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Sample records for ahr agonist activity

  1. Characterization of AhR agonists reveals antagonistic activity in European herring gull (Larus argentatus) eggs.

    PubMed

    Muusse, Martine; Christensen, Guttorm; Gomes, Tânia; Kočan, Anton; Langford, Katherine; Tollefsen, Knut Erik; Vaňková, Lenka; Thomas, Kevin V

    2015-05-01

    European herring gull (Larus argentatus) eggs from two Norwegian islands, Musvær in the south east and Reiaren in Northern Norway, were screened for dioxins, furans, and dioxin-like and selected non-dioxin-like polychlorinated biphenyls (PCBs), and subjected to non-target analysis to try to identify the aryl hydrocarbon receptor (AhR) agonists, responsible for elevated levels measured using the dioxin responsive chemically activated luciferase expression (DR-CALUX) assay. Eggs from Musvær contained chemically calculated toxic equivalent (WHO TEQ) levels of between 109 and 483 pg TEQ/g lw, and between 82 and 337 pg TEQ/g lw was determined in eggs from Reiaren. In particular PCB126 contributed highly to the total TEQ (69-82%). In 19 of the 23 samples the calculated WHO TEQ was higher than the TEQCALUX. Using CALUX specific relative effect potencies (REPs), the levels were lower at between 77 and 292 pg/g lw in eggs from Musvær and between 55 and 223 pg/g lw in eggs from Reiaren, which was higher than the TEQCALUX in 16 of the 23 samples. However, the means of the REP values and the TEQCALUX were not significantly different. This suggests the presence of compounds that can elicit antagonist effects, with a low binding affinity to the AhR. Non-target analysis identified the presence of hexachlorobenzene (HCB) (quantified at 9.6-185 pg/g lw) but neither this compound nor high concentrations of PCB126 and non-dioxin-like PCBs could explain the differences between the calculated TEQ or REP values and the TEQCALUX. Even though, for most AhR agonists, the sensitivity of herring gulls is not known, the reported levels can be considered to represent a risk for biological effects in the developing embryo, compared to LC50 values in chicken embryos. For human consumers of herring gull eggs, these eggs contain TEQ levels up to four times higher than the maximum tolerable weekly intake.

  2. TOXICITY OF AHR AGONISTS TO FISH EARLY LIFE STAGES

    EPA Science Inventory

    Fish early life stages are exceptionally sensitive to the lethal toxicity of chemicals that act as arylhydrocarbon receptor (AhR) agonists. Toxicity characterizations based on 2,3,7,8-tetrachlorodibenzo-p-dioxin, generally the most potent AhR agonist, support the toxicity equiva...

  3. Polychlorinated biphenyls (PCBs) contamination and aryl hydrocarbon receptor (AhR) agonist activity of Omega-3 polyunsaturated fatty acid supplements: implications for daily intake of dioxins and PCBs.

    PubMed

    Bourdon, J A; Bazinet, T M; Arnason, T T; Kimpe, L E; Blais, J M; White, P A

    2010-11-01

    Omega-3 polyunsaturated fatty acid (n-3 PUFA) rich oils derived primarily from fish are frequently consumed as supplements. Due to the tendency of persistent organic pollutants (POPs) to accumulate in exposed organisms, n-3 PUFA supplements can contain sufficient POPs to present a risk to consumers. Here we investigated PCB concentrations and aryl hydrocarbon receptor (AhR) agonist activity in 17 n-3 PUFA supplements available in Canada. PCBs ranged from <0.8 to 793 ng g(-1) oil, with salmon- and seal-derived products yielding the highest values. AhR agonist activity from a reporter gene assay ranged from 1.3 to 72.2 pg TEQ g(-1) oil, with salmon and tuna yielding the highest values. When consumed at the recommended doses and as a supplement to the average Canadian diet, seal-derived oil can contribute to exceedance of the tolerable daily intake of 20 ng PCBs kg-BW(-1)day(-1), and salmon-, tuna-, and sea herring-derived oils can contribute to exceedance of the tolerable daily intake limit of 2.3 pg TEQ kg-BW(-1)day(-1). The beneficial properties of fish and n-3 PUFA supplements, and the results of this study suggest that it is prudent to consume supplements derived from small, cold-water fatty fish. Further research will be necessary to draw firm conclusions.

  4. Combination effects of AHR agonists and Wnt/β-catenin modulators in zebrafish embryos: Implications for physiological and toxicological AHR functions

    SciTech Connect

    Wincent, Emma; Stegeman, John J.; Jönsson, Maria E.

    2015-04-15

    Wnt/β-catenin signaling regulates essential biological functions and acts in developmental toxicity of some chemicals. The aryl hydrocarbon receptor (AHR) is well-known to mediate developmental toxicity of persistent dioxin-like compounds (DLCs). Recent studies indicate a crosstalk between β-catenin and the AHR in some tissues. However the nature of this crosstalk in embryos is poorly known. We observed that zebrafish embryos exposed to the β-catenin inhibitor XAV939 display effects phenocopying those of the dioxin-like 3,3′,4,4′,5-pentachlorobiphenyl (PCB126). This led us to investigate the AHR interaction with β-catenin during development and ask whether developmental toxicity of DLCs involves antagonism of β-catenin signaling. We examined phenotypes and transcriptional responses in zebrafish embryos exposed to XAV939 or to a β-catenin activator, 1-azakenpaullone, alone or with AHR agonists, either PCB126 or 6-formylindolo[3,2-b]carbazole (FICZ). Alone 1-azakenpaullone and XAV939 both were embryo-toxic, and we found that in the presence of FICZ, the toxicity of 1-azakenpaullone decreased while the toxicity of XAV939 increased. This rescue of 1-azakenpaullone effects occurred in the time window of Ahr2-mediated toxicity and was reversed by morpholino-oligonucleotide knockdown of Ahr2. Regarding PCB126, addition of either 1-azakenpaullone or XAV939 led to lower mortality than with PCB126 alone but surviving embryos showed severe edemas. 1-Azakenpaullone induced transcription of β-catenin-associated genes, while PCB126 and FICZ blocked this induction. The data indicate a stage-dependent antagonism of β-catenin by Ahr2 in zebrafish embryos. We propose that the AHR has a physiological role in regulating β-catenin during development, and that this is one point of intersection linking toxicological and physiological AHR-governed processes.

  5. Tissue specificity of aryl hydrocarbon receptor (AhR) mediated responses and relative sensitivity of white sturgeon (Acipenser transmontanus) to an AhR agonist.

    PubMed

    Doering, Jon A; Wiseman, Steve; Beitel, Shawn C; Tendler, Brett J; Giesy, John P; Hecker, Markus

    2012-06-15

    Sturgeons are endangered in some parts of the world. Due to their benthic nature and longevity sturgeon are at greater risk of exposure to bioaccumulative contaminants such as dioxin-like compounds that are associated with sediments. Despite their endangered status, little research has been conducted to characterize the relative responsiveness of sturgeon to dioxin-like compounds. In an attempt to study the biological effects and possible associated risks of exposure to dioxin-like compounds in sturgeon, the molecular and biochemical responses of white sturgeon (Acipenser transmontanus) to a model aryl hydrocarbon receptor (AhR) agonist, β-naphthoflavone (βNF) were investigated. White sturgeon were injected intraperitoneally with one of three doses of βNF (0, 50, or 500mg/kg, bw). Rainbow trout (Oncorhynchus mykiss) were used as a reference species since their responses have been well characterized in the past. Three days following injection with βNF, fish were euthanized and livers, gills, and intestines collected for biochemical and molecular analyses. White sturgeon exposed to βNF had significantly greater ethoxyresorufin O-deethylase (EROD) activity in liver (up to 37-fold), gill (up to 41-fold), and intestine (up to 36-fold) than did unexposed controls. Rainbow trout injected with βNF exhibited EROD activity that was significantly greater in liver (88-fold), than that of controls, but was undetectable in gills or intestine. Abundance of CYP1A transcript displayed a comparable pattern of tissue-specific induction with intestine (up to 189-fold), gills (up to 53-fold), and liver (up to 21-fold). Methoxyresorufin O-deethylase (MROD) and pentoxyresorufin O-deethylase (PROD) activities were undetectable in unexposed white sturgeon tissues while exposed tissues displayed MROD activity that was only moderately greater than the activity that could be detected. Differential inducibility among liver, gill, and intestine following exposure to an AhR agonist is

  6. Zebrafish Cardiotoxicity: The Effects of CYP1A Inhibition and AHR2 Knockdown Following Exposure to Weak Aryl Hydrocarbon Receptor Agonists

    PubMed Central

    Clark, Bryan William; Van Tiem Garner, Lindsey; Di Giulio, Richard Thomas

    2014-01-01

    The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that mediates many of the toxic effects of dioxin-like compounds (DLCs) and some polycyclic aromatic hydrocarbons (PAHs). Strong AHR agonists, such as certain polychlorinated biphenyls and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), cause severe cardiac teratogenesis in fish embryos. Moderately strong AHR agonists, such as benzo[a]pyrene and β-naphthoflavone, have been shown to cause similar cardiotoxic effects when coupled with a cytochrome P450 1A (CYP1A) inhibitor, such as fluoranthene (FL). We sought to determine if weak AHR agonists, when combined with a CYP1A inhibitor (FL) or CYP1A morpholino gene knockdown, are capable of causing cardiac deformities similar to moderately strong AHR agonists (Wassenberg and Di Giulio 2004; Wassenberg and Di Giulio 2004; Billiard, Timme-Laragy et al. 2006; Van Tiem and Di Giulio 2011). The weak AHR agonists included the following: carbaryl, phenanthrene, 2-methylindole, 3-methylindole, indigo, and indirubin. The results showed a complex pattern of cardiotoxic response to weak agonist inhibitor exposure and morpholino-knockdown. Danio rerio (zebrafish) embryos were first exposed to weak AHR agonists at equimolar concentrations. The agonists were assessed for their relative potency as inducers of CYP1 enzyme activity, measured by the ethoxyresorufin-o-deethylase (EROD) assay, and cardiac deformities. Carbaryl, 2-methylindole, and 3-methylindole induced the highest CYP1A activity in zebrafish. Experiments were then conducted to determine the individual cardiotoxicity of each compound. Next, zebrafish were co-exposed to each agonist (at concentrations below those determined to be cardiotoxic) and FL in combination to assess if CYP1A inhibition could induce cardiac deformities. Carbaryl, 2-methylindole, 3-methylindole, and phenanthrene significantly increased pericardial edema relative to controls when combined with FL. To further evaluate the

  7. Mono-Substituted Isopropylated Triaryl Phosphate, a Major Component of Firemaster 550, is an AHR Agonist that Exhibits AHR-Independent Cardiotoxicity in Zebrafish

    PubMed Central

    Gerlach, Cory V.; Das, Siba R.; Volz, David C.; Bisson, William H.; Kolluri, Siva K.; Tanguay, Robert L.

    2014-01-01

    Firemaster 550 (FM550) is an additive flame retardant mixture used within polyurethane foam and is increasingly found in house dust and the environment due to leaching. Despite the widespread use of FM550, very few studies have investigated the potential toxicity of its ingredients during early vertebrate development. In the current study, we sought to specifically investigate mono-substituted isopropylated triaryl phosphate (mITP), a component comprising approximately 32% of FM550, which has been shown to cause cardiotoxicity during zebrafish embryogenesis. Previous research showed that developmental defects are rescued using an aryl hydrocarbon receptor (AHR) antagonist (CH223191), suggesting that mITP-induced toxicity was AHR-dependent. As zebrafish have three known AHR isoforms, we used a functional AHR2 knockout line along with AHR1A-and AHR1B-specific morpholinos to determine which AHR isoform, if any, mediates mITP-induced cardiotoxicity. As in silico structural homology modeling predicted that mITP may bind favorably to both AHR2 and AHR1B isoforms, we evaluated AHR involvement in vivo by measuring CYP1A mRNA and protein expression following exposure to mITP in the presence or absence of CH223191 or AHR-specific morpholinos. Based on these studies, we found that mITP interacts with both AHR2 and AHR1B isoforms to induce CYP1A expression. However, while CH223191 blocked mITP-induced CYP1A induction and cardiotoxicity, knockdown of all three AHR isoforms failed to block mITP-induced cardiotoxicity in the absence of detectable CYP1A induction. Overall, these results suggest that, while mITP is an AHR agonist, mITP causes AHR-independent cardiotoxicity through a pathway that is also antagonized by CH223191. PMID:24865613

  8. Mono-substituted isopropylated triaryl phosphate, a major component of Firemaster 550, is an AHR agonist that exhibits AHR-independent cardiotoxicity in zebrafish.

    PubMed

    Gerlach, Cory V; Das, Siba R; Volz, David C; Bisson, William H; Kolluri, Siva K; Tanguay, Robert L

    2014-09-01

    Firemaster 550 (FM550) is an additive flame retardant mixture used within polyurethane foam and is increasingly found in house dust and the environment due to leaching. Despite the widespread use of FM550, very few studies have investigated the potential toxicity of its ingredients during early vertebrate development. In the current study, we sought to specifically investigate mono-substituted isopropylated triaryl phosphate (mITP), a component comprising approximately 32% of FM550, which has been shown to cause cardiotoxicity during zebrafish embryogenesis. Previous research showed that developmental defects are rescued using an aryl hydrocarbon receptor (AHR) antagonist (CH223191), suggesting that mITP-induced toxicity was AHR-dependent. As zebrafish have three known AHR isoforms, we used a functional AHR2 knockout line along with AHR1A- and AHR1B-specific morpholinos to determine which AHR isoform, if any, mediates mITP-induced cardiotoxicity. As in silico structural homology modeling predicted that mITP may bind favorably to both AHR2 and AHR1B isoforms, we evaluated AHR involvement in vivo by measuring CYP1A mRNA and protein expression following exposure to mITP in the presence or absence of CH223191 or AHR-specific morpholinos. Based on these studies, we found that mITP interacts with both AHR2 and AHR1B isoforms to induce CYP1A expression. However, while CH223191 blocked mITP-induced CYP1A induction and cardiotoxicity, knockdown of all three AHR isoforms failed to block mITP-induced cardiotoxicity in the absence of detectable CYP1A induction. Overall, these results suggest that, while mITP is an AHR agonist, mITP causes AHR-independent cardiotoxicity through a pathway that is also antagonized by CH223191.

  9. RELATIONSHIPS BETWEEN RESIDUES OF AHR AGONISTS IN FISH AND CONCENTRATIONS IN WATER AND SEDIMENTS

    EPA Science Inventory

    Relationships between Residues of AhR Agonists in Fish and Concentrations in Water and Sediment. Cook, PM*, Burkhard, LP, Mount, DR, US-EPA, NHEERL, MED, Duluth, MN. The bioaccumulation visualization approach of Burkhard et al. (2002) can be effectively used to describe the bioa...

  10. Teratogenic impact of dioxin-activated AHR in laboratory animals

    EPA Science Inventory

    AHR and ARNT are expressed in mouse and human palatal shelves and in the urinary tract of the mouse fetus. AHR expression, translocation to the nucleus, binding to DRE, and activation are required for mediation of TCDD-induction of CP and HN. Although the human palate requires a ...

  11. Combined chemical and toxicological long-term monitoring for AhR agonists with SPMD-based virtual organisms in drinking water Danjiangkou Reservoir, China.

    PubMed

    Wang, Jingxian; Song, Guoqiang; Li, Aimin; Henkelmann, Bernhard; Pfister, Gerd; Tong, Anthony Z; Schramm, Karl-Werner

    2014-08-01

    SPMD-based virtual organisms (VOs) were employed for time-integrating, long-term sampling combined biological and chemical analyses for exposure assessment of hydrophobic organic pollutants (HOPs) in a drinking water reservoir, China. The SPMDs were deployed at four and five sites in the Danjiangkou (DJK) reservoir over two periods of 26 and 31 d to sequester the hydrophobic contaminants in water. The chosen bioassay response for the extracts of the SPMDs, the induction of 7-ethoxyresorufin-o-deethylase (EROD) was assayed using a rat hepatoma cell line (H4IIE). The known aryl hydrocarbon receptor (AhR) agonists PAHs and PCBs were analyzed by HRGC/HRMS instrument. The cause-effect relationship between the observed AhR activities and chemical concentrations of detected AhR agonists was examined. The results show that the extracts from the SPMD samples could induce AhR activity significantly, whereas the chemically derived 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) equivalent (TEQcal) was not correlated with the bioassay-derived TCDD equivalent (TEQbio). The known AhR agonists could only account for 2-10% of the observed AhR responses among which the contribution of PCBs could almost be neglected. Unidentified AhR-active compounds represented a greater proportion of the TCDD equivalent (TCDD-EQ) in SPMD samples from DJK. Based on the first assessment, the VO followed by the combination of chemical and biological analyses emerges as a resource efficient water monitoring device in ecotoxicological assessment for toxicologically relevant compounds which are readily available for uptake by resident aquatic biota in drinking water resources.

  12. The activation mechanism of the aryl hydrocarbon receptor (AhR) by molecular chaperone HSP90

    PubMed Central

    Tsuji, Noriko; Fukuda, Kana; Nagata, Yuhtaroh; Okada, Hirotaka; Haga, Asami; Hatakeyama, Shiori; Yoshida, Shiho; Okamoto, Tomoya; Hosaka, Miki; Sekine, Kazuhiro; Ohtaka, Kei; Yamamoto, Soh; Otaka, Michiro; Grave, Ewa; Itoh, Hideaki

    2014-01-01

    The aryl hydrocarbon receptor is a member of the nuclear receptor superfamily that associates with the molecular chaperone HSP90 in the cytoplasm. The activation mechanism of the AhR is not yet fully understood. It has been proposed that after binding of ligands such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 3methylcholanthrene (3-MC), or β-naphthoflavone (β-NF), the AhR dissociates from HSP90 and translocates to the nucleus. It has also been hypothesized that the AhR translocates to the nucleus and forms a complex with HSP90 and other co-chaperones. There are a few reports about the direct association or dissociation of AhR and HSP90 due to difficulties in purifying AhR. We constructed and purified the PAS domain from AhR. Binding of the AhR-PAS domain to β-NF affinity resin suggested that it possesses ligand-binding affinity. We demonstrated that the AhR-PAS domain binds to HSP90 and the association is not affected by ligand binding. The ligand 17-DMAG inhibited binding of HSP90 to GST-PAS. In an immunoprecipitation assay, HSP90 was co-immunoprecipitated with AhR both in the presence or absence of ligand. Endogenous AhR decreased in the cytoplasm and increased in the nucleus of HeLa cells 15 min after treatment with ligand. These results suggested that the ligand-bound AhR is translocated to nucleus while in complex with HSP90. We used an in situ proximity ligation assay to confirm whether AhR was translocated to the nucleus alone or together with HSP90. HSP90 was co-localized with AhR after the nuclear translocation. It has been suggested that the ligand-bound AhR was translocated to the nucleus with HSP90. Activated AhR acts as a transcription factor, as shown by the transcription induction of the gene CYP1A1 8 h after treatment with β-NF. PMID:25349783

  13. Molecular Determinants of Species-Specific Agonist and Antagonist Activity of A Substituted Flavone towards the Aryl Hydrocarbon Receptor*

    PubMed Central

    Henry, E. C.; Gasiewicz, T. A.

