive events. We report here that the NR4A nuclear receptor AHR38 plays a critical role in mediating these actions of 20E and EcR. AHR38 RNA interference (RNAi) depletion in female mosquitoes blocked the transcriptional activation of CM genes encoding phosphoglucomutase (PGM) and trehalose-6-phophate synthase (TPS); it caused an increase of glycogen accumulation and a decrease of the circulating sugar trehalose. This treatment also resulted in a dramatic reduction in fecundity. Considering that these phenotypes resulting from AHR38 RNAi depletion are similar to those of EcR RNAi, we investigated a possible connection between these transcription factors in CM regulation. EcR RNAi inhibits the AHR38 gene expression. Moreover, the 20E-induced EcR complex directly activates AHR38 by binding to the ecdysone response element (EcRE) in the upstream regulatory region of this gene. The present work has implicated AHR38 in the 20E-mediated control of CM and provided new insight into mechanisms of 20E regulation of metabolism during female mosquito reproduction.

Generative artificial intelligence offers a fresh view on molecular design. We present the first-time prospective application of a deep learning model for designing new druglike compounds with desired activities. For this purpose, we trained a recurrent neural network to capture the constitution of a large set of known bioactive compounds represented as SMILES strings. By transfer learning, this general model was fine-tuned on recognizing retinoid X and peroxisome proliferator-activated receptor agonists. We synthesized five top-ranking compounds designed by the generative model. Four of the compounds revealed nanomolar to low-micromolar receptor modulatory activity in cell-based assays. Apparently, the computational model intrinsically captured relevant chemical and biological knowledge without the need for explicit rules. The results of this study advocate generative artificial intelligence for prospective de novo molecular design, and demonstrate the potential of these methods for future medicinal chemistry

The human body has developed a defence system to prevent the accumulation of endogenous (bile acids, steroids, cholesterol metabolites, neurotransmitters, etc.) as well as exogenous (xenobiotics, clinical drugs, etc.) small molecules at toxic levels. This task is accomplished by ‘drug metabolism and disposition (DMD) machinery' which entails phase I and phase II enzymes, and phase III transporter proteins. The components of this machinery act in a coordinated manner to biotransform and facilitate the elimination of small toxic molecules from the cellular milieu. Constitutive androstane receptor (CAR), a member of the nuclear receptor superfamily, acts as one of the major transcriptional regulators of the DMD machinery. Prescription of combination therapy is a common regimen during the treatment of diverse metabolic disorders and infectious diseases. In such combination therapies one drug may modulate the expression of genes of DMD, influencing the metabolism of another co-administered drug. This leads to decreased bioavailability or increased toxicity of the latter. Evaluation of drug-drug interactions (DDIs) has now become a major safety concern during drug discovery and development processes. Pre-assessment of the small molecules for modulatory effects on CAR and induction of the components of DMD can resolve the safety concerns, treatment failures and drug withdrawals due to the harmful DDIs. In the present study, we have followed a ‘reverse approach' to assess CAR activation by drugs previously withdrawn from clinical practices. We selected three redundant members of thiazolidinedione family of anti-diabetic drugs and examined their potential in regulation of CAR and its target gene CYP2B6. These drugs showed differential transcriptional activation of CAR. Two of the TZD i.e., rosiglitazone and pioglitazone enhanced CAR activity by behaving as receptor ligands while the other (troglitazone) did not influence the receptor function and was justly withdrawn since i

The human body has developed a defence system to prevent the accumulation of endogenous (bile acids, steroids, cholesterol metabolites, neurotransmitters, etc.) as well as exogenous (xenobiotics, clinical drugs, etc.) small molecules at toxic levels. This task is accomplished by ‘drug metabolism and disposition (DMD) machinery' which entails phase I and phase II enzymes, and phase III transporter proteins. The components of this machinery act in a coordinated manner to biotransform and facilitate the elimination of small toxic molecules from the cellular milieu. Constitutive androstane receptor (CAR), a member of the nuclear receptor superfamily, acts as one of the major transcriptional regulators of the DMD machinery. Prescription of combination therapy is a common regimen during the treatment of diverse metabolic disorders and infectious diseases. In such combination therapies one drug may modulate the expression of genes of DMD, influencing the metabolism of another co-administered drug. This leads to decreased bioavailability or increased toxicity of the latter. Evaluation of drug-drug interactions (DDIs) has now become a major safety concern during drug discovery and development processes. Pre-assessment of the small molecules for modulatory effects on CAR and induction of the components of DMD can resolve the safety concerns, treatment failures and drug withdrawals due to the harmful DDIs. In the present study, we have followed a ‘reverse approach' to assess CAR activation by drugs previously withdrawn from clinical practices. We selected three redundant members of thiazolidinedione family of anti-diabetic drugs and examined their potential in regulation of CAR and its target gene CYP2B6. These drugs showed differential transcriptional activation of CAR. Two of the TZD i.e., rosiglitazone and pioglitazone enhanced CAR activity by behaving as receptor ligands while the other (troglitazone) did not influence the receptor function and was justly withdrawn since i