    2008-01-01

    The aryl hydrocarbon receptor (AhR) mediates the toxicity of dioxins and related xenobiotics. Other chemicals also bind the AhR to elicit either agonist or antagonist responses. Here we used site-directed mutagenesis within the ligand binding domain of murine AhR to probe for specific residues that might interact differentially with the antagonist 3’-methoxy-4’-nitroflavone (MNF) compared with the prototypical agonist TCDD. Reduced 3 H-TCDD binding, dioxin-response element (DRE) binding, and transcriptional activity were observed for several point mutants. One mutation, R355I, changed the response to MNF from antagonist to agonist. Notably, Ile is the residue found in the guinea pig AhR, towards which MNF has partial agonist activity in contrast to its strong antagonist activity in mouse. A similar reversal of response to MNF was observed in chimeric AhRs in which the C-terminal region of mAhR was replaced with the guinea pig C-terminal region. These data demonstrate that different amino acids can be important in binding of different AhR ligands and can mediate distinct responses. The ultimate response of the AhR also depends on how other portions of the receptor protein are functionally coupled to the initial ligand binding event. PMID:18294953

  14. A constitutive active MAPK/ERK pathway due to BRAFV600E positively regulates AHR pathway in PTC

    PubMed Central

    Regazzo, Daniela; Bertazza, Loris; Galuppini, Francesca; Guzzardo, Vincenza; Jaffrain-Rea, Marie Lise; Vianello, Federica; Ciato, Denis; Ceccato, Filippo; Watutantrige-Fernando, Sara; Bisognin, Andrea; Bortoluzzi, Stefania; Pennelli, Gianmaria; Boscaro, Marco; Scaroni, Carla; Mian, Caterina

    2015-01-01

    The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor mediating the toxicity and tumor-promoting properties of dioxin. AHR has been reported to be overexpressed and constitutively active in a variety of solid tumors, but few data are currently available concerning its role in thyroid cancer. In this study we quantitatively explored a series of 51 paired-normal and papillary thyroid carcinoma (PTC) tissues for AHR-related genes. We identified an increased AHR expression/activity in PTC, independently from its nuclear dimerization partner and repressor but strictly related to a constitutive active MAPK/ERK pathway. The AHR up-regulation followed by an increased expression of AHR target genes was confirmed by a meta-analysis of published microarray data, suggesting a ligand-independent active AHR pathway in PTC. In-vitro studies using a PTC-derived cell line (BCPAP) and HEK293 cells showed that BRAFV600E may directly modulate AHR localization, induce AHR expression and activity in an exogenous ligand-independent manner. The AHR pathway might represent a potential novel therapeutic target for PTC in the clinical practice. PMID:26392334

  15. A constitutive active MAPK/ERK pathway due to BRAFV600E positively regulates AHR pathway in PTC.

    PubMed

    Occhi, Gianluca; Barollo, Susi; Regazzo, Daniela; Bertazza, Loris; Galuppini, Francesca; Guzzardo, Vincenza; Jaffrain-Rea, Marie Lise; Vianello, Federica; Ciato, Denis; Ceccato, Filippo; Watutantrige-Fernando, Sara; Bisognin, Andrea; Bortoluzzi, Stefania; Pennelli, Gianmaria; Boscaro, Marco; Scaroni, Carla; Mian, Caterina

    2015-10-13

    The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor mediating the toxicity and tumor-promoting properties of dioxin. AHR has been reported to be overexpressed and constitutively active in a variety of solid tumors, but few data are currently available concerning its role in thyroid cancer. In this study we quantitatively explored a series of 51 paired-normal and papillary thyroid carcinoma (PTC) tissues for AHR-related genes. We identified an increased AHR expression/activity in PTC, independently from its nuclear dimerization partner and repressor but strictly related to a constitutive active MAPK/ERK pathway. The AHR up-regulation followed by an increased expression of AHR target genes was confirmed by a meta-analysis of published microarray data, suggesting a ligand-independent active AHR pathway in PTC. In-vitro studies using a PTC-derived cell line (BCPAP) and HEK293 cells showed that BRAFV600E may directly modulate AHR localization, induce AHR expression and activity in an exogenous ligand-independent manner. The AHR pathway might represent a potential novel therapeutic target for PTC in the clinical practice.

  16. New CYP1 genes in the frog Xenopus (Silurana) tropicalis: Induction patterns and effects of AHR agonists during development

    SciTech Connect

    Joensson, Maria E.; Berg, Cecilia; Goldstone, Jared V.; Stegeman, John J.

    2011-01-15

    The Xenopus tropicalis genome shows a single gene in each of the four cytochrome P450 1 (CYP1) subfamilies that occur in vertebrates, designated as CYP1A, CYP1B1, CYP1C1, and CYP1D1. We cloned the cDNAs of these genes and examined their expression in untreated tadpoles and in tadpoles exposed to waterborne aryl hydrocarbon receptor agonists, 3,3',4,4',5-pentachlorobiphenyl (PCB126), {beta}-naphthoflavone ({beta}NF), or indigo. We also examined the effects of PCB126 on expression of genes involved in stress response, cell proliferation, thyroid homeostasis, and prostaglandin synthesis. PCB126 induced CYP1A, CYP1B1, and CYP1C1 but had little effect on CYP1D1 (77-, 1.7-, 4.6- and 1.4-fold induction versus the control, respectively). {beta}NF induced CYP1A and CYP1C1 (26- and 2.5-fold), while, under conditions used, indigo tended to induce only CYP1A (1.9-fold). The extent of CYP1 induction by PCB126 and {beta}NF was positively correlated to the number of putative dioxin response elements 0-20 kb upstream of the start codons. No morphological effect was observed in tadpoles exposed to 1 nM-10 {mu}M PCB126 at two days post-fertilization (dpf) and screened 20 days later. However, in 14-dpf tadpoles a slight up-regulation of the genes for PCNA, transthyretin, HSC70, Cu-Zn SOD, and Cox-2 was observed two days after exposure to 1 {mu}M PCB126. This study of the full suite of CYP1 genes in an amphibian species reveals gene- and AHR agonist-specific differences in response, as well as a much lower sensitivity to CYP1 induction and short-term toxicity by PCB126 compared with in fish larvae. The single genes in each CYP1 subfamily may make X. tropicalis a useful model for mechanistic studies of CYP1 functions.

  17. New CYP1 genes in the frog Xenopus (Silurana) tropicalis: Induction patterns and effects of AHR agonists during development

    PubMed Central

    Jönsson, Maria E.; Berg, Cecilia; Goldstone, Jared V.; Stegeman, John J.

    2010-01-01

    The Xenopus tropicalis genome shows a single gene in each of the four cytochrome P450 1 (CYP1) subfamilies that occur in vertebrates, designated as CYP1A, CYP1B1, CYP1C1, and CYP1D1. We cloned the cDNAs of these genes and examined their expression in untreated tadpoles and in tadpoles exposed to waterborne aryl hydrocarbon receptor agonists, 3,3',4,4',5-pentachlorobiphenyl (PCB126), β-naphthoflavone (βNF), or indigo. We also examined the effects of PCB126 on expression of genes involved in stress response, cell proliferation, thyroid homeostasis, and prostaglandin synthesis. PCB126 induced CYP1A, CYP1B1, and CYP1C1 but had little effect on CYP1D1 (77-, 1.7-, 4.6- and 1.4-fold induction versus the control, respectively). βNF induced CYP1A and CYP1C1 (26- and 2.5-fold), while, under conditions used, indigo tended to induce only CYP1A (1.9-fold). The extent of CYP1 induction by PCB126 and βNF was positively correlated to the number of putative dioxin response elements 0–20 kb upstream of the start codons. No morphological effect was observed in tadpoles exposed to 1 nM-10 µM PCB126 at two days post-fertilization (dpf) and screened 20 days later. However, in 14-dpf tadpoles a slight up-regulation of the genes for PCNA, transthyretin, HSC70, Cu-Zn SOD, and Cox-2 was observed two days after exposure to 1 µM PCB126. This study of the full suite of CYP1 genes in an amphibian species reveals gene- and AHR agonist-specific differences in response, as well as a much lower sensitivity to CYP1 induction and short-term toxicity by PCB126 compared with in fish larvae. The single genes in each CYP1 subfamily may make X. tropicalis a useful model for mechanistic studies of CYP1 functions. PMID:20965207

  18. 3-Methylcholanthrene and other aryl hydrocarbon receptor agonists directly activate estrogen receptor alpha.

    PubMed

    Abdelrahim, Maen; Ariazi, Eric; Kim, Kyounghyun; Khan, Shaheen; Barhoumi, Rola; Burghardt, Robert; Liu, Shengxi; Hill, Denise; Finnell, Richard; Wlodarczyk, Bogdan; Jordan, V Craig; Safe, Stephen

    2006-02-15

    3-Methylcholanthrene (3MC) is an aryl hydrocarbon receptor (AhR) agonist, and it has been reported that 3MC induces estrogenic activity through AhR-estrogen receptor alpha (ER alpha) interactions. In this study, we used 3MC and 3,3',4,4',5-pentachlorobiphenyl (PCB) as prototypical AhR ligands, and both compounds activated estrogen-responsive reporter genes/gene products (cathepsin D) in MCF-7 breast cancer cells. The estrogenic responses induced by these AhR ligands were inhibited by the antiestrogen ICI 182780 and by the transfection of a small inhibitory RNA for ER alpha but were not affected by the small inhibitory RNA for AhR. These results suggest that 3MC and PCB directly activate ER alpha, and this was confirmed in a competitive ER alpha binding assay and in a fluorescence resonance energy transfer experiment in which PCB and 3MC induced CFP-ER alpha/YFP-ER alpha interactions. In a chromatin immunoprecipitation assay, PCB and 3MC enhanced ER alpha (but not AhR) association with the estrogen-responsive region of the pS2 gene promoter. Moreover, in AhR knockout mice, 3MC increased uterine weights and induced expression of cyclin D1 mRNA levels. These results show that PCB and 3MC directly activate ER alpha-dependent transactivation and extend the number of ligands that activate both AhR and ER alpha.

  19. A binding site for activation by the Bacillus subtilis AhrC protein, a repressor/activator of arginine metabolism.

    PubMed

    Klingel, U; Miller, C M; North, A K; Stockley, P G; Baumberg, S

    1995-08-21

    In Bacillus subtilis, the AhrC protein represses genes encoding enzymes of arginine biosynthesis and activates those mediating its catabolism. To determine how this repressor also functions as an activator, we attempted to clone catabolic genes by searching for insertions of the Tn917-lacZ transposon that express AhrC-dependent, arginine-inducible beta-galactosidase activity. One such isolate was obtained. The region upstream of lacZ was subcloned in Escherichia coli in such a way that it could be replaced in the B. subtilis chromosome after appropriate manipulation. Analysis of exonuclease III-derived deletions located an AhrC-dependent, arginine-inducible promoter to within a ca. 1.9 kb fragment. The sequence revealed: the 3' end of an ORF homologous to gdh genes encoding glutamate dehydrogenase, with highest homology to the homologue from Clostridium difficile; the 5' end of an ORF homologous to a Saccharomyces cerevisiae gene encoding delta 1-pyrroline 5-carboxylate dehydrogenase (P5CDH), an enzyme of arginine catabolism; and just upstream of the latter, a sequence with homology to known AhrC binding sites in the upstream part of the biosynthetic argCJBD-cpa-F cluster. The same region has also been sequenced by others as part of the B. subtilis genome sequencing project, revealing that the P5CDH gene is the first in a cluster termed rocABC. Restriction fragments containing the putative AhrC-binding sequence, but not those lacking it, showed retarded electrophoretic mobility in the presence of purified AhrC. A 277 bp AhrC-binding fragment also showed anomalous mobility in the absence of AhrC, consistent with its being intrinsically bent. DNAse I footprinting localized AhrC binding to bp -16/-22 to +1 (the transcription startpoint). Such a location for an activator binding site, i.e. overlapping the transcription start, is unusual.

  20. Effects of scorched food leachates with or without activated charcoal pretreatment on AhR activation in cultured cells.

    PubMed

    Takahashi, Satoshi; Morita, Koji; Kinoshita, Makoto; Fujimori, Shin; Ishikawa, Toshio

    2015-12-01

    Aryl hydrocarbon receptor (AhR) is a transcription factor activated by xenobiotics, including dioxins and polycyclic aromatic hydrocarbons (PAHs). Although AhR is also activated by some dietary constituents, it has not been completely clarified in what circumstances AhR ligands are ingested in our daily life. Because PAHs are formed by the incomplete combustion of organic materials, we hypothesized that scorched foods might contain and leach out AhR ligands sufficient to stimulate AhR in vitro. To test this hypothesis, scorched foods (bread, cheese, etc.) were mixed vigorously with water, and the supernatants were retrieved as samples. The samples were added to HepG2 cells stably expressing an AhR-responsive reporter gene. Also, expression of CYP1A1, an endogenous AhR-responsive gene, was analyzed by RT-PCR in different cell lines treated with the samples. We further tested whether pretreatment of the samples with activated charcoal would alter their AhR-stimulating activity. All the supernatant samples tested induced AhR-dependent reporter gene activity and CYP1A1 mRNA expression. In some samples, these inductions were inhibited by pretreatment with activated charcoal. Our findings indicate that scorched food leachates stimulate AhR in cultured cells and that activated charcoal adsorbs the AhR-stimulating substances in some leachates. Thus, people who habitually eat scorched foods are exposed to AhR ligands on a regular basis. Further studies are needed to elucidate whether burnt foods actually exert biological effects on our health.

  1. Gestational exposure to the AhR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin induces BRCA-1 promoter hypermethylation and reduces BRCA-1 expression in mammary tissue of rat offspring: preventive effects of resveratrol.

    PubMed

    Papoutsis, Andreas J; Selmin, Ornella I; Borg, Jamie L; Romagnolo, Donato F

    2015-04-01

    Studies with murine models suggest that maternal exposure to aromatic hydrocarbon receptor (AhR) agonists may impair mammary gland differentiation and increase the susceptibility to mammary carcinogenesis in offspring. However, the molecular mechanisms responsible for these perturbations remain largely unknown. Previously, we reported that the AhR agonists 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induced CpG methylation of the breast cancer-1 (BRCA-1) gene and reduced BRCA-1 expression in breast cancer cell lines. Based on the information both the human and rat BRCA-1 genes harbor xenobiotic responsive elements (XRE = 5'-GCGTG-3'), which are binding targets for the AhR, we extended our studies to the analysis of offspring of pregnant Sprague-Dawley rats treated during gestation with TCDD alone or in combination with the dietary AhR antagonist resveratrol (Res). We report that the in utero exposure to TCDD increased the number of terminal end buds (TEB) and reduced BRCA-1 expression in mammary tissue of offspring. The treatment with TCDD induced occupancy of the BRCA-1 promoter by DNA methyltransferase-1 (DNMT-1), CpG methylation of the BRCA-1 promoter, and expression of cyclin D1 and cyclin-dependent kinase-4 (CDK4). These changes were partially overridden by pre-exposure to Res, which stimulated the expression of the AhR repressor (AhRR) and its recruitment to the BRCA-1 gene. These findings point to maternal exposure to AhR agonists as a risk factor for breast cancer in offspring through epigenetic inhibition of BRCA-1 expression, whereas dietary antagonists of the AhR may exert protective effects.

  2. AhR activation underlies the CYP1A autoinduction by A-998679 in rats

    PubMed Central

    Liguori, Michael J.; Lee, Chih-Hung; Liu, Hong; Ciurlionis, Rita; Ditewig, Amy C.; Doktor, Stella; Andracki, Mark E.; Gagne, Gerard D.; Waring, Jeffrey F.; Marsh, Kennan C.; Gopalakrishnan, Murali; Blomme, Eric A. G.; Yang, Yi

    2012-01-01

    Xenobiotic-mediated induction of cytochrome P450 (CYP) drug metabolizing enzymes (DMEs) is frequently encountered in drug discovery and can influence disposition, pharmacokinetic, and toxicity profiles. The CYP1A subfamily of DMEs plays a central role in the biotransformation of several drugs and environmental chemicals. Autoinduction of drugs through CYP3A enzymes is a common mechanism for their enhanced clearance. However, autoinduction via CYP1A is encountered less frequently. In this report, an experimental compound, A-998679 [3-(5-pyridin-3-yl-1,2,4-oxadiazol-3-yl) benzonitrile], was shown to enhance its own clearance via induction of Cyp1a1 and Cyp1a2. Rats were dosed for 5 days with 30, 100, and 200 mg/kg/day A-998679. During the dosing period, the compound's plasma AUC decreased at 30 mg/kg (95%) and 100 mg/kg (80%). Gene expression analysis and immunohistochemistry of the livers showed a large increase in the mRNA and protein levels of Cyp1a, which was involved in the biotransformation of A-998679. Induction of CYP1A was confirmed in primary rat, human, and dog hepatocytes. The compound also weakly inhibited CYP1A2 in human liver microsomes. A-998679 activated the aryl hydrocarbon receptor (AhR) in a luciferase gene reporter assay in HepG2 cells, upregulated expression of genes associated with AhR activation in rat liver and enhanced nuclear migration of AhR in HepG2 cells. Collectively these results demonstrate that A-998679 is an AhR activator that induces Cyp1a1 and Cyp1a2 expression, resulting in an autoinduction phenomenon. The unique properties of A-998679, along with its novel structure distinct from classical polycyclic aromatic hydrocarbons (PAHs), may warrant its further evaluation as a tool compound for use in studies involving AhR biology and CYP1A-related mechanisms of drug metabolism and toxicity. PMID:23112805

  3. Ligand Promiscuity of Aryl Hydrocarbon Receptor Agonists and Antagonists Revealed by Site-Directed Mutagenesis

    PubMed Central

    Soshilov, Anatoly A.

    2014-01-01

    The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that can be activated by structurally diverse chemicals. To examine the mechanisms responsible for the promiscuity in AhR ligand binding, we determined the effects of mutations within the AhR ligand-binding domain (LBD) on the activity of diverse AhR ligands. Site-directed mutagenesis identified Ile319 of the mouse AhR and, to a lesser extent, Phe318 as residues involved in ligand-selective modulation of AhR transformation using a panel of 12 AhR ligands. These ligands could be categorized into four distinct structurally related groups based on their ability to activate AhR mutants at position 319 in vitro. The mutation I319K was selectively activated by FICZ and not by other examined ligands in vitro and in cell culture. F318L and F318A mutations resulted in the conversion of AhR agonists β-naphthoflavone and 3-methylcholanthrene, respectively, into partial agonists/antagonists. Hsp90 binding to the AhR was decreased with several mutations and was inversely correlated with AhR ligand-binding promiscuity. Together, these data define overlapping amino acid residues within the AhR LBD involved in the selectivity of ligand binding, the agonist or antagonist mode of ligand binding, and hsp90 binding and provide insights into the ligand diversity of AhR activators. PMID:24591650

  4. Assessment of energetic costs of AhR activation by β-naphthoflavone in rainbow trout (Oncorhynchus mykiss) hepatocytes using metabolic flux analysis

    SciTech Connect

    Nault, Rance; Abdul-Fattah, Hiba; Mironov, Gleb G.; Berezovski, Maxim V.; Moon, Thomas W.

    2013-08-15

    Exposure to environmental contaminants such as activators of the aryl hydrocarbon receptor (AhR) leads to the induction of defense and detoxification mechanisms. While these mechanisms allow organisms to metabolize and excrete at least some of these environmental contaminants, it has been proposed that these mechanisms lead to significant energetic challenges. This study tests the hypothesis that activation of the AhR by the model agonist β-naphthoflavone (βNF) results in increased energetic costs in rainbow trout (Oncorhynchus mykiss) hepatocytes. To address this hypothesis, we employed traditional biochemical approaches to examine energy allocation and metabolism including the adenylate energy charge (AEC), protein synthesis rates, Na{sup +}/K{sup +}-ATPase activity, and enzyme activities. Moreover, we have used for the first time in a fish cell preparation, metabolic flux analysis (MFA) an in silico approach for the estimation of intracellular metabolic fluxes. Exposure of trout hepatocytes to 1 μM βNF for 48 h did not alter hepatocyte AEC, protein synthesis, or Na{sup +}/K{sup +}-ATPase activity but did lead to sparing of glycogen reserves and changes in activities of alanine aminotransferase and citrate synthase suggesting altered metabolism. Conversely, MFA did not identify altered metabolic fluxes, although we do show that the dynamic metabolism of isolated trout hepatocytes poses a significant challenge for this type of approach which should be considered in future studies. - Highlights: • Energetic costs of AhR activation by βNF was examined in rainbow trout hepatocytes. • Metabolic flux analysis was performed on a fish cell preparation for the first time. • Exposure to βNF led to sparing of glycogen reserves and altered enzyme activities. • Adenylate energy charge was maintained despite temporal changes in metabolism.

  5. NcoA2-Dependent Inhibition of HIF-1α Activation Is Regulated via AhR.

    PubMed

    Tsai, Chi-Hao; Li, Ching-Hao; Liao, Po-Lin; Cheng, Yu-Wen; Lin, Cheng-Hui; Huang, Shih-Hsuan; Kang, Jaw-Jou

    2015-12-01

    High endogenous levels of aryl hydrocarbon receptor (AhR) contribute to hypoxia signaling pathway inhibition following exposure to the potent AhR ligand benzo[a]pyrene (B[a]P) and could alter cellular homeostasis and disease condition. Increasing evidence indicates that AhR might compete with AhR nuclear translocator (ARNT) for complex formation with hypoxia-inducible factor-1α (HIF-1α) for transactivation, which could alter several physiological variables. Nuclear receptor coactivator 2 (NcoA2) is a transcription coactivator that regulates transcription factor activation and inhibition of basic helix-loop-helix Per (Period)-ARNT-SIM (single-minded) (bHLH-PAS) family proteins, such as HIF-1α, ARNT, and AhR, through protein-protein interactions. In this study, we demonstrated that both hypoxia and hypoxia-mimic conditions decreased NcoA2 protein expression in HEK293T cells. Hypoxia response element (HRE) and xenobiotic-responsive element (XRE) transactivation also were downregulated with NcoA2 knockdown under hypoxic conditions. In addition, B[a]P significantly decreased NcoA2 protein expression be accompanied with AhR degradation. We next evaluated whether the absence of AhR could affect NcoA2 protein function under hypoxia-mimetic conditions. NcoA2 and HIF-1α nuclear localization decreased in both B[a]P-pretreated and AhR-knockdown HepG2 cells under hypoxia-mimic conditions. Interestingly, NcoA2 overexpression downregulated HRE transactivation by competing with HIF-1α and AhR to form protein complexes with ARNT. Both NcoA2 knockdown and overexpression inhibited endothelial cell tube formation in vitro. We also demonstrated using the in vivo plug assay that NcoA2-regulated vascularization decreased in mice. Taken together, these results revealed a biphasic role of NcoA2 between AhR and hypoxic conditions, thus providing a novel mechanism underlying the cross talk between AhR and hypoxia that affects disease development and progression.

  6. Role of zebrafish cytochrome P450 CYP1C genes in the reduced mesencephalic vein blood flow caused by activation of AHR2

    SciTech Connect

    Kubota, Akira; Stegeman, John J.; Woodin, Bruce R.; Iwanaga, Toshihiko; Harano, Ryo; Peterson, Richard E.; Hiraga, Takeo; Teraoka, Hiroki

    2011-06-15

    2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) causes various signs of toxicity in early life stages of vertebrates through activation of the aryl hydrocarbon receptor (AHR). We previously reported a sensitive and useful endpoint of TCDD developmental toxicity in zebrafish, namely a decrease in blood flow in the dorsal midbrain, but downstream genes involved in the effect are not known. The present study addressed the role of zebrafish cytochrome P450 1C (CYP1C) genes in association with a decrease in mesencephalic vein (MsV) blood flow. The CYP1C subfamily was recently discovered in fish and includes the paralogues CYP1C1 and CYP1C2, both of which are induced via AHR2 in zebrafish embryos. We used morpholino antisense oligonucleotides (MO or morpholino) to block initiation of translation of the target genes. TCDD-induced mRNA expression of CYP1Cs and a decrease in MsV blood flow were both blocked by gene knockdown of AHR2. Gene knockdown of CYP1C1 by two different morpholinos and CYP1C2 by two different morpholinos, but not by their 5 nucleotide-mismatch controls, was effective in blocking reduced MsV blood flow caused by TCDD. The same CYP1C-MOs prevented reduction of blood flow in the MsV caused by {beta}-naphthoflavone (BNF), representing another class of AHR agonists. Whole-mount in situ hybridization revealed that mRNA expression of CYP1C1 and CYP1C2 was induced by TCDD most strongly in branchiogenic primordia and pectoral fin buds. In situ hybridization using head transverse sections showed that TCDD increased the expression of both CYP1Cs in endothelial cells of blood vessels, including the MsV. These results indicate a potential role of CYP1C1 and CYP1C2 in the local circulation failure induced by AHR2 activation in the dorsal midbrain of the zebrafish embryo. - Research Highlights: > We examine the roles of zebrafish CYP1C1 and CYP1C2 in TCDD developmental toxicity. > TCDD induces mRNA expression of both CYP1Cs in the mesencephalic vein. > Knockdown of each

  7. Constitutive AhR activation leads to concomitant ABCG2-mediated multidrug resistance in cisplatin-resistant esophageal carcinoma cells.

    PubMed

    To, Kenneth K W; Yu, Le; Liu, Shuwen; Fu, Jianhua; Cho, Chi Hin

    2012-06-01

    Esophageal squamous cell carcinoma (ESCC) is a highly malignant disease that is generally not responding to chemotherapy. It is particularly predominant in China. Although ESCC is significantly associated with cigarette smoking, the relationship between its molecular pathogenesis and responsiveness to chemotherapy and cigarette smoke remains elusive. This study reported the constitutive activation of aryl hydrocarbon receptor (AhR), leading to ABCG2 upregulation and the multidrug resistance (MDR) phenotype, in ESCC cell lines with acquired cisplatin resistance. Reporter gene assay, chromatin immunoprecipitation analysis and specific gene knockdown confirmed that the enhanced AhR binding to a xenobiotic response element (XRE) within the ABCG2 promoter is responsible for ABCG2 overexpression. A HSP90 inhibitor (17-AAG) and two AhR antagonists (kaempferol and salicylamide) were shown to inhibit ABCG2 upregulation, thereby reversing the ABCG2-mediated MDR. Our data therefore advocate the use of these inhibitors as novel chemosensitizers for the treatment of esophageal cancer.

  8. Benzo[ghi]perylene activates the AHR pathway to exert biological effects on the NL-20 human bronchial cell line.

    PubMed

    Zaragoza-Ojeda, Montserrat; Eguía-Aguilar, Pilar; Perezpeña-Díazconti, Mario; Arenas-Huertero, Francisco

    2016-08-10

    Polycyclic aromatic hydrocarbons (PAH) are produced by incomplete combustion of organic material. In the Mexico City atmosphere, the most abundant PAH is benzo[ghi]perylene (BghiP), a gasoline combustion marker. At present, there are no reports of the effects of BghiP on human bronchial cells, so the aim of the study was to evaluate the effects in vitro of BghiP on the NL-20 cell line. Results showed that BghiP induced the formation of small vesicles throughout the cytoplasm, with absence of nuclear fragmentation. At 48h exposition, damage in cell membrane increased significantly at 1.24μg/mL of BghiP (p<0.05). Immunocytochemistry revealed that BghiP provokes nuclear translocation of AhR receptor, which indicates that this compound can induce transcription of genes via receptor binding (AhR pathway activation). BghiP induced a two-fold increase (p<0.05) in the expression of AhR and CYP4B1 (a lung-specific pathway effector). In the presence of the receptor antagonist CH-223191, the loss of viability, the nuclear translocation and the overexpression of genes decreased, though this did not prevent the formation of vesicles. BghiP induced oxidative stress and in presence of the receptor antagonist this increased significantly. In conclusion, BghiP can activate the overexpression of AhR and CYP4B1, and the effects are abated by the AhR receptor antagonist. This is the first report to prove that BghiP utilizes the AhR pathway to exert its toxic effects on the NL-20 human bronchial cell line .

  9. Newspapers and newspaper ink contain agonists for the ah receptor.

    PubMed

    Bohonowych, Jessica E S; Zhao, Bin; Timme-Laragy, Alicia; Jung, Dawoon; Di Giulio, Richard T; Denison, Michael S

    2008-04-01

    Ligand-dependent activation of the aryl hydrocarbon receptor (AhR) pathway leads to a diverse array of biological and toxicological effects. The best-studied ligands for the AhR include polycyclic and halogenated aromatic hydrocarbons, the most potent of which is 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). However, as new AhR ligands are identified and characterized, their structural and physiochemical diversity continues to expand. Our identification of AhR agonists in crude extracts from diverse materials raises questions as to the magnitude and extent of human exposure to AhR ligands through normal daily activities. We have found that solvent extracts of newspapers from countries around the world stimulate the AhR signaling pathway. AhR agonist activity was observed for dimethyl sulfoxide (DMSO), ethanol, and water extracts of printed newspaper, unprinted virgin paper, and black printing ink, where activation of luciferase reporter gene expression was transient, suggesting that the AhR active chemical(s) was metabolically labile. DMSO and ethanol extracts also stimulated AhR transformation and DNA binding, and also competed with [(3)H]TCDD for binding to the AhR. In addition, DMSO extracts of printed newspaper induced cytochrome P450 1A associated 7-ethoxyresorufin-O-deethylase activity in zebrafish embryos in vivo. Although the responsible bioactive chemical(s) remain to be identified, our results demonstrate that newspapers and printing ink contain relatively potent metabolically labile agonists of the AhR. Given the large amount of recycling and reprocessing of newspapers throughout the world, release of these easily extractable AhR agonists into the environment should be examined and their potential effects on aquatic organisms assessed.

  10. Newspapers and Newspaper Ink Contain Agonists for the Ah Receptor

    PubMed Central

    Bohonowych, Jessica E. S.; Zhao, Bin; Timme-Laragy, Alicia; Jung, Dawoon; Di Giulio, Richard T.; Denison, Michael S.

    2010-01-01

    Ligand-dependent activation of the aryl hydrocarbon receptor (AhR) pathway leads to a diverse array of biological and toxicological effects. The best-studied ligands for the AhR include polycyclic and halogenated aromatic hydrocarbons, the most potent of which is 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). However, as new AhR ligands are identified and characterized, their structural and physiochemical diversity continues to expand. Our identification of AhR agonists in crude extracts from diverse materials raises questions as to the magnitude and extent of human exposure to AhR ligands through normal daily activities. We have found that solvent extracts of newspapers from countries around the world stimulate the AhR signaling pathway. AhR agonist activity was observed for dimethyl sulfoxide (DMSO), ethanol, and water extracts of printed newspaper, unprinted virgin paper, and black printing ink, where activation of luciferase reporter gene expression was transient, suggesting that the AhR active chemical(s) was metabolically labile. DMSO and ethanol extracts also stimulated AhR transformation and DNA binding, and also competed with [3H]TCDD for binding to the AhR. In addition, DMSO extracts of printed newspaper induced cytochrome P450 1A associated 7-ethoxyresorufin-O-deethylase activity in zebrafish embryos in vivo. Although the responsible bioactive chemical(s) remain to be identified, our results demonstrate that newspapers and printing ink contain relatively potent metabolically labile agonists of the AhR. Given the large amount of recycling and reprocessing of newspapers throughout the world, release of these easily extractable AhR agonists into the environment should be examined and their potential effects on aquatic organisms assessed. PMID:18203687

  11. The Effects of Chronic Lifelong Activation of the AHR Pathway by Industrial Chemical Pollutants on Female Human Reproduction

    PubMed Central

    Vacca, Margherita; Nardelli, Claudia; Castegna, Alessandra; Arnesano, Fabio; Carella, Nicola; Depalo, Raffaella

    2016-01-01

    Environmental chemicals, such as heavy metals, affect female reproductive function. A biological sensor of the signals of many toxic chemical compounds seems to be the aryl hydrocarbon receptor (AHR). Previous studies demonstrated the environmental of heavy metals in Taranto city (Italy), an area that has been influenced by anthropogenic factors such as industrial activities and waste treatments since 1986. However, the impact of these elements on female fertility in this geographic area has never been analyzed. Thus, in the present study, we evaluated the AHR pathway, sex steroid receptor pattern and apoptotic process in granulosa cells (GCs) retrieved from 30 women, born and living in Taranto, and 30 women who are living in non-contaminated areas (control group), who were undergoing in vitro fertilization (IVF) protocol. In follicular fluids (FFs) of both groups the toxic and essential heavy metals, such as chromiun (Cr), Manganese (Mn), iron (Fe), cobalt (Co), nickel (Ni), copper (Cu), zinc (Zn), cadmium (Cd) and lead (Pb), were also analyzed. Higher levels of Cr, Fe, Zn and Pb were found in the FFs of the women from Taranto as compared to the control group, as were the levels of AHR and AHR-dependent cytochrome P450 1A1 and 1B1; while CYP19A1 expression was decreased. The anti-apoptotic process found in the GCs of women fromTaranto was associated with the highest levels of progesterone receptor membrane component 1 (PGRMC1), a novel progesterone receptor, the expression of which is subjected to AHR activated by its highest affinity ligands (e.g., dioxins) or indirectly by other environmental pollutants, such as heavy metals. In conclusion, decreased production of estradiol and decreased number of retrieved mature oocytes found in women from Taranto could be due to chronic exposure to heavy metals, in particular to Cr and Pb. PMID:27008165

  12. Agonist-activated ion channels

    PubMed Central

    Colquhoun, David

    2006-01-01

    This paper looks at ion channels as an example of the pharmacologist's stock in trade, the action of an agonist on a receptor to produce a response. Looked at in this way, ion channels have been helpful because they are still the only system which is simple enough for quantitative investigation of transduction mechanisms. A short history is given of attempts to elucidate what happens between the time when agonist first binds, and the time when the channel opens. PMID:16402101

  13. The AhR Ligand, TCDD, Regulates Androgen Receptor Activity Differently in Androgen-Sensitive versus Castration-Resistant Human Prostate Cancer Cells

    PubMed Central

    Ghotbaddini, Maryam; Powell, Joann B.

    2015-01-01

    The reported biological effects of TCDD include induction of drug metabolizing enzymes, wasting syndrome and tumor promotion. TCDD elicits most of its effects through binding the aryl hydrocarbon receptor (AhR). TCDD induced degradation of AhR has been widely reported and requires ubiquitination of the protein. The rapid depletion of AhR following TCDD activation serves as a mechanism to modulate AhR mediated gene induction. In addition to inducing AhR degradation, TCDD has been reported to induce degradation of hormone receptors. The studies reported here, evaluate the effect of TCDD exposure on androgen receptor (AR) expression and activity in androgen-sensitive LNCaP and castration-resistant C4-2 prostate cancer cells. Our results show that TCDD exposure does not induce AhR or AR degradation in C4-2 cells. However, both AhR and AR are degraded in LNCaP cells following TCDD exposure. In addition, TCDD enhances AR phosphorylation and induces expression of AR responsive genes in LNCaP cells. Our data reveals that TCDD effect on AR expression and activity differs in androgen-sensitive and castration-resistant prostate cancer cell models. PMID:26154658

  14. AhR signaling activation disrupts migration and dendritic growth of olfactory interneurons in the developing mouse

    PubMed Central

    Kimura, Eiki; Ding, Yunjie; Tohyama, Chiharu

    2016-01-01

    Perinatal exposure to a low level of dioxin, a ubiquitous environmental pollutant, has been shown to induce abnormalities in learning and memory, emotion, and sociality in laboratory animals later in adulthood. However, how aryl hydrocarbon receptor (AhR) signaling activation disrupts the higher brain function remains unclear. Therefore, we studied the possible effects of excessive activation of AhR signaling on neurodevelopmental processes, such as cellular migration and neurite growth, in mice. To this end, we transfected a constitutively active-AhR plasmid into stem cells in the lateral ventricle by in vivo electroporation on postnatal day 1. Transfection was found to induce tangential migration delay and morphological abnormalities in neuronal precursors in the rostral migratory stream at 6 days post-electroporation (dpe) as well as disrupt radial migration in the olfactory bulb and apical and basal dendritic growth of the olfactory interneurons in the granule cell layer at 13 and 20 dpe. These results suggest that the retarded development of interneurons by the excessive AhR signaling may at least in part explain the dioxin-induced abnormal behavioral alterations previously reported in laboratory animals. PMID:27197834

  15. Pityriazepin and other potent AhR ligands isolated from Malassezia furfur yeast.

    PubMed

    Mexia, Nikitia; Gaitanis, Georgios; Velegraki, Aristea; Soshilov, Anatoly; Denison, Michael S; Magiatis, Prokopios

    2015-04-01

    Malassezia furfur yeast strains isolated from diseased human skin preferentially biosynthesize indole alkaloids which can be detected in the human skin and are highly potent activators of the aryl hydrocarbon receptor (AhR) and AhR-dependent gene expression. Chemical analysis of an EtOAc extract of a M. furfur strain obtained from diseased human skin and grown on l-tryptophan agar revealed several known AhR active tryptophan metabolites along with a previously unidentified compound, pityriazepin. While its structure resembled that of the known alkaloid pityriacitrin, the comprised pyridine ring had been transformed into an azepinone. The indoloazepinone scaffold of pityriazepin is extremely rare in nature and has only been reported once previously. Pityriazepin, like the other isolated compounds, was found to be a potent activator of the AhR-dependent reporter gene assay in recombinant cell lines derived from four different species, although significant species differences in relative potency were observed. The ability of pityriazepin to competitively bind to the AhR and directly stimulate AhR DNA binding classified it as a new naturally-occurring potent AhR agonist. M. furfur produces an expanded collection of extremely potent naturally occurring AhR agonists, which produce their biological effects in a species-specific manner.