27-Hydroxycholesterol (27OH) is a strong suppressor of cholesterol synthesis and a weak activator of LXR in vitro. The regulatory importance of 27OH in vivo is controversial. Here we utilized male mice with increased levels of 27OH either due to increased production (CYP27A1 transgenic mice) or reduced metabolism (Cyp7b1-/- mice). We also used mice lacking 27OH due to a knockout of Cyp27a1. The latter mice were treated with cholic acid to compensate for reduced bile acid synthesis. The effects of the different levels of 27OH on Srebp- and other LXR-regulated genes in the liver were investigated. In the liver of CYP27tg mice we found a modest increase of the mRNA levels corresponding to the LXR target genes Cyp7b1 and Abca1. A number of other LXR-regulated genes were not affected. The effect on Abca1 mRNA was not seen in the liver of Cyp7b1-/- mice. There were little or no effects on cholesterol synthesis. In the liver of the Cyp27-/- mice treated with 0.025% cholic acid there was no significant effect of the knockout on the LXR target genes. In a previous work triple-knockout mice deficient in the biosynthesis of 24S-hydroxycholesterol, 25-hydroxycholesterol and 27OH were shown to have impaired response to dietary cholesterol, suggesting side-chain oxidized oxysterols to be mediators in cholesterol-induced effects on LXR target genes at a transcriptional level (Chen W. et al., Cell Metab. 5 (2007) 73-79). The hydroxylated oxysterol responsible for the effect was not defined. We show here that treatment of wildtype mice with dietary cholesterol under the same conditions as in the above study induced the LXR target genes Lpl, Abcg8 and Srebp1c in wild type mice but failed to activate the same genes in mice lacking 27-hydroxycholesterol due to a knockout of Cyp27. We failed to demonstrate the above effects at the protein level (Abcg8) or at the activity level (Lpl). The results suggest that 27OH is not an important regulator of Srebp- or LXR regulated genes under basal, Special Issue: SI

The placenta and tumors share important characteristics, including a requirement to establish effective angiogenesis. In the case of the placenta, optimal angiogenesis is required to sustain the blood flow required to maintain a successful pregnancy, whereas in tumors establishing new blood supplies is considered a key step in supporting metastases. Therefore the development of novel angiogenesis inhibitors has been an area of active research in oncology. A subset of the molecular processes regulating angiogenesis are well understood in the context of both early placentation and tumorigenesis. In this review we focus on the well-established role of androgen regulation of angiogenesis in cancer and relate these mechanisms to placental angiogenesis. The physiological actions of androgens are mediated by the androgen receptor (AR), a ligand dependent transcription factor. Androgens and the AR are essential for normal male embryonic development, puberty and lifelong health. Defects in androgen signalling are associated with a diverse range of clinical disorders in men and women including disorders of sex development (DSD), polycystic ovary syndrome in women and many cancers. We summarize the diverse molecular mechanisms of androgen regulation of angiogenesis and infer the potential significance of these pathways to normal and pathogenic placental function. Finally, we offer potential research applications of androgen-targeting molecules developed to treat cancer as investigative tools to help further delineate the role of androgen signalling in placental function and maternal and offspring health in animal models.

Cholesterol homeostasis is maintained through concerted action of the SREBPs and LXRs. Here, we report that RNF145, a previously uncharacterized ER membrane ubiquitin ligase, participates in crosstalk between these critical signaling pathways. RNF145 expression is induced in response to LXR activation and high-cholesterol diet feeding. Transduction of RNF145 into mouse liver inhibits the expression of genes involved in cholesterol biosynthesis and reduces plasma cholesterol levels. Conversely, acute suppression of RNF145 via shRNA-mediated knockdown, or chronic inactivation of RNF145 by genetic deletion, potentiates the expression of cholesterol biosynthetic genes and increases cholesterol levels both in liver and plasma. Mechanistic studies show that RNF145 triggers ubiquitination of SCAP on lysine residues within a cytoplasmic loop essential for COPII binding, potentially inhibiting its transport to Golgi and subsequent processing of SREBP-2. These findings define an additional mechanism linking hepatic sterol levels to the reciprocal actions of the SREBP-2 and LXR pathways.