  16. A Structural Switch between Agonist and Antagonist Bound Conformations for a Ligand-Optimized Model of the Human Aryl Hydrocarbon Receptor Ligand Binding Domain

    PubMed Central

    Perkins, Arden; Phillips, Jessica L.; Kerkvliet, Nancy I.; Tanguay, Robert L.; Perdew, Gary H.; Kolluri, Siva K.; Bisson, William H.

    2014-01-01

    The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that regulates the expression of a diverse group of genes. Exogenous AHR ligands include the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which is a potent agonist, and the synthetic AHR antagonist N-2-(1H-indol-3yl)ethyl)-9-isopropyl-2-(5-methylpyridin-3-yl)-9H-purin-6-amine (GNF351). As no experimentally determined structure of the ligand binding domain exists, homology models have been utilized for virtual ligand screening (VLS) to search for novel ligands. Here, we have developed an “agonist-optimized” homology model of the human AHR ligand binding domain, and this model aided in the discovery of two human AHR agonists by VLS. In addition, we performed molecular dynamics simulations of an agonist TCDD-bound and antagonist GNF351-bound version of this model in order to gain insights into the mechanics of the AHR ligand-binding pocket. These simulations identified residues 307–329 as a flexible segment of the AHR ligand pocket that adopts discrete conformations upon agonist or antagonist binding. This flexible segment of the AHR may act as a structural switch that determines the agonist or antagonist activity of a given AHR ligand. PMID:25329374

  17. Identification of benzothiazole derivatives and polycyclic aromatic hydrocarbons as aryl hydrocarbon receptor agonists present in tire extracts.

    PubMed

    He, Guochun; Zhao, Bin; Denison, Michael S

    2011-08-01

    Leachate from rubber tire material contains a complex mixture of chemicals previously shown to produce toxic and biological effects in aquatic organisms. The ability of these leachates to induce Ah receptor (AhR)-dependent cytochrome P4501A1 expression in fish indicated the presence of AhR active chemicals, but the responsible chemicals and their direct interaction with the AhR signaling pathway were not examined. Using a combination of AhR-based bioassays, we have demonstrated the ability of tire extract to stimulate both AhR DNA binding and AhR-dependent gene expression and confirmed that the responsible chemicals were metabolically labile. The application of CALUX (chemical-activated luciferase gene expression) cell bioassay-driven toxicant identification evaluation not only revealed that tire extract contained a variety of known AhR-active polycyclic aromatic hydrocarbons but also identified 2-methylthiobenzothiazole and 2-mercaptobenzothiazole as AhR agonists. Analysis of a structurally diverse series of benzothiazoles identified many that could directly stimulate AhR DNA binding and transiently activate the AhR signaling pathway and identified benzothiazoles as a new class of AhR agonists. In addition to these compounds, the relatively high AhR agonist activity of a large number of fractions strongly suggests that tire extract contains a large number of physiochemically diverse AhR agonists whose identities and toxicological/biological significances are unknown.

  18. Identification of Benzothiazole Derivatives and Polycyclic Aromatic Hydrocarbons as Aryl Hydrocarbon Receptor Agonists Present in Tire Extracts

    PubMed Central

    He, Guochun; Zhao, Bin; Denison, Michael S.

    2012-01-01

    Leachate from rubber tire material contains a complex mixture of chemicals previously shown to produce toxic and biological effects in aquatic organisms. While the ability of these leachates to induce Ah receptor (AhR)-dependent cytochrome P4501A1 expression in fish indicated the presence of AhR active chemicals, the responsible chemical(s) and their direct interaction with the AhR signaling pathway were not examined. Using a combination of AhR-based bioassays, we have demonstrated the ability of tire extract to stimulate both AhR DNA binding and AhR-dependent gene expression and confirmed that the responsible chemical(s) was metabolically labile. The application of CALUX (Chemical-Activated LUciferase gene eXpression) cell bioassay-driven toxicant identification evaluation not only revealed that tire extract contained a variety of known AhR-active polycyclic aromatic hydrocarbons, but also identified 2-methylthiobenzothiazole and 2-mercaptobenzothiazole as AhR agonists. Analysis of a structurally diverse series of benzothiazoles identified many that could directly stimulate AhR DNA binding and transiently activate the AhR signaling pathway and identified benzothiazoles as a new class of AhR agonists. In addition to these compounds, the relatively high AhR agonist activity of a large number of fractions strongly suggests that tire extract contains a large number of physiochemically diverse AhR agonists whose identities and toxicological/biological significances are unknown. PMID:21590714

  19. Developmental Activation of the AHR Increases Effector CD4+ T Cells and Exacerbates Symptoms in Autoimmune Disease-Prone Gnaq+/− Mice

    PubMed Central

    Boule, Lisbeth A.; Burke, Catherine G.; Fenton, Bruce M.; Thevenet-Morrison, Kelly; Jusko, Todd A.; Lawrence, B. Paige

    2015-01-01

    Perinatal environmental exposures are potentially important contributors to the increase in autoimmune diseases. Yet, the mechanisms by which these exposures increase self-reactive immune responses later in life are poorly understood. Autoimmune diseases require CD4+ T cells for initiation, progression, and/or clinical symptoms; thus, developmental exposures that cause durable changes in CD4+ T cells may play a role. Early life activation of the aryl hydrocarbon receptor (AHR) causes persistent changes in the response of CD4+ T cells to infection later in life but whether CD4+ T cells are affected by developmental exposure in the context of an autoimmune disease is unknown. Gnaq+/− mice develop symptoms of autoimmune disease similar to those measured clinically, and therefore can be used to evaluate gene-environment interactions during development on disease progression. Herein, we examined the effect of AHR activation in utero and via lactation, or solely via lactation, on disease onset and severity in adult Gnaq+/− offspring. Developmental activation of the AHR-accelerated disease in Gnaq+/− mice, and this correlates with increases in effector CD4+ T-cell populations. Increased symptom onset and cellular changes due to early life AHR activation were more evident in female Gnaq+/− mice compared with males. These observations suggest that developmental AHR activation by pollutants, and other exogenous ligands, may increase the likelihood that genetically predisposed individuals will develop clinical symptoms of autoimmune disease later in life. PMID:26363170

  20. Developmental Activation of the AHR Increases Effector CD4+ T Cells and Exacerbates Symptoms in Autoimmune Disease-Prone Gnaq+/- Mice.

    PubMed

    Boule, Lisbeth A; Burke, Catherine G; Fenton, Bruce M; Thevenet-Morrison, Kelly; Jusko, Todd A; Lawrence, B Paige

    2015-12-01

    Perinatal environmental exposures are potentially important contributors to the increase in autoimmune diseases. Yet, the mechanisms by which these exposures increase self-reactive immune responses later in life are poorly understood. Autoimmune diseases require CD4(+) T cells for initiation, progression, and/or clinical symptoms; thus, developmental exposures that cause durable changes in CD4(+) T cells may play a role. Early life activation of the aryl hydrocarbon receptor (AHR) causes persistent changes in the response of CD4(+) T cells to infection later in life but whether CD4(+) T cells are affected by developmental exposure in the context of an autoimmune disease is unknown. Gnaq(+/-) mice develop symptoms of autoimmune disease similar to those measured clinically, and therefore can be used to evaluate gene-environment interactions during development on disease progression. Herein, we examined the effect of AHR activation in utero and via lactation, or solely via lactation, on disease onset and severity in adult Gnaq(+/-) offspring. Developmental activation of the AHR-accelerated disease in Gnaq(+/-) mice, and this correlates with increases in effector CD4(+) T-cell populations. Increased symptom onset and cellular changes due to early life AHR activation were more evident in female Gnaq(+/-) mice compared with males. These observations suggest that developmental AHR activation by pollutants, and other exogenous ligands, may increase the likelihood that genetically predisposed individuals will develop clinical symptoms of autoimmune disease later in life.

  1. In Vitro Transformation of Chlorinated Parabens by the Liver S9 Fraction: Kinetics, Metabolite Identification, and Aryl Hydrocarbon Receptor Agonist Activity.

    PubMed

    Terasaki, Masanori; Wada, Takeshi; Nagashima, Satoshi; Makino, Masakazu; Yasukawa, Hiro

    2016-01-01

    We investigated the kinetics of in vitro transformation of a dichlorinated propyl paraben (2-propyl 3,5-dichloro-4-hydroxybenzoate; Cl2PP) by the rat liver S9 fraction and assessed the aryl hydrocarbon receptor (AhR) agonist activity of the metabolite products identified in HPLC and GC/MS analysis and by metabolite syntheses. The results indicated that the chlorination of Cl2PP reduced its degradation rate by approximately 40-fold. Two hydroxylated metabolite products showed AhR agonist activity of up to 39% of that of the parent Cl2PP when assessed in a yeast (YCM3) reporter gene assay. The determination of the metabolic properties of paraben bioaccumulation presented here provides further information on the value of risk assessments of chlorinated parabens as a means to ensure human health and environmental safety.

  2. Baicalein induces G1 arrest in oral cancer cells by enhancing the degradation of cyclin D1 and activating AhR to decrease Rb phosphorylation

    SciTech Connect

    Cheng, Ya-Hsin; Li, Lih-Ann; Lin, Pinpin; Cheng, Li-Chuan; Hung, Chein-Hui; Chang, Nai Wen; Lin, Chingju

    2012-09-15

    Baicalein is a flavonoid, known to have anti-inflammatory and anti-cancer effects. As an aryl hydrocarbon receptor (AhR) ligand, baicalein at high concentrations blocks AhR-mediated dioxin toxicity. Because AhR had been reported to play a role in regulating the cell cycle, we suspected that the anti-cancer effect of baicalein is associated with AhR. This study investigated the molecular mechanism involved in the anti-cancer effect of baicalein in oral cancer cells HSC-3, including whether such effect would be AhR-mediated. Results revealed that baicalein inhibited cell proliferation and increased AhR activity in a dose-dependent manner. Cell cycle was arrested at the G1 phase and the expression of CDK4, cyclin D1, and phosphorylated retinoblastoma (pRb) was decreased. When the AhR was suppressed by siRNA, the reduction of pRb was partially reversed, accompanied by a decrease of cell population at G1 phase and an increase at S phase, while the reduction of cyclin D1 and CDK4 did not change. This finding suggests that the baicalein activation of AhR is indeed associated with the reduction of pRb, but is independent of the reduction of cyclin D1 and CDK4. When cells were pre-treated with LiCl, the inhibitor of GSK-3β, the decrease of cyclin D1 was blocked and the reduction of pRb was recovered. The data indicates that in HSC-3 the reduction of pRb is both mediated by baicalein through activation of AhR and facilitation of cyclin D1 degradation, which causes cell cycle arrest at the G1 phase, and results in the inhibition of cell proliferation. -- Highlights: ► Baicalein causes the G1 phase arrest by decreasing Rb phosphorylation. ► Baicalein modulates AhR-mediated cell proliferation. ► Both AhR activation and cyclin D1 degradation results in hypophosphorylation of Rb. ► Baicalein facilitates cyclin D1 degradation by signalling the GSK-3β pathway.

  3. Measured and predicted affinities of binding and relative potencies to activate the AhR of PAHs and their alkylated analogues.

    PubMed

    Lee, Sangwoo; Shin, Woong-Hee; Hong, Seongjin; Kang, Habyeong; Jung, Dawoon; Yim, Un Hyuk; Shim, Won Joon; Khim, Jong Seong; Seok, Chaok; Giesy, John P; Choi, Kyungho

    2015-11-01

    Polycyclic aromatic hydrocarbons (PAHs) and their alkylated forms are important components of crude oil. Both groups of PAHs have been reported to cause dioxin-like responses, mediated by aryl hydrocarbon receptor (AhR). Thus, characterization of binding affinity to the AhR of unsubstituted or alkylated PAHs is important to understand the toxicological consequences of oil contamination on ecosystems. We investigated the potencies of major PAHs of crude oil, e.g., chrysene, phenanthrene and dibenzothiophene, and their alkylated forms (n=17) to upregulate expression of AhR-mediated processes by use of the H4IIE-luc transactivation bioassay. In addition, molecular descriptors of different AhR activation potencies among PAHs were investigated by use of computational molecular docking models. Based on responses of the H4IIE-luc in vitro assay, it was shown that potencies of PAHs were determined by alkylation in addition to the number and conformation of rings. Potencies of AhR-mediated processes were generally greater when a chrysene group was substituted, especially in 1-methyl-chrysene. Significant negative correlations were observed between the in vitro dioxin-like potency measured in H4IIE-luc cells and the binding distance estimated from the in silico modeling. The difference in relative potency for AhR activation observed among PAHs and their alkylated forms could be explained by differences among binding distances in the ligand binding domain of the AhR caused by alkylation. The docking model developed in the present study may have utility in predicting risks of environmental contaminants of which toxicities are mediated by AhR binding.

  4. The Role of AhR in Breast Cancer Development

    DTIC Science & Technology

    2006-07-01

    other cell types, galangin is a potent inhibitor of the aryl hydrocarbon receptor (AhR), an environmental carcinogen-responsive transcription factor...constitutively active AhR. Constitutive and environmental chemical-inducible AhR activity was profoundly suppressed by galangin as was cell growth...However, the failure of a-naphthoflavone or FhAhRR transfection to block growth indicated that galangin -mediated AhR inhibition was either insufficient

  5. Occurrence of aryl hydrocarbon receptor agonists and genotoxic compounds in the river systems in Southern Taiwan.

    PubMed

    Chou, Pei-Hsin; Liu, Tong-Cun; Ko, Fung-Chi; Liao, Mong-Wei; Yeh, Hsiao-Mei; Yang, Tse-Han; Wu, Chun-Ting; Chen, Chien-Hsun; Tsai, Tsung-Ya

    2014-07-01

    Water and sediment samples from river systems located in Southern Taiwan were investigated for the presence of aryl hydrocarbon receptor (AhR) agonists and genotoxicants by a combination of recombinant cell assays and gas chromatography-mass spectrometry analysis. AhR agonist activity and genotoxic response were frequently detected in samples collected during different seasons. In particular, dry-season water and sediment samples from Erren River showed strong AhR agonist activity (201-1423 ng L(-1) and 1374-5631 ng g(-1) β-naphthoflavone equivalents) and high genotoxic potential. Although no significant correlation was found between AhR agonist activity and genotoxicity, potential genotoxicants in sample extracts were suggested to be causative agents for yeast growth inhibition in the AhR-responsive reporter gene assay. After high performance liquid chromatography fractionation, AhR agonist candidates were detected in several fractions of Erren River water and sediment extracts, while possible genotoxicants were only found in water extracts. In addition, polycyclic aromatic hydrocarbons, the typical contaminants showing high AhR binding affinity, were only minor contributors to the AhR agonist activity detected in Erren River sediment extracts. Our findings displayed the usefulness of bioassays in evaluating the extent of environmental contamination, which may be helpful in reducing the chances of false-negative results obtained from chemical analysis of conventional contaminants. Further research will be undertaken to identify major candidates for xenobiotic AhR agonists and genotoxicants to better protect the aquatic environments in Taiwan.

  6. Mixed-ligand copper(II) complexes activate aryl hydrocarbon receptor AhR and induce CYP1A genes expression in human hepatocytes and human cell lines.

    PubMed

    Kubešová, Kateřina; Dořičáková, Aneta; Trávníček, Zdeněk; Dvořák, Zdeněk

    2016-07-25

    The effects of four copper(II) mixed-ligand complexes [Cu(qui1)(L)]NO3·H2O (1-3) and [Cu(qui2)(phen)]NO3 (4), where qui1=2-phenyl-3-hydroxy-4(1H)-quinolinone, Hqui2=2-(4-amino-3,5-dichlorophenyl)-N-propyl-3-hydroxy-4(1H)-quinolinone-7-carboxamide, L=1,10-phenanthroline (phen) (1), 5-methyl-1,10-phenanthroline (mphen) (2), bathophenanthroline (bphen) (3), on transcriptional activities of steroid receptors, nuclear receptors and xenoreceptors have been studied. The complexes (1-4) did not influence basal or ligand-inducible activities of glucocorticoid receptor, androgen receptor, thyroid receptor, pregnane X receptor and vitamin D receptor, as revealed by gene reporter assays. The complexes 1 and 2 dose-dependently induced luciferase activity in stable gene reporter AZ-AhR cell line, and this induction was reverted by resveratrol, indicating involvement of aryl hydrocarbon receptor (AhR) in the process. The complexes 1, 2 and 3 induced CYP1A1 mRNA in LS180 cells and CYP1A1/CYP1A2 in human hepatocytes through AhR. Electrophoretic mobility shift assay EMSA showed that the complexes 1 and 2 transformed AhR in its DNA-binding form. Collectively, we demonstrate that the complexes 1 and 2 activate AhR and induce AhR-dependent genes in human hepatocytes and cancer cell lines. In conclusion, the data presented here might be of toxicological importance, regarding the multiple roles of AhR in human physiology and pathophysiology.

  7. Feedback control of AHR signalling regulates intestinal immunity.

    PubMed

    Schiering, Chris; Wincent, Emma; Metidji, Amina; Iseppon, Andrea; Li, Ying; Potocnik, Alexandre J; Omenetti, Sara; Henderson, Colin J; Wolf, C Roland; Nebert, Daniel W; Stockinger, Brigitta

    2017-02-09

    The aryl hydrocarbon receptor (AHR) recognizes xenobiotics as well as natural compounds such as tryptophan metabolites, dietary components and microbiota-derived factors, and it is important for maintenance of homeostasis at mucosal surfaces. AHR activation induces cytochrome P4501 (CYP1) enzymes, which oxygenate AHR ligands, leading to their metabolic clearance and detoxification. Thus, CYP1 enzymes have an important feedback role that curtails the duration of AHR signalling, but it remains unclear whether they also regulate AHR ligand availability in vivo. Here we show that dysregulated expression of Cyp1a1 in mice depletes the reservoir of natural AHR ligands, generating a quasi AHR-deficient state. Constitutive expression of Cyp1a1 throughout the body or restricted specifically to intestinal epithelial cells resulted in loss of AHR-dependent type 3 innate lymphoid cells and T helper 17 cells and increased susceptibility to enteric infection. The deleterious effects of excessive AHR ligand degradation on intestinal immune functions could be counter-balanced by increasing the intake of AHR ligands in the diet. Thus, our data indicate that intestinal epithelial cells serve as gatekeepers for the supply of AHR ligands to the host and emphasize the importance of feedback control in modulating AHR pathway activation.

  8. Polycyclic aromatic hydrocarbons (PAHs) mediate transcriptional activation of the ATP binding cassette transporter ABCB6 gene via the aryl hydrocarbon receptor (AhR).

    PubMed

    Chavan, Hemantkumar; Krishnamurthy, Partha

    2012-09-14

    Liver is endowed with a mechanism to induce hepatic cytochromes P450 (CYP450s) in response to therapeutic drugs and environmental contaminants, leading to increased detoxification and elimination of the xenobiotics. Each CYP450 is composed of an apoprotein moiety and a heme prosthetic group, which is required for CYP450 activity. Thus, under conditions of CYP450 induction, there is a coordinate increase in heme biosynthesis to compensate for the increased expression of CYP450s. ABCB6, a mitochondrial ATP binding cassette transporter, which regulates coproporphyrinogen transport from the cytoplasm into the mitochondria to complete heme biosynthesis, represents a previously unrecognized rate-limiting step in heme biosynthesis. However, it is not known if exposure to drugs and environmental contaminants induces ABCB6 expression, to assure an adequate and apparently coordinated supply of heme for the generation of functional cytochrome holoprotein. In the present study, we demonstrate that polycyclic aromatic hydrocarbons (PAHs), the widely distributed environmental toxicants shown to induce porphyrin accumulation causing hepatic porphyria, up-regulate ABCB6 expression in both mice and humans. Using siRNA technology and Abcb6 knock-out mice, we demonstrate that PAH-mediated increase in hepatic porphyrins is compromised in the absence of ABCB6. Moreover, in vivo studies in aryl hydrocarbon receptor (AhR) knock-out mice demonstrate that PAH induction of ABCB6 is mediated by AhR. Promoter activation studies combined with electrophoretic mobility shift assay and chromatin immunoprecipitation assay demonstrate direct interactions between the AhR binding sites in the ABCB6 promoter and the AhR receptor, implicating drug activation mechanisms for ABCB6 similar to those found in inducible cytochrome P450s. These studies are the first to describe direct transcriptional activation of both mouse and human ABCB6 by xenobiotics.

  9. A maternal Ahr null genotype sensitizes embryos to chemical teratogenesis.

    PubMed

    Thomae, Tami L; Glover, Edward; Bradfield, Christopher A

    2004-07-16

    The aryl hydrocarbon receptor (encoded by the Ahr locus) is a ligand-activated transcription factor that mediates the toxicology and teratology of 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin). In an effort to understand the role of the maternal compartment in dioxin teratology, we designed a breeding strategy that allowed us to compare the teratogenic response in embryos from Ahr(-/-) (null) and Ahr(+/+) (wild-type) dams. Using this strategy, we demonstrate that embryos from the Ahr(-/-) dams are 5-fold more sensitive to dioxin-induced cleft palate and hydronephrosis as compared with embryos from an Ahr(+/+) dam. Moreover, this increased teratogenic sensitivity extends beyond dioxin, because embryos from Ahr(-/-) dams exhibited a 9-fold increase in their sensitivity to the fetotoxic effects of the glucocorticoid, dexamethasone. In searching for an explanation for this increased sensitivity, we found that more dioxin and dexamethasone reached the embryos from Ahr(-/-) dams as compared with embryos from Ahr(+/+) dams. We propose that increased deposition of teratogens/fetotoxicants to the embryonic compartment is the result of porto-systemic shunting and/or blocked P4501A induction in Ahr(-/-) dams. In addition to demonstrating the importance of maternal AHR in teratogenesis, these data may have implications that reach beyond the mechanism of action of dioxin. In this regard, the Ahr(-/-) mouse may provide a system that allows pharmacological agents and toxicants to be more easily studied in a model where first pass clearance is a significant obstacle.

  10. AHR-11797: a novel benzodiazepine antagonist

    SciTech Connect

    Johnson, D.N.; Kilpatrick, B.F.; Hannaman, P.K.

    1986-03-01

    AHR-11797(5,6-dihydro-6-methyl-1-phenyl-/sup 3/H-pyrrolo(3,2,1-ij)quinazolin-3-one) displaced /sup 3/H-flunitrazepam (IC/sub 50/ = 82 nM) and /sup 3/H-Ro 15-1877 (IC/sub 50/ = 104 nM) from rat brain synaptosomes. AHR-11797 did not protect mice from seizures induced by maximal electroshock or subcutaneous Metrazol (scMET), nor did it induce seizures in doses up to the lethal dose. However, at 31.6 mg/kg, IP, it significantly increased the anticonvulsant ED/sub 50/ of chlordiazepoxide (CDPX) from 1.9 to 31.6 mg/kg, IP. With 56.7 mg/kg, IP, of AHR-11797, CDPX was inactive in doses up to 100 mg/kg, IP. AHR-11797 did not significantly increase punished responding in the Geller and Seifter conflict procedure, but it did attenuate the effects of diazepam. Although the compound is without anticonvulsant or anxiolytic activity, it did have muscle relaxant properties. AHR-11797 blocked morphine-induced Straub tail in mice (ED/sub 50/ = 31 mg/kg, IP) and it selectively suppressed the polysnaptic linguomandibular reflex in barbiturate-anesthetized cats. The apparent muscle relaxant activity of AHR-11797 suggests that different receptor sites are involved for muscle relaxant vs. anxiolytic/anticonvulsant activities of the benzodiazepines.

  11. Role of DNA methylation of AHR1 and AHR2 promoters in differential sensitivity to PCBs in Atlantic Killifish, Fundulus heteroclitus.

    PubMed

    Aluru, Neelakanteswar; Karchner, Sibel I; Hahn, Mark E

    2011-01-17

    Atlantic killifish (Fundulus heteroclitus) inhabiting the PCB-contaminated Superfund site in New Bedford Harbor (MA, USA) have evolved genetic resistance to the toxic effects of these compounds. They also lack induction of cytochrome P4501A (CYP1A) and other aryl hydrocarbon receptor (AHR)-dependent responses after exposure to AHR agonists, suggesting an overall down-regulation of the AHR signaling pathway. In this study, we hypothesized that the genetic resistance is due to altered AHR expression resulting from hypermethylation of DNA in the promoter region of AHR genes in fish inhabiting New Bedford Harbor. To test this hypothesis, we cloned and sequenced AHR1 and AHR2 promoter regions and employed bisulfite conversion-polymerase chain reaction (BS-PCR) followed by clonal analysis to compare the methylation status of CpG islands of AHR1 and AHR2 in livers of adult killifish collected from New Bedford Harbor and a reference site (Scorton Creek, MA). No significant differences in methylation profiles were observed in either AHR1 or AHR2 promoter regions between NBH and SC fish. However, hypermethylation of the AHR1 promoter correlated with low expression of transcripts in the liver in both populations. In comparison to AHR1, hepatic mRNA expression of AHR2 is high and its promoter is hypomethylated. Taken together, our results suggest that genetic resistance to contaminants in NBH fish is not due to altered methylation of AHR promoter regions, but that promoter methylation may control tissue-specific expression of AHR genes in killifish.

  12. Exposure to Diesel Exhaust Particle Extracts (DEPe) Impairs Some Polarization Markers and Functions of Human Macrophages through Activation of AhR and Nrf2

    PubMed Central

    Jaguin, Marie; Fardel, Olivier; Lecureur, Valérie

    2015-01-01

    Macrophages (MΦ), well-known to play an important role in immune response, also respond to environmental toxic chemicals such as diesel exhaust particles (DEP). Potential effects of DEPs towards MΦ polarization, a key hall-mark of MΦ physiology, remain however poorly documented. This study was therefore designed to evaluate the effects of a reference DEP extract (DEPe) on human MΦ polarization. Human blood monocytes-derived MΦ were incubated with IFNγ+LPS or IL-4 to obtain M1 and M2 subtypes, respectively; a 24 h exposure of polarizing MΦ to 10 μg/ml DEPe was found to impair expression of some macrophagic M1 and M2 markers, without however overall inhibition of M1 and M2 polarization processes. Notably, DEPe treatment increased the secretion of the M1 marker IL-8 and the M2 marker IL-10 in both MΦ subtypes, whereas it reduced lipopolysaccharide-induced IL-6 and IL-12p40 secretion in M1 MΦ. In M2 MΦ, DEPe exposure led to a reduction of CD200R expression and of CCL17, CCL18 and CCL22 secretion, associated with a lower chemotaxis of CCR4-positive cells. DEPe activated the Nrf2 and AhR pathways and induced expression of their reference target genes such as Hmox-1 and cytochrome P-4501B1 in M1 and M2 MΦ. Nrf2 or AhR silencing through RNA interference prevented DEPe-related down-regulation of IL-6. AhR silencing also inhibited the down-secretion of IL-12p40 and CCL18 in M1- and M2-DEPe-exposed MΦ, respectively. DEPs are therefore likely to alter expression of some M1 and M2 markers in an AhR- and Nrf2-dependent manner; such regulations may contribute to deleterious immune effects of atmospheric DEP. PMID:25710172

  13. Quantifying agonist activity at G protein-coupled receptors.

    PubMed

    Ehlert, Frederick J; Suga, Hinako; Griffin, Michael T

    2011-12-26

    When an agonist activates a population of G protein-coupled receptors (GPCRs), it elicits a signaling pathway that culminates in the response of the cell or tissue. This process can be analyzed at the level of a single receptor, a population of receptors, or a downstream response. Here we describe how to analyze the downstream response to obtain an estimate of the agonist affinity constant for the active state of single receptors. Receptors behave as quantal switches that alternate between active and inactive states (Figure 1). The active state interacts with specific G proteins or other signaling partners. In the absence of ligands, the inactive state predominates. The binding of agonist increases the probability that the receptor will switch into the active state because its affinity constant for the active state (K(b)) is much greater than that for the inactive state (K(a)). The summation of the random outputs of all of the receptors in the population yields a constant level of receptor activation in time. The reciprocal of the concentration of agonist eliciting half-maximal receptor activation is equivalent to the observed affinity constant (K(obs)), and the fraction of agonist-receptor complexes in the active state is defined as efficacy (ε) (Figure 2). Methods for analyzing the downstream responses of GPCRs have been developed that enable the estimation of the K(obs) and relative efficacy of an agonist. In this report, we show how to modify this analysis to estimate the agonist K(b) value relative to that of another agonist. For assays that exhibit constitutive activity, we show how to estimate K(b) in absolute units of M(-1). Our method of analyzing agonist concentration-response curves consists of global nonlinear regression using the operational model. We describe a procedure using the software application, Prism (GraphPad Software, Inc., San Diego, CA). The analysis yields an estimate of the product of K(obs) and a parameter proportional to efficacy (

  14. Flavonoids as aryl hydrocarbon receptor agonists/antagonists: effects of structure and cell context.

    PubMed Central

    Zhang, Shu; Qin, Chunhua; Safe, Stephen H

    2003-01-01

    Chemoprotective phytochemicals exhibit multiple activities and interact with several cellular receptors, including the aryl hydrocarbon (Ah) receptor (AhR). In this study we investigated the AhR agonist/antagonist activities of the following flavonoids: chrysin, phloretin, kaempferol, galangin, naringenin, genistein, quercetin, myricetin, luteolin, baicalein, daidzein, apigenin, and diosmin. We also investigated the AhR-dependent activities of cantharidin and emodin (in herbal extracts) in Ah-responsive MCF-7 human breast cells, HepG2 human liver cancer cells, and mouse Hepa-1 cells transiently or stably transfected with plasmids expressing a luciferase reporter gene linked to multiple copies of a consensus dioxin-responsive element. The AhR agonist activities of the compounds (1 and 10 micro M) were as high as 25% of the maximal response induced by 5 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and their potencies were dependent on cell context. Galangin, genistein, daidzein, and diosmin were active only in Hepa-1 cells, and cantharidin induced activity only in human HepG2 and MCF-7 cells. Western blot analysis confirmed that baicalein and emodin also induced CYP1A1 protein in the human cancer cell lines. The AhR antagonist activities of four compounds inactive as agonists in MCF-7 and HepG2 cells (kaempferol, quercetin, myricetin, and luteolin) were also investigated. Luteolin was an AhR antagonist in both cell lines, and the inhibitory effects of the other compound were dependent on cell context. These data suggest that dietary phytochemicals exhibit substantial cell context-dependent AhR agonist as well as antagonist activities. Moreover, because phytochemicals and other AhR-active compounds in food are present in the diet at relatively high concentrations, risk assessment of dietary toxic equivalents of TCDD and related compounds should also take into account AhR agonist/antagonist activities of phytochemicals. PMID:14644660

  15. Aryl hydrocarbon receptor agonists trigger avoidance of novel food in rats.

    PubMed

    Mahiout, Selma; Pohjanvirta, Raimo

    2016-12-01

    The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that mediates the toxicity of dioxins, but also plays important physiological roles, which are only beginning to unfold. Previous studies have surprisingly unveiled that low doses of the potent AHR agonist TCDD induce a strong and persistent avoidance of novel food items in rats. Here, we further examined the involvement of the AHR in the avoidance response in Sprague-Dawley rats with three established AHR agonists: 6-formylindolo(3,2-b)carbazole (FICZ), β-naphthoflavone (BNF) and benzo[a]pyrene (BaP); with a novel selective AHR modulator (C2); and with an activator of another nuclear receptor, CAR: 2,4,6-tryphenyldioxane-1,3 (TPD). As sensitive indices of AHR or CAR activity, we used Cyp1a1 and Cyp2b1 gene expression, as they are, respectively, the drug-metabolizing enzymes specifically regulated by them. We further attempted to address the roles played by enhanced neophobia and conditioned taste aversion (CTA) in the avoidance behaviour. All AHR agonists triggered practically total avoidance of novel chocolate, but the durations varied. Likewise, acutely subtoxic doses of C2, differing by 25-fold, all elicited a similar outcome. In contrast, TPD did not influence chocolate consumption at all. If rats were initially accustomed to chocolate for 6h after single FICZ or BNF exposure, avoidance was still clearly present two weeks later when chocolate was offered again. Hence, the avoidance response appears to specifically involve the AHR instead of being triggered by induction of intestinal or hepatic nuclear receptor signalling in general. It is also shared by both endogenous and exogenous AHR activators. Moreover, this behavioural change in rats seems to contain elements of both CTA and enhanced neophobia, but further clarification of this is still required.

  16. The cardiovascular effects of peroxisome proliferator-activated receptor agonists.

    PubMed

    Friedland, Sayuri N; Leong, Aaron; Filion, Kristian B; Genest, Jacques; Lega, Iliana C; Mottillo, Salvatore; Poirier, Paul; Reoch, Jennifer; Eisenberg, Mark J

    2012-02-01

    Although peroxisome proliferator-activated receptor agonists are prescribed to improve cardiovascular risk factors, their cardiovascular safety is controversial. We therefore reviewed the literature to identify landmark randomized controlled trials evaluating the effect of peroxisome proliferator-activated receptor gamma agonists (pioglitazone and rosiglitazone), alpha agonists (fenofibrate and gemfibrozil), and pan agonists (bezafibrate, muraglitazar, ragaglitazar, tesaglitazar, and aleglitazar) on cardiovascular outcomes. Pioglitazone may modestly reduce cardiovascular events but also may increase the risk of bladder cancer. Rosiglitazone increases the risk of myocardial infarction and has been withdrawn in European and restricted in the United States. Fibrates improve cardiovascular outcomes only in select subgroups: fenofibrate in diabetic patients with metabolic syndrome, gemfibrozil in patients with dyslipidemia, and bezafibrate in patients with diabetes or metabolic syndrome. The cardiovascular safety of the new pan agonist aleglitazar, currently in phase II trials, remains to be determined. The heterogenous effects of peroxisome proliferator-activated receptor agonists to date highlight the importance of postmarketing surveillance. The critical question of why peroxisome proliferator-activated receptor agonists seem to improve cardiovascular risk factors without significantly improving cardiovascular outcomes requires further investigation.

  17. The constitutively active Ah receptor (CA-Ahr) mouse as a potential model for dioxin exposure--effects in vital organs.

    PubMed

    Brunnberg, Sara; Andersson, Patrik; Lindstam, Maria; Paulson, Ivar; Poellinger, Lorenz; Hanberg, Annika

    2006-07-25

    The dioxin/aryl hydrocarbon receptor (AhR) mediates most, if not all, toxic effects of dioxins and functions as a ligand-activated transcription factor regulating transcription of a battery of genes. In order to study the mechanisms behind the toxicity of ligands of the Ah receptor we have created a transgenic mouse model expressing a constitutively active Ah receptor (CA-AhR). The mutant Ah receptor is expressed and functionally active in all organs studied. The purpose of the present study was to characterize histopathologically, the phenotype of the CA-AhR with regard to the liver, kidney, lung, heart, spleen and thymus of male and female transgenic CA-AhR mice. Moreover, cell-specific activity of the CA-AhR using up-regulation of the AhR target gene CYP1A1 as a marker, was also examined. The relative weight of liver, kidney and heart were increased while relative thymus weight was decreased. Furthermore, slight morphological lesions of the liver, kidney and spleen was seen. Expression of CYP1A1 was found in cells corresponding to endothelial cells in all of the organs studied. In some tissues additional cell types, such as hepatocytes, renal tubuli cell and Clara cells expressed CYP1A1. Both the effects on organ weights and the cellular expression of CYP1A1 in CA-AhR mice correspond well to observations in TCDD-exposed mice. In conclusion, this characterization further support that the CA-AhR mouse is a useful model for life-long continuous low-level activity of the AhR, i.e. the dioxin exposure situation of humans of the general population.

  18. Genetic and pharmacological analysis identifies a physiological role for the AHR in epidermal differentiation

    PubMed Central

    van den Bogaard, Ellen; Podolsky, Michael; Smits, Jos; Cui, Xiao; John, Christian; Gowda, Krishne; Desai, Dhimant; Amin, Shantu; Schalkwijk, Joost; Perdew, Gary H.

    2015-01-01

    Stimulation of the aryl hydrocarbon receptor (AHR) by xenobiotics is known to affect epidermal differentiation and skin barrier formation. The physiological role of endogenous AHR signaling in keratinocyte differentiation is not known. We used murine and human skin models to address the hypothesis that AHR activation is required for normal keratinocyte differentiation. Using transcriptome analysis of Ahr-/- and Ahr+/+ murine keratinocytes, we found significant enrichment of differentially expressed genes linked to epidermal differentiation. Primary Ahr-/- keratinocytes showed a significant reduction in terminal differentiation gene and protein expression, similar to Ahr+/+ keratinocytes treated with AHR antagonists GNF351 and CH223191, or the selective AHR modulator (SAhRM), SGA360. In vitro keratinocyte differentiation led to increased AHR levels and subsequent nuclear translocation, followed by induced CYP1A1 gene expression. Monolayer cultured primary human keratinocytes treated with AHR antagonists also showed an impaired terminal differentiation program. Inactivation of AHR activity during human skin equivalent development severely impaired epidermal stratification, terminal differentiation protein expression and stratum corneum formation. As disturbed epidermal differentiation is a main feature of many skin diseases, pharmacological agents targeting AHR signaling or future identification of endogenous keratinocyte-derived AHR ligands should be considered as potential new drugs in dermatology. PMID:25602157

  19. Cytochrome P4501A induction in avian hepatocyte cultures exposed to polychlorinated biphenyls: Comparisons with AHR1-mediated reporter gene activity and in ovo toxicity

    SciTech Connect

    Manning, Gillian E.; Mundy, Lukas J.; Crump, Doug; Jones, Stephanie P.; Chiu, Suzanne; Klein, Jeff; Konstantinov, Alex; Potter, Dave; Kennedy, Sean W.

    2013-01-01

    Avian-specific toxic equivalency factors (TEFs) were developed by the World Health Organization to simplify environmental risk assessments of dioxin-like compounds (DLCs), but TEFs do not account for differences in the toxic and biochemical potencies of DLCs among species of birds. Such variability may be due to differences in species sensitivity to individual DLCs. The sensitivity of avian species to DLCs was recently associated with the identity of amino acids 324 and 380 in the aryl hydrocarbon receptor 1 (AHR1) ligand binding domain. A luciferase reporter gene (LRG) assay, measuring AHR1-mediated induction of a cytochrome P450 1A5 (CYP1A5) reporter gene, in combination with a species' AHR1 ligand binding domain sequence, were also shown to predict avian species sensitivity to polychlorinated biphenyls (PCBs) and PCB relative potency in a given species. The goals of the present study were to (1) characterize the concentration-dependent effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin and PCBs 126, 77, 105 and 118 on induction of ethoxyresorufin O-deethylase (EROD) activity and CYP1A4/5 mRNA in chicken, ring-necked pheasant and Japanese quail embryo hepatocytes and (2) compare these in vitro results to those previously generated by the LRG assay and in ovo toxicity studies. EROD activity and CYP1A4/5 mRNA expression data support and complement the findings of the LRG assay. CYP1A enzyme activity and mRNA expression were significantly correlated both with luciferase activity and in ovo toxicity induced by PCBs. Relative potency values were generally similar between the LRG and EROD assays and indicate that the relative potency of some PCBs may differ among species. -- Highlights: ► The chicken isn't the most sensitive species to CYP1A induction by PCB 105 and 118. ► The relative potency of PCBs differs between avian species. ► EROD activity was correlated with luciferase activity from the LRG assay. ► EROD activity was a better predictor of toxicity than CYP

  20. Cooperation of structurally different aryl hydrocarbon receptor agonists and β-catenin in the regulation of CYP1A expression.

    PubMed

    Vaas, Sebastian; Kreft, Luisa; Schwarz, Michael; Braeuning, Albert

    2014-11-05

    The ligand-activated nuclear receptor AhR (aryl hydrocarbon receptor) mediates the response of hepatocytes to various exogenous compounds. AhR is classically activated by planar, aromatic hydrocarbons, but also by other, structurally rather unrelated compounds. Recent data show that the canonical Wnt/β-catenin signaling pathway is also involved in the regulation of hepatic zonal gene expression and drug metabolism in mammalian liver. Previous studies indicate that the loss of β-catenin in hepatocytes diminishes the response to the AhR agonists 3-methylcholanthrene (3MC) in vivo and to 2,3,7,8-tetrachlorodibenzo-[p]-dioxin in vitro. The knockout of β-catenin also impairs the zonal pattern of AhR target gene induction by 3MC. However, it is presently unknown whether the chemical nature of the AhR agonist influences the AhR/β-catenin interaction. Moreover, no information is available about the dose-response curves of AhR activation in the absence or presence of Wnt/β-catenin signaling. In the present study, we have analyzed AhR-dependent responses to different concentrations of structurally unrelated AhR agonists in vivo and in vitro. The results demonstrate that β-catenin is essential to obtain the maximum AhR response. Moreover, using transgenic mouse models which allow for the ablation of β-catenin at different age of mice, we demonstrate that the presence of β-catenin, not postnatal developmental effects in β-catenin-deficient livers, is responsible for the observed interplay of β-catenin and the AhR.

  1. Embryonic cardiotoxicity of weak aryl hydrocarbon receptor agonists and CYP1A inhibitor fluoranthene in the Atlantic killifish (Fundulus heteroclitus).

    PubMed

    Brown, D R; Clark, B W; Garner, L V T; Di Giulio, R T

    2016-10-01

    High affinity aryl hydrocarbon receptor (AHR) ligands, such as certain polychlorinated biphenyls and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), cause severe cardiac teratogenesis in fish embryos. Moderately strong AHR agonists, for example benzo[a]pyrene and β-naphthoflavone, are capable of causing similar cardiotoxic effects, particularly when coupled with cytochrome P450 1A (CYP1A) inhibitors (e.g., fluoranthene (FL). Additionally, some weaker AHR agonists (carbaryl, 2-methylindole, 3-methylindole, and phenanthrene) are known to also cause cardiotoxicity in zebrafish (Danio rerio) embryos when coupled with FL; however, the cardiotoxic effects were not mediated specifically by AHR stimulation. This study was performed to determine if binary exposure to weak AHR agonists and FL were also capable of causing cardiotoxicity in Atlantic killifish Fundulus heteroclitus embryos. Binary exposures were performed in both naïve and PAH-adapted killifish embryos to examine resistance to weak agonists and FL binary exposures. Weak agonists used in this study included the following: carbaryl, phenanthrene, 2-methylindole, 3-methylindole, indigo, and indirubin. Carbaryl, indigo, and indirubin induced the highest CYP1 activity levels in naïve killifish embryos, but no significant CYP1 induction was observed in the PAH-adapted killifish. Embryos were coexposed to subteratogenic levels of each agonist and 500μg/L FL to assess if binary administration could cause cardiotoxicity. Indigo and indirubin coupled with FL caused cardiac teratogenesis in naïve killifish, but coexposures did not produce cardiac chamber abnormalities in the PAH-adapted population. Knockdown of AHR2 in naïve killifish embryos did not prevent cardiac teratogenesis. The data suggest a unique mechanism of cardiotoxicity that is not driven by AHR2 activation.

  2. Synthesis and activity of small molecule GPR40 agonists.

    PubMed

    Garrido, Dulce M; Corbett, David F; Dwornik, Kate A; Goetz, Aaron S; Littleton, Thomas R; McKeown, Steve C; Mills, Wendy Y; Smalley, Terrence L; Briscoe, Celia P; Peat, Andrew J

    2006-04-01

    The first report on the identification and structure-activity relationships of a novel series of GPR40 agonists based on a 3-(4-{[N-alkyl]amino}phenyl)propanoic acid template is described. Structural modifications to the original screening hit yielded compounds with a 100-fold increase in potency at the human GPR40 receptor and pEC(50)s in the low nanomolar range. The carboxylic acid moiety is not critical for activity but typically elicits an agonistic response higher than those observed with carboxamide replacements. These compounds may prove useful in unraveling the therapeutic potential of this receptor for the treatment of Type 2 diabetes.

  3. A Dominant Negative Zebrafish Ahr2 Partially Protects Developing Zebrafish from Dioxin Toxicity

    PubMed Central

    Lanham, Kevin A.; Prasch, Amy L.; Weina, Kasia M.; Peterson, Richard E.; Heideman, Warren

    2011-01-01

    The toxicity by 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD) is thought to be caused by activation of the aryl hydrocarbon receptor (AHR). However, our understanding of how AHR activation by TCDD leads to toxic effects is poor. Ideally we would like to manipulate AHR activity in specific tissues and at specific times. One route to this is expressing dominant negative AHRs (dnAHRs). This work describes the construction and characterization of dominant negative forms of the zebrafish Ahr2 in which the C-terminal transactivation domain was either removed, or replaced with the inhibitory domain from the Drosophila engrailed repressor protein. One of these dnAhr2s was selected for expression from the ubiquitously active e2fα promoter in transgenic zebrafish. We found that these transgenic zebrafish expressing dnAhr2 had reduced TCDD induction of the Ahr2 target gene cyp1a, as measured by 7-ethoxyresorufin-O-deethylase activity. Furthermore, the cardiotoxicity produced by TCDD, pericardial edema, heart malformation, and reduced blood flow, were all mitigated in the zebrafish expressing the dnAhr2. These results provide in vivo proof-of-principle results demonstrating the effectiveness of dnAHRs in manipulating AHR activity in vivo, and demonstrating that this approach can be a means for blocking TCDD toxicity. PMID:22194803

  4. Naturally-Occurring Marine Brominated Indoles are Aryl Hydrocarbon Receptor Ligands/Agonists

    PubMed Central

    DeGroot, Danica E.; Franks, Diana G.; Higa, Tatsuo; Tanaka, Junichi; Hahn, Mark E.; Denison, Michael S.

    2015-01-01

    The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that mediates the toxic and biological effects of structurally diverse chemicals, including the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). As part of a larger effort to identify the full spectrum of chemicals that can bind to and activate the AhR, we have examined the ability of several naturally-occurring marine-derived brominated indoles and brominated (methylthio)indoles (collectively referred to as “brominated indoles”) to bind to the AhR and stimulate AhR-dependent gene expression. Incubation of mouse, rat and guinea pig recombinant cell lines containing a stably transfected AhR-responsive luciferase reporter gene with eight brominated indoles revealed that all compounds stimulated luciferase reporter gene activity, although some species-specific differences were observed. All compounds induced significantly more luciferase activity when incubated with cells for 4 h as compared to 24 h, demonstrating that these compounds are transient activators of the AhR signaling pathway. Three of the brominated indoles induced CYP1A1 mRNA in human HepG2 cells in vitro and Cyp1a mRNA in zebrafish embryos in vivo. The identification of the brominated indoles as direct ligands and activators/agonists of the AhR was confirmed by their ability to compete with [3H]TCDD for binding to the AhR and to stimulate AhR transformation and DNA binding in vitro. Taken together, these marine-derived brominated indoles are members of a new class of naturally-occurring AhR agonists. PMID:26001051

  5. Hepatic stellate cells increase the immunosuppressive function of natural Foxp3+ regulatory T cells via IDO-induced AhR activation.

    PubMed

    Kumar, Sudhir; Wang, Jiang; Thomson, Angus W; Gandhi, Chandrashekhar R

    2017-02-01

    Immunosuppressive, naturally occurring CD4(+)CD25(+)forkhead box p3(+) (Foxp3(+)) regulatory T cells (nTregs) offer potential for the treatment of immune-mediated inflammatory disorders. However, potential instability of ex vivo-expanded nTregs following their adoptive transfer may be a significant limitation. LPS-stimulated hepatic stellate cells (HSCs) induce expansion and enhance the suppressive function and stability of allogeneic nTregs We aimed to delineate mechanisms underlying HSC-induced expansion and increased potency of nTregs HSCs and nTregs were isolated from mouse livers and spleens, respectively. Following coculture with LPS-pretreated allogeneic HSCs (LPS/HSCs), proliferation of nTregs was measured by CFSE dilution, and Foxp3 expression and acetylation were determined by immunoprecipitation (IP) and Western blotting analysis. Expression of various genes associated with immunologic tolerance was determined by quantitative RT-PCR (qRT-PCR). LPS stimulation increased the expression and activity of the immunoregulatory enzyme IDO1 in HSCs, and LPS/HSCs stimulated aryl hydrocarbon receptor (AhR) signaling in cocultured nTregs Reciprocally, Tregs increased IDO1 expression in HSCs. IDO1(-/-) LPS/HSCs were inferior to WT LPS/HSCs in stimulating nTreg expansion. Pharmacologic inhibition of IDO1 in HSCs by 1-methyltryptophan (1MT) inhibited LPS/HSC-induced AhR signaling in nTregs, which was responsible for their expansion, Foxp3 expression, and stabilization of Foxp3 by increasing acetylation of lysine residues. Finally, HSCs cryopreserved, following 2-3 passages, were as potent as primary-cultured HSCs in expanding nTregs In conclusion, LPS/HSCs expand allogeneic nTregs through an IDO-dependent, AhR-mediated mechanism and increase their stability through lysine-acetylation of Foxp3. nTregs expanded by cryopreserved HSCs may have potential for clinical use.

  6. Covalent agonists for studying G protein-coupled receptor activation

    PubMed Central

    Weichert, Dietmar; Kruse, Andrew C.; Manglik, Aashish; Hiller, Christine; Zhang, Cheng; Hübner, Harald; Kobilka, Brian K.; Gmeiner, Peter

    2014-01-01

    Structural studies on G protein-coupled receptors (GPCRs) provide important insights into the architecture and function of these important drug targets. However, the crystallization of GPCRs in active states is particularly challenging, requiring the formation of stable and conformationally homogeneous ligand-receptor complexes. Native hormones, neurotransmitters, and synthetic agonists that bind with low affinity are ineffective at stabilizing an active state for crystallogenesis. To promote structural studies on the pharmacologically highly relevant class of aminergic GPCRs, we here present the development of covalently binding molecular tools activating Gs-, Gi-, and Gq-coupled receptors. The covalent agonists are derived from the monoamine neurotransmitters noradrenaline, dopamine, serotonin, and histamine, and they were accessed using a general and versatile synthetic strategy. We demonstrate that the tool compounds presented herein display an efficient covalent binding mode and that the respective covalent ligand-receptor complexes activate G proteins comparable to the natural neurotransmitters. A crystal structure of the β2-adrenoreceptor in complex with a covalent noradrenaline analog and a conformationally selective antibody (nanobody) verified that these agonists can be used to facilitate crystallogenesis. PMID:25006259

  7. The aryl hydrocarbon receptor and glucocorticoid receptor interact to activate human metallothionein 2A

    SciTech Connect

    Sato, Shoko; Shirakawa, Hitoshi; Tomita, Shuhei; Tohkin, Masahiro; Gonzalez, Frank J.; Komai, Michio

    2013-11-15

    Although the aryl hydrocarbon receptor (AHR) and glucocorticoid receptor (GR) play essential roles in mammalian development, stress responses, and other physiological events, crosstalk between these receptors has been the subject of much debate. Metallothioneins are classic glucocorticoid-inducible genes that were reported to increase upon treatment with AHR agonists in rodent tissues and cultured human cells. In this study, the mechanism of human metallothionein 2A (MT2A) gene transcription activation by AHR was investigated. Cotreatment with 3-methylcholanthrene and dexamethasone, agonists of AHR and GR respectively, synergistically increased MT2A mRNA levels in HepG2 cells. MT2A induction was suppressed by RNA interference against AHR or GR. Coimmunoprecipitation experiments revealed a physical interaction between AHR and GR proteins. Moreover, chromatin immunoprecipitation assays indicated that AHR was recruited to the glucocorticoid response element in the MT2A promoter. Thus, we provide a novel mechanism whereby AHR modulates expression of human MT2A via the glucocorticoid response element and protein–protein interactions with GR. - Highlights: • Aryl hydrocarbon receptor forms a complex with glucocorticoid receptor in cells. • Human metallothionein gene is regulated by the AHR and GR interaction. • AHR–GR complex binds to glucocorticoid response element in metallothionein gene. • We demonstrated a novel transcriptional mechanism via AHR and GR interaction.

  8. AHR-related activities in a creosote-adapted population of adult atlantic killifish, Fundulus heteroclitus, two decades post-EPA superfund status at the Atlantic Wood Site, Portsmouth, VA USA.

    PubMed

    Wojdylo, Josephine V; Vogelbein, Wolfgang; Bain, Lisa J; Rice, Charles D

    2016-08-01

    Atlantic killifish, Fundulus heteroclitus, are adapted to creosote-based PAHs at the US EPA Superfund site known as Atlantic Wood (AW) on the southern branch of the Elizabeth River, VA USA. Subsequent to the discovery of the AW population in the early 1990s, these fish were shown to be recalcitrant to CYP1A induction by PAHs under experimental conditions, and even to the time of this study, killifish embryos collected from the AW site are resistant to developmental deformities typically associated with exposure to PAHs in reference fish. Historically, however, 90 +% of the adult killifish at this site have proliferative hepatic lesions including cancer of varying severity. Several PAHs at this site are known to be ligands for the aryl hydrocarbon receptor (AHR). In this study, AHR-related activities in AW fish collected between 2011 and 2013 were re-examined nearly 2 decades after first discovery. This study shows that CYP1A mRNA expression is three-fold higher in intestines of AW killifish compared to a reference population. Using immunohistochemistry, CYP1A staining in intestines was uniformly positive compared to negative staining in reference fish. Livers of AW killifish were examined by IHC to show that CYP1A and AHR2 protein expression reflect lesions-specific patterns, probably representing differences in intrinsic cellular physiology of the spectrum of proliferative lesions comprising the hepatocarcinogenic process. We also found that COX2 mRNA expression levels were higher in AW fish livers compared to those in the reference population, suggesting a state of chronic inflammation. Overall, these findings suggest that adult AW fish are responsive to AHR signaling, and do express CYP1A and AHR2 proteins in intestines at a level above what was observed in the reference population.

  9. AHR promoter variant modulates its transcription and downstream effectors by allele-specific AHR-SP1 interaction functioning as a genetic marker for vitiligo.

    PubMed

    Wang, Xiaowen; Li, Kai; Liu, Ling; Shi, Qiong; Song, Pu; Jian, Zhe; Guo, Sen; Wang, Gang; Li, Chunying; Gao, Tianwen

    2015-09-15

    Vitiligo is an acquired depigmentation disorder largely caused by defective melanocyte- or autoimmunity-induced melanocyte destruction. The aryl hydrocarbon receptor (AHR) is essential for melanocyte homeostasis and immune process, and abnormal AHR was observed in vitiligo. We previously identified the T allele of AHR -129C > T variant as a protective factor against vitiligo. However, biological characterization underlying such effects is not fully certain, further validation by mechanistic research is warranted and was conducted in the present study. We showed that -129T allele promoted AHR transcriptional activity through facilitating its interaction with SP1 transcription factor (SP1) compared with -129C allele. We subsequently found reduced peripheral AHR and SP1 transcript expressions in vitiligo and a negative correlation of AHR level with disease duration. We also investigated AHR-related cytokines and observed increased serum TNF-α concentration and diminished serum levels of IL-10 and TGF-β1 in vitiligo. Further genetic analysis showed that -129T carriers possessed higher levels of AHR and IL-10 than -129C carriers. Therefore, our study indicates that the modulation of AHR transcription by a promoter variant has a profound influence on vitiligo, not only advancing our understanding on AHR function but also providing novel insight into the pathogenesis of degenerative or autoimmune diseases including vitiligo.

  10. Biased signaling by peptide agonists of protease activated receptor 2.

    PubMed

    Jiang, Yuhong; Yau, Mei-Kwan; Kok, W Mei; Lim, Junxian; Wu, Kai-Chen; Liu, Ligong; Hill, Timothy A; Suen, Jacky Y; Fairlie, David P

    2017-02-07

    Protease activated receptor 2 (PAR2) is associated with metabolism, obesity, inflammatory, respiratory and gastrointestinal disorders, pain, cancer and other diseases. The extracellular N-terminus of PAR2 is a common target for multiple proteases, which cleave it at different sites to generate different N-termini that activate different PAR2-mediated intracellular signaling pathways. There are no synthetic PAR2 ligands that reproduce the same signaling profiles and potencies as proteases. Structure-activity relationships here for 26 compounds spanned a signaling bias over 3 log units, culminating in three small ligands as biased agonist tools for interrogating PAR2 functions. DF253 (2f-LAAAAI-NH2) triggered PAR2-mediated calcium release (EC50 2 μM) but not ERK1/2 phosphorylation (EC50 > 100 μM) in CHO cells transfected with hPAR2. AY77 (Isox-Cha-Chg-NH2) was a more potent calcium-biased agonist (EC50 40 nM, Ca2+; EC50 2 μM, ERK1/2), while its analogue AY254 (Isox-Cha-Chg-A-R-NH2) was an ERK-biased agonist (EC50 2 nM, ERK1/2; EC50 80 nM, Ca2+). Signaling bias led to different functional responses in human colorectal carcinoma cells (HT29). AY254, but not AY77 or DF253, attenuated cytokine-induced caspase 3/8 activation, promoted scratch-wound healing and induced IL-8 secretion, all via PAR2-ERK1/2 signaling. Different ligand components were responsible for different PAR2 signaling and functions, clues that can potentially lead to drugs that modulate different pathway-selective cellular and physiological responses.

  11. The bioflavonoid galangin blocks aryl hydrocarbon receptor activation and polycyclic aromatic hydrocarbon-induced pre-B cell apoptosis.

    PubMed

    Quadri, S A; Qadri, A N; Hahn, M E; Mann, K K; Sherr, D H

    2000-09-01

    Bioflavonoids are plant compounds touted for their potential to treat or prevent several diseases including cancers induced by common environmental chemicals. Much of the biologic activity of one such class of pollutants, polycyclic aromatic hydrocarbons (PAH), is mediated by the aryl hydrocarbon receptor/transcription factor (AhR). For example, the AhR regulates PAH immunotoxicity that manifests as pre-B cell apoptosis in models of B cell development. Because bioflavonoids block PAH-induced cell transformation and are structurally similar to AhR ligands, it was postulated that some of them would suppress PAH-induced, AhR-dependent immunotoxicity, possibly through a direct AhR blockade. This hypothesis was tested using a model of B cell development in which pre-B cells are cultured with and are dependent on bone marrow stromal or hepatic parenchymal cell monolayers. Of seven bioflavonoids screened, galangin (3,5,7-trihydroxyflavone) blocked PAH-induced but not C(2)-ceramide- or H(2)O(2)-induced pre-B cell apoptosis. Because galangin blocked AhR-dependent reporter gene expression, AhR complex-DNA binding, and AhR nuclear translocation, inhibition of a relatively early step in AhR signaling was implicated. This hypothesis was supported by the ability of galangin to bind the AhR and stabilize AhR-90-kDa heat shock protein complexes in the presence of AhR agonists. These studies demonstrate the utility of pre-B cell culture systems in identifying compounds capable of blocking PAH immunotoxicity, define at least one mechanism of galangin activity (i.e., repression of AhR activation), and motivate the use of this and similar dietary bioflavonoids as relatively nontoxic inhibitors of AhR agonist activity and as pharmacologic agents with which to dissect AhR signaling pathways.

  12. Modeling of the Aryl Hydrocarbon Receptor (AhR) ligand binding domain and its utility in virtual ligand screening to predict new AhR ligands

    PubMed Central

    Bisson, William; Koch, Daniel; O’Donnell, Edmond; Khalil, Sammy M.; Kerkvliet, Nancy; Tanguay, Robert; Abagyan, Ruben; Kolluri, Siva Kumar

    2012-01-01

    The Aryl Hydrocarbon Receptor (AhR) is a ligand-activated transcription factor; the AhR Per-AhR/Arnt-Sim (PAS) domain binds ligands. We developed homology models of the AhR PAS domain to characterize previously observed intra- and inter-species differences in ligand binding using Molecular Docking. In silico structure-based virtual ligand screening using our model resulted in the identification of pinocembrin and 5-hydroxy-7-methoxyflavone, which promoted nuclear translocation and transcriptional activation of AhR and AhR-dependent induction of endogenous target genes. PMID:19719119

  13. Ahr function in lymphocytes: emerging concepts

    PubMed Central

    Zhou, Liang

    2015-01-01

    The aryl hydrocarbon receptor (Ahr) is an important regulator of the development and function of both innate and adaptive immune cells through roles associated with Ahr's ability to respond to cellular and dietary ligands. Recent findings have revealed tissue and context-specific functions for Ahr in both homeostasis and in during an immune response. I review these findings here, and integrate them into the current understanding of the mechanisms that regulate Ahr transcription and function. I propose a conceptual framework in which Ahr function is determined by three factors: the amount of Ahr in any given cell, the abundance and potency of Ahr ligands within certain tissues, and the tissue microenvironment wherein Ahr+ cells reside. This complexity emphasizes the necessity cell-type specific genetic approaches towards the study of Ahr function. PMID:26700314

  14. The emerging roles of AhR in physiology and immunity.

    PubMed

    Hao, Nan; Whitelaw, Murray L

    2013-09-01

    The aryl hydrocarbon receptor (AhR) is traditionally defined as a transcriptional regulator involved in adaptive xenobiotic response, however, emerging evidence supports physiological functions of AhR in normal cell development and immune response. The role of AhR in immunomodulation is multi-dimensional. On the one hand, activation of AhR by TCDD and other ligands leads to profound immunosuppression, potentially via skewed Th1/Th2 cell balance toward Th1 dominance, and boosted Treg cell differentiation. On the other hand, activation of AhR can also induce Th17 cell polarization and increase the severity of autoimmune disease. In addition to T lymphocytes, the AhR also appears to play a vital role in B cell maturation, and regulates the activity of macrophages, dendritic cells and neutrophils following lipopolysaccharide challenge or influenza virus infection. In these scenarios, activation of AhR is associated with decreased host response and reduced survival. Furthermore, gene knock out studies suggest that AhR is indispensable for the postnatal maintenance of intestinal intraepithelial lymphocytes and skin-resident dendritic epidermal gamma delta T cells, providing a potential link between AhR and gut immunity and wound healing. It is well accepted that the magnitude and the type of immune response is dependent on the local cytokine milieu and the AhR appears to be one of the key factors involved in the fine turning of this cytokine balance.

  15. The aryl hydrocarbon receptor AhR links atopic dermatitis and air pollution via induction of the neurotrophic factor artemin.

    PubMed

    Hidaka, Takanori; Ogawa, Eisaku; Kobayashi, Eri H; Suzuki, Takafumi; Funayama, Ryo; Nagashima, Takeshi; Fujimura, Taku; Aiba, Setsuya; Nakayama, Keiko; Okuyama, Ryuhei; Yamamoto, Masayuki

    2017-01-01

    Atopic dermatitis is increasing worldwide in correlation with air pollution. Various organic components of pollutants activate the transcription factor AhR (aryl hydrocarbon receptor). Through the use of AhR-CA mice, whose keratinocytes express constitutively active AhR and that develop atopic-dermatitis-like phenotypes, we identified Artn as a keratinocyte-specific AhR target gene whose product (the neurotrophic factor artemin) was responsible for epidermal hyper-innervation that led to hypersensitivity to pruritus. The activation of AhR via air pollutants induced expression of artemin, alloknesis, epidermal hyper-innervation and inflammation. AhR activation and ARTN expression were positively correlated in the epidermis of patients with atopic dermatitis. Thus, AhR in keratinocytes senses environmental stimuli and elicits an atopic-dermatitis pathology. We propose a mechanism of air-pollution-induced atopic dermatitis via activation of AhR.

  16. The flavonoid galangin is an inhibitor of CYP1A1 activity and an agonist/antagonist of the aryl hydrocarbon receptor.

    PubMed

    Ciolino, H P; Yeh, G C

    1999-03-01

    The effect of the dietary flavonoid galangin on the metabolism of 7,12-dimethylbenz[a]anthracene (DMBA), the activity of cytochrome P450 1A1 (CYP1A1), and the expression of CYP1A1 in MCF-7 human breast carcinoma cells was investigated. Galangin inhibited the catabolic breakdown of DMBA, as measured by thin-layer chromatography, in a dose-dependent manner. Galangin also inhibited the formation of DMBA-DNA adducts, and prevented DMBA-induced inhibition of cell growth. Galangin caused a potent, dose-dependent inhibition of CYP1A1 activity, as measured by ethoxyresorufin-O-deethylase activity, in intact cells and in microsomes isolated from DMBA-treated cells. Analysis of the inhibition kinetics by double-reciprocal plot demonstrated that galangin inhibited CYP1A1 activity in a noncompetitive manner. Galangin caused an increase in the level of CYP1A1 mRNA, indicating that it may be an agonist of the aryl hydrocarbon receptor, but it inhibited the induction of CYP1A1 mRNA by DMBA or by 2,3,5,7-tetrachlorodibenzo-p-dioxin (TCDD). Galangin also inhibited the DMBA- or TCDD-induced transcription of a reporter vector containing the CYP1A1 promoter. Thus, galangin is a potent inhibitor of DMBA metabolism and an agonist/antagonist of the AhR, and may prove to be an effective chemopreventive agent.

  17. Constitutive Activation of the Aromatic Hydrocarbon Receptor

    PubMed Central

    Chang, Ching-Yi; Puga, Alvaro

    1998-01-01

    The ligand-activated aromatic hydrocarbon receptor (AHR) dimerizes with the AHR nuclear translocator (ARNT) to form a functional complex that transactivates expression of the cytochrome P-450 CYP1A1 gene and other genes in the dioxin-inducible [Ah] gene battery. Previous work from this laboratory has shown that the activity of the CYP1A1 enzyme negatively regulates this process. To study the relationship between CYP1A1 activity and Ah receptor activation we used CYP1A1-deficient mouse hepatoma c37 cells and CYP1A1- and AHR-deficient African green monkey kidney CV-1 cells. Using gel mobility shift and luciferase reporter gene expression assays, we found that c37 cells that had not been exposed to exogenous Ah receptor ligands already contained transcriptionally active AHR-ARNT complexes, a finding that we also observed in wild-type Hepa-1 cells treated with Ellipticine, a CYP1A1 inhibitor. In CV-1 cells, transient expression of AHR and ARNT leads to high levels of AHR–ARNT-dependent luciferase gene expression even in the absence of an agonist. Using a green fluorescent protein-tagged AHR, we showed that elevated reporter gene expression correlates with constitutive nuclear localization of the AHR. Transcriptional activation of the luciferase reporter gene observed in CV-1 cells is significantly decreased by (i) expression of a functional CYP1A1 enzyme, (ii) competition with chimeric or truncated AHR proteins containing the AHR ligand-binding domain, and (iii) treatment with the AHR antagonist α-naphthoflavone. These results suggest that a CYP1A1 substrate, which accumulates in cells lacking CYP1A1 enzymatic activity, is an AHR ligand responsible for endogenous activation of the Ah receptor. PMID:9418899

  18. Dioxin-dependent and dioxin-independent gene batteries: comparison of liver and kidney in AHR-null mice.

    PubMed

    Boutros, Paul C; Bielefeld, Kirsten A; Pohjanvirta, Raimo; Harper, Patricia A

    2009-11-01

    The aryl hydrocarbon receptor (AHR) is a widely expressed ligand-dependent transcription factor that mediates cellular responses to dioxins and other planar aromatic hydrocarbons. Ahr-null mice are refractory to the toxic effects of dioxin exposure. Although some mechanistic aspects of AHR activity are well understood, the tissue specificity of AHR effects remains unclear, both during development and following administration of exogenous ligands. To address the latter issue, we defined and compared transcriptional responses to dioxin exposure in the liver and kidney of wild-type and Ahr-null adult C57BL/6J mice treated with either 2,3,7,8-tetrachlorodibenzo-p-dioxin or corn-oil vehicle. In both tissues, essentially all effects of dioxin on hepatic mRNA levels were mediated by the AHR. Although 297 genes were altered by dioxin exposure in the liver, only 17 were changed in the kidney, including a number of well-established AHR target genes. Ahr genotype had a large effect in both tissues, profoundly remodeling both the renal and hepatic transcriptomes. Surprisingly, a large number of genes were affected by Ahr genotype in both tissues, suggesting the presence of a basal AHR gene battery. Alterations of the renal transcriptome in Ahr-null animals were associated with perturbation of specific functional pathways and enrichment of specific DNA motifs. Our results demonstrate the importance of intertissue comparisons, highlight the basal role of the AHR in liver and kidney, and support a role in development or normal physiology.

  19. Intracellular calcium strongly potentiates agonist-activated TRPC5 channels

    PubMed Central

    Blair, Nathaniel T.; Kaczmarek, J. Stefan

    2009-01-01

    TRPC5 is a calcium (Ca2+)-permeable nonselective cation channel expressed in several brain regions, including the hippocampus, cerebellum, and amygdala. Although TRPC5 is activated by receptors coupled to phospholipase C, the precise signaling pathway and modulatory signals remain poorly defined. We find that during continuous agonist activation, heterologously expressed TRPC5 currents are potentiated in a voltage-dependent manner (∼5-fold at positive potentials and ∼25-fold at negative potentials). The reversal potential, doubly rectifying current–voltage relation, and permeability to large cations such as N-methyl-d-glucamine remain unchanged during this potentiation. The TRPC5 current potentiation depends on extracellular Ca2+: replacement by Ba2+ or Mg2+ abolishes it, whereas the addition of 10 mM Ca2+ accelerates it. The site of action for Ca2+ is intracellular, as simultaneous fura-2 imaging and patch clamp recordings indicate that potentiation is triggered at ∼1 µM [Ca2+]. This potentiation is prevented when intracellular Ca2+ is tightly buffered, but it is promoted when recording with internal solutions containing elevated [Ca2+]. In cell-attached and excised inside-out single-channel recordings, increases in internal [Ca2+] led to an ∼10–20-fold increase in channel open probability, whereas single-channel conductance was unchanged. Ca2+-dependent potentiation should result in TRPC5 channel activation preferentially during periods of repetitive firing or coincident neurotransmitter receptor activation. PMID:19398778

  20. Mechanical stress activates NMDA receptors in the absence of agonists.

    PubMed

    Maneshi, Mohammad Mehdi; Maki, Bruce; Gnanasambandam, Radhakrishnan; Belin, Sophie; Popescu, Gabriela K; Sachs, Frederick; Hua, Susan Z

    2017-01-03

    While studying the physiological response of primary rat astrocytes to fluid shear stress in a model of traumatic brain injury (TBI), we found that shear stress induced Ca(2+) entry. The influx was inhibited by MK-801, a specific pore blocker of N-Methyl-D-aspartic acid receptor (NMDAR) channels, and this occurred in the absence of agonists. Other NMDA open channel blockers ketamine and memantine showed a similar effect. The competitive glutamate antagonists AP5 and GluN2B-selective inhibitor ifenprodil reduced NMDA-activated currents, but had no effect on the mechanically induced Ca(2+) influx. Extracellular Mg(2+) at 2 mM did not significantly affect the shear induced Ca(2+) influx, but at 10 mM it produced significant inhibition. Patch clamp experiments showed mechanical activation of NMDAR and inhibition by MK-801. The mechanical sensitivity of NMDARs may play a role in the normal physiology of fluid flow in the glymphatic system and it has obvious relevance to TBI.

  1. Mechanical stress activates NMDA receptors in the absence of agonists

    PubMed Central

    Maneshi, Mohammad Mehdi; Maki, Bruce; Gnanasambandam, Radhakrishnan; Belin, Sophie; Popescu, Gabriela K.; Sachs, Frederick; Hua, Susan Z.

    2017-01-01

    While studying the physiological response of primary rat astrocytes to fluid shear stress in a model of traumatic brain injury (TBI), we found that shear stress induced Ca2+ entry. The influx was inhibited by MK-801, a specific pore blocker of N-Methyl-D-aspartic acid receptor (NMDAR) channels, and this occurred in the absence of agonists. Other NMDA open channel blockers ketamine and memantine showed a similar effect. The competitive glutamate antagonists AP5 and GluN2B-selective inhibitor ifenprodil reduced NMDA-activated currents, but had no effect on the mechanically induced Ca2+ influx. Extracellular Mg2+ at 2 mM did not significantly affect the shear induced Ca2+ influx, but at 10 mM it produced significant inhibition. Patch clamp experiments showed mechanical activation of NMDAR and inhibition by MK-801. The mechanical sensitivity of NMDARs may play a role in the normal physiology of fluid flow in the glymphatic system and it has obvious relevance to TBI. PMID:28045032

  2. The AhR and NF-κB/Rel Proteins Mediate the Inhibitory Effect of 2,3,7,8-Tetrachlorodibenzo-p-Dioxin on the 3' Immunoglobulin Heavy Chain Regulatory Region.

    PubMed

    Salisbury, Richard L; Sulentic, Courtney E W

    2015-12-01

    Transcriptional regulation of the murine immunoglobulin (Ig) heavy chain gene (Igh) involves several regulatory elements including the 3'Igh regulatory region (3'IghRR), which is composed of at least 4 enhancers (hs3A, hs1.2, hs3B, and hs4). The hs1.2 and hs4 enhancers exhibit the greatest transcriptional activity and contain binding sites for several transcription factors including nuclear factor kappaB/Rel (NF-κB/Rel) proteins and the aryl hydrocarbon receptor (AhR). Interestingly, the environmental immunosuppressant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which potently inhibits antibody secretion, also profoundly inhibits 3'IghRR and hs1.2 enhancer activation induced by the B-lymphocyte activator lipopolysaccharide (LPS), but enhances LPS-induced activation of the hs4 enhancer. Within the hs1.2 and hs4 enhancers, the AhR binding site is in close proximity or overlaps an NF-κB/Rel binding site suggesting a potential reciprocal modulation of the 3'IghRR by AhR and NF-κB/Rel. The objective of the current study was to evaluate the role of NF-κB/Rel and the AhR on the 3'IghRR and its enhancers using the AhR ligand TCDD, the AhR antagonist CH223191, and toll-like receptor agonists LPS, Resiquimod (R848), or cytosine-phosphate-guanine-oligodeoxynucleotides (CpG). Utilizing the CH12.LX B-lymphocyte cell line and variants expressing either a 3'IghRR-regulated transgene reporter or an inducible IκBα (inhibitor kappa B-alpha protein) superrepressor (IκBαAA), we demonstrate an AhR- and NF-κB/Rel-dependent modulation of 3'IghRR and hs4 activity. Additionally, in mouse splenocytes or CH12.LX cells, binding within the hs1.2 and hs4 enhancer of the AhR and the NF-κB/Rel proteins RelA and RelB was differentially altered by the cotreatment of LPS and TCDD. These results suggest that the AhR and NF-κB/Rel protein binding profile within the 3'IghRR mediates the inhibitory effects of TCDD on Ig expression and therefore antibody levels.

  3. The AhR and NF-κB/Rel Proteins Mediate the Inhibitory Effect of 2,3,7,8-Tetrachlorodibenzo-p-Dioxin on the 3′ Immunoglobulin Heavy Chain Regulatory Region

    PubMed Central

    Salisbury, Richard L.; Sulentic, Courtney E. W.

    2015-01-01

    Transcriptional regulation of the murine immunoglobulin (Ig) heavy chain gene (Igh) involves several regulatory elements including the 3′Igh regulatory region (3′IghRR), which is composed of at least 4 enhancers (hs3A, hs1.2, hs3B, and hs4). The hs1.2 and hs4 enhancers exhibit the greatest transcriptional activity and contain binding sites for several transcription factors including nuclear factor kappaB/Rel (NF-κB/Rel) proteins and the aryl hydrocarbon receptor (AhR). Interestingly, the environmental immunosuppressant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which potently inhibits antibody secretion, also profoundly inhibits 3′IghRR and hs1.2 enhancer activation induced by the B-lymphocyte activator lipopolysaccharide (LPS), but enhances LPS-induced activation of the hs4 enhancer. Within the hs1.2 and hs4 enhancers, the AhR binding site is in close proximity or overlaps an NF-κB/Rel binding site suggesting a potential reciprocal modulation of the 3′IghRR by AhR and NF-κB/Rel. The objective of the current study was to evaluate the role of NF-κB/Rel and the AhR on the 3′IghRR and its enhancers using the AhR ligand TCDD, the AhR antagonist CH223191, and toll-like receptor agonists LPS, Resiquimod (R848), or cytosine-phosphate-guanine-oligodeoxynucleotides (CpG). Utilizing the CH12.LX B-lymphocyte cell line and variants expressing either a 3′IghRR-regulated transgene reporter or an inducible IκBα (inhibitor kappa B-alpha protein) superrepressor (IκBαAA), we demonstrate an AhR- and NF-κB/Rel-dependent modulation of 3′IghRR and hs4 activity. Additionally, in mouse splenocytes or CH12.LX cells, binding within the hs1.2 and hs4 enhancer of the AhR and the NF-κB/Rel proteins RelA and RelB was differentially altered by the cotreatment of LPS and TCDD. These results suggest that the AhR and NF-κB/Rel protein binding profile within the 3′IghRR mediates the inhibitory effects of TCDD on Ig expression and therefore antibody levels. PMID:26377645

  4. Aryl hydrocarbon receptor activity modulates prolactin expression in the pituitary

    PubMed Central

    Moran, Tyler B.; Brannick, Katherine E.; Raetzman, Lori T.

    2012-01-01

    Pituitary tumors account for 15% of intracranial neoplasms, however the extent to which environmental toxicants contribute to the proliferation and hormone expression of pituitary cells is unknown. Aryl-hydrocarbon receptor (AhR) interacting protein (AIP) loss of function mutations cause somatotroph and lactotroph adenomas in humans. AIP sequesters AhR and inhibits its transcriptional function. Because of the link between AIP and pituitary tumors, we hypothesize that exposure to dioxins, potent exogenous ligands for AhR that are persistent in the environment, may predispose to pituitary dysfunction through activation of AhR. In the present study, we examined the effect of AhR activation on proliferation and endogenous pituitary hormone expression in the GH3 rat somato-lactotrope tumor cell line and the effect of loss of AhR action in knockout mice. GH3 cells respond to nM doses of the reversible AhR agonist β-naphthoflavone with a robust induction of Cyp1a1. Although mRNA levels of the anti-proliferative signaling cytokine TGFbeta1 are suppressed upon β-naphthoflavone treatment, we did not observe an alteration in cell proliferation. AhR activation with β-naphthoflavone suppresses Ahr expression and impairs expression of prolactin (PRL), but not growth hormone (GH) mRNA in GH3 cells. In mice, loss of Ahr similarly leads to a reduction in Prl mRNA at P3, while Gh is unaffected. Additionally, there is a significant reduction pituitary hormones Lhb and Fshb in the absence of Ahr. Overall, these results demonstrate that AhR is important for pituitary hormone expression and suggests environmental dioxins can exert endocrine disrupting effects at the pituitary. PMID:22975028

  5. Dehydroepiandrosterone Derivatives as Potent Antiandrogens with Marginal Agonist Activity

    DTIC Science & Technology

    2012-07-01

    July 2011 - 30 June 2012 4 . TITLE AND SUBTITLE Dehydroepiandrosterone Derivatives as Potent Antiandrogens with Marginal Agonist Activity 5a...Introduction…………………………………………………………….………..….. 1 Body………………………………………………………………………………….. 1 Key Research Accomplishments………………………………………….…….. 4 ...Reportable Outcomes……………………………………………………………… 4 Conclusion…………………………………………………………………………… 8 References……………………………………………………………………………. 9

  6. Different skeletal effects of the peroxisome proliferator activated receptor (PPAR)α agonist fenofibrate and the PPARγ agonist pioglitazone

    PubMed Central

    Syversen, Unni; Stunes, Astrid K; Gustafsson, Björn I; Obrant, Karl J; Nordsletten, Lars; Berge, Rolf; Thommesen, Liv; Reseland, Janne E

    2009-01-01

    Background All the peroxisome proliferator activated receptors (PPARs) are found to be expressed in bone cells. The PPARγ agonist rosiglitazone has been shown to decrease bone mass in mice and thiazolidinediones (TZDs) have recently been found to increase bone loss and fracture risk in humans treated for type 2 diabetes mellitus. The aim of the study was to examine the effect of the PPARα agonist fenofibrate (FENO) and the PPARγ agonist pioglitazone (PIO) on bone in intact female rats. Methods Rats were given methylcellulose (vehicle), fenofibrate or pioglitazone (35 mg/kg body weight/day) by gavage for 4 months. BMC, BMD, and body composition were measured by DXA. Histomorphometry and biomechanical testing of excised femurs were performed. Effects of the compounds on bone cells were studied. Results The FENO group had higher femoral BMD and smaller medullary area at the distal femur; while trabecular bone volume was similar to controls. Whole body BMD, BMC, and trabecular bone volume were lower, while medullary area was increased in PIO rats compared to controls. Ultimate bending moment and energy absorption of the femoral shafts were reduced in the PIO group, while similar to controls in the FENO group. Plasma osteocalcin was higher in the FENO group than in the other groups. FENO stimulated proliferation and differentiation of, and OPG release from, the preosteoblast cell line MC3T3-E1. Conclusion We show opposite skeletal effects of PPARα and γ agonists in intact female rats. FENO resulted in significantly higher femoral BMD and lower medullary area, while PIO induced bone loss and impairment of the mechanical strength. This represents a novel effect of PPARα activation. PMID:19331671

  7. Disruption of CLOCK-BMAL1 Transcriptional Activity Is Responsible for Aryl Hydrocarbon Receptor–Mediated Regulation of Period1 Gene

    PubMed Central

    Xu, Can-Xin; Krager, Stacey L.; Liao, Duan-Fang; Tischkau, Shelley A.

    2010-01-01

    The aryl hydrocarbon receptor (AhR) is a period-aryl hydrocarbon receptor nuclear transporter-simple minded domain transcription factor that shares structural similarity with circadian clock genes and readily interacts with components of the molecular clock. Activation of AhR by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) alters behavioral circadian rhythms and represses the Period1 (Per1) gene in murine hematopoietic stem and progenitor cells. Per1 expression is driven by circadian locomotor activity cycles kaput-brain muscle ARNT-like (CLOCK-BMAL1)–dependent activation of Eboxes in the Per1 promoter. We hypothesized that the effects of AhR activation on the circadian clock are mediated by disruption of CLOCK-BMAL1 function and subsequent Per1 gene suppression. Effects of AhR activation on rhythmic Per1 transcripts were examined in livers of mice after treatment with the AhR agonist, TCDD; the molecular mechanisms of Per1 repression by AhR were determined in hepatoma cells using TCDD and β-napthoflavone as AhR activators. This study reports, for the first time, that AhR activation by TCDD alters the Per1 rhythm in the mouse liver and that Per1 gene suppression depends upon the presence of AhR. Furthermore, AhR interaction with BMAL1 attenuates CLOCK-BMAL1 activity and decreases CLOCK binding at Ebox1 and Ebox3 in the Per1 promoter. Taken together, these data suggest that AhR activation represses Per1 through disrupting CLOCK-BMAL1 activity, producing dysregulation of rhythmic Per1 gene expression. These data define alteration of the Per1 rhythm as novel signaling events downstream of AhR activation. Downregulation of Per1 could contribute to metabolic disease, cancer, and other detrimental effects resulting from exposure to certain environmental pollutants. PMID:20106950

  8. The aryl hydrocarbon receptor and glucocorticoid receptor interact to activate human metallothionein 2A.

    PubMed

    Sato, Shoko; Shirakawa, Hitoshi; Tomita, Shuhei; Tohkin, Masahiro; Gonzalez, Frank J; Komai, Michio

    2013-11-15

    Although the aryl hydrocarbon receptor (AHR) and glucocorticoid receptor (GR) play essential roles in mammalian development, stress responses, and other physiological events, crosstalk between these receptors has been the subject of much debate. Metallothioneins are classic glucocorticoid-inducible genes that were reported to increase upon treatment with AHR agonists in rodent tissues and cultured human cells. In this study, the mechanism of human metallothionein 2A (MT2A) gene transcription activation by AHR was investigated. Cotreatment with 3-methylcholanthrene and dexamethasone, agonists of AHR and GR respectively, synergistically increased MT2A mRNA levels in HepG2 cells. MT2A induction was suppressed by RNA interference against AHR or GR. Coimmunoprecipitation experiments revealed a physical interaction between AHR and GR proteins. Moreover, chromatin immunoprecipitation assays indicated that AHR was recruited to the glucocorticoid response element in the MT2A promoter. Thus, we provide a novel mechanism whereby AHR modulates expression of human MT2A via the glucocorticoid response element and protein-protein interactions with GR.

  9. 3-Methylcholanthrene, an AhR Agonist, Caused Cell-Cycle Arrest by Histone Deacetylation through a RhoA-Dependent Recruitment of HDAC1 and pRb2 to E2F1 Complex

    PubMed Central

    Yang, Nian-Jie; Lee, Yi-Hsuan; Juan, Shu-Hui

    2014-01-01

    We previously showed that treating vascular endothelial cells with 3-methylcholanthrene (3MC) caused cell-cycle arrest in the Go/G1 phase; this resulted from the induction of p21 and p27 and a decreased level and activity of the cyclin-dependent kinase, Cdk2. We further investigated the molecular mechanisms that modulate cell-cycle regulatory proteins through the aryl-hydrocarbon receptor (AhR)/Ras homolog gene family, member A (RhoA) dependent epigenetic modification of histone. AhR/RhoA activation mediated by 3MC was essential for the upregulation of retinoblastoma 2 (pRb2) and histone deacetylase 1 (HDAC1), whereas their nuclear translocation was primarily modulated by RhoA activation. The combination of increased phosphatase and tensin homolog (PTEN) activity and decreased phosphatidylinositide 3-kinase (PI3K) activation by 3MC led to the inactivation of the Ras-cRaf pathway, which contributed to pRb2 hypophosphorylation. Increased HDAC1/pRb2 recruitment to the E2F1 complex decreased E2F1-transactivational activity and H3/H4 deacetylation, resulting in the downregulation of cell-cycle regulatory proteins (Cdk2/4 and Cyclin D3/E). Co-immunoprecipitation and electrophoretic mobility shift assay (EMSA) results showed that simvastatin prevented the 3MC-increased binding activities of E2F1 proteins in their promoter regions. Additionally, RhoA inhibitors (statins) reversed the effect of 3MC in inhibiting DNA synthesis by decreasing the nuclear translocation of pRb2/HDAC1, leading to a recovery of the levels of cell-cycle regulatory proteins. In summary, 3MC decreased cell proliferation by the epigenetic modification of histone through an AhR/RhoA-dependent mechanism that can be rescued by statins. PMID:24658119

  10. 3-Methylcholanthrene, an AhR agonist, caused cell-cycle arrest by histone deacetylation through a RhoA-dependent recruitment of HDAC1 and pRb2 to E2F1 complex.

    PubMed

    Chang, Chih-Cheng; Sue, Yuh-Mou; Yang, Nian-Jie; Lee, Yi-Hsuan; Juan, Shu-Hui

    2014-01-01

    We previously showed that treating vascular endothelial cells with 3-methylcholanthrene (3MC) caused cell-cycle arrest in the Go/G1 phase; this resulted from the induction of p21 and p27 and a decreased level and activity of the cyclin-dependent kinase, Cdk2. We further investigated the molecular mechanisms that modulate cell-cycle regulatory proteins through the aryl-hydrocarbon receptor (AhR)/Ras homolog gene family, member A (RhoA) dependent epigenetic modification of histone. AhR/RhoA activation mediated by 3MC was essential for the upregulation of retinoblastoma 2 (pRb2) and histone deacetylase 1 (HDAC1), whereas their nuclear translocation was primarily modulated by RhoA activation. The combination of increased phosphatase and tensin homolog (PTEN) activity and decreased phosphatidylinositide 3-kinase (PI3K) activation by 3MC led to the inactivation of the Ras-cRaf pathway, which contributed to pRb2 hypophosphorylation. Increased HDAC1/pRb2 recruitment to the E2F1 complex decreased E2F1-transactivational activity and H3/H4 deacetylation, resulting in the downregulation of cell-cycle regulatory proteins (Cdk2/4 and Cyclin D3/E). Co-immunoprecipitation and electrophoretic mobility shift assay (EMSA) results showed that simvastatin prevented the 3MC-increased binding activities of E2F1 proteins in their promoter regions. Additionally, RhoA inhibitors (statins) reversed the effect of 3MC in inhibiting DNA synthesis by decreasing the nuclear translocation of pRb2/HDAC1, leading to a recovery of the levels of cell-cycle regulatory proteins. In summary, 3MC decreased cell proliferation by the epigenetic modification of histone through an AhR/RhoA-dependent mechanism that can be rescued by statins.

  11. Specific Ligand Binding Domain Residues Confer Low Dioxin Responsiveness to AHR1β of Xenopus laevis

    PubMed Central

    Odio, Camila; Holzman, Sarah A.; Denison, Michael S.; Fraccalvieri, Domenico; Bonati, Laura; Franks, Diana G.; Hahn, Mark E.; Powell, Wade H.

    2013-01-01

    The aryl hydrocarbon receptor (AHR) is a PAS-family protein that mediates the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in vertebrates. Frogs are remarkably insensitive to TCDD, and AHRs from Xenopus laevis bind TCDD with low affinity. We sought to identify structural features of X. laevis AHR1β associated with low TCDD sensitivity. Substitution of the entire ligand-binding domain (LBD) with the corresponding sequence from mouse AHRb-1 dramatically increased TCDD responsiveness in transactivation assays. To identify amino acid residues responsible, we constructed a comparative model of the AHR1β LBD using homologous domains of PAS proteins HIF2α and ARNT. The model revealed an internal cavity of similar dimensions to the putative binding cavity of mouse AHRb-1, suggesting the importance of side-chain interactions over cavity size. Of residues with side chains clearly pointing into the cavity, only two differed from the mouse sequence. When A354, located within a conserved β-strand, was changed to serine, the corresponding mouse residue, the EC50 for TCDD decreased more than 15-fold. When N325 was changed to serine, EC50 declined 3-fold. When the mutations were combined, the EC50 declined from 18.6 nM to 0.8 nM, nearly matching mouse AHR for TCDD sensitivity. Velocity sedimentation analysis confirmed that mutant frog AHRs exhibited correspondingly increased TCDD binding. We also assayed mutant AHRs for responsiveness to a candidate endogenous ligand, 6-formylindolo[3,2b]carbazole (FICZ). Mutations that increased TCDD sensitivity also increased sensitivity to FICZ. This comparative study represents a novel approach to discerning fundamental information about the structure of AHR and its interactions with biologically important agonists. PMID:23394719

  12. Transactivation activity of human, zebrafish, and rainbow trout aryl hydrocarbon receptors expressed in COS-7 cells: greater insight into species differences in toxic potency of polychlorinated dibenzo-p-dioxin, dibenzofuran, and biphenyl congeners.

    PubMed

    Abnet, C C; Tanguay, R L; Heideman, W; Peterson, R E

    1999-08-15

    Transactivation assays were used to compare the potency and efficacy of polychlorinated dibenzo-p-dioxin (PCDD), dibenzofuran (PCDF), and biphenyl (PCB) congeners in activating aryl hydrocarbon receptors (AhRs) from rainbow trout (rtAhR2alpha and rtAhR2beta), zebrafish (zfAhR2), and human (huAhR), respectively. All AhRs were expressed with their species-specific AhR nuclear translocator (ARNT) in COS-7 cells. Transactivation activity was determined for two PCDD, two PCDF, and seven PCB congeners with each of the four AhR/ARNT pairs using prt1Aluc, a luciferase reporter driven by two dioxin-responsive enhancer elements (DREs) from the rainbow trout cyp1A gene. Maximal-fold induction, EC50, and relative potency values were calculated for congeners that exhibited dose-related activity in the assay. Of the four AhR/ARNT pairs tested with PCDD, PCDF, and non-ortho PCB congeners, three exhibited high activity (rainbow trout AhR2alpha, zebrafish AhR2, and human AhR), while rainbow trout AhR2beta had very weak or no activity. Comparisons between these AhRs showed that while mono-ortho PCBs were able to activate the human AhR, they were generally ineffective in activating rainbow trout and zebrafish AhR2s. This supports the hypothesis that structural differences between mammalian and fish AhRs may account for differences in relative potencies of the mono-ortho PCBs between mammals and fish. Another important finding was a significant difference in transactivation activity between the two rainbow trout AhR2 isoforms despite the fact that they are 95% identical at the amino acid level. For all PCDD, PCDF, and PCB agonists tested, rainbow trout AhR2alpha was significantly more active than AhR2beta. However, rainbow trout AhR2beta is active as a 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-activated transcription factor, with enhancer elements from the mouse cyp1A gene. This suggests that AhR2beta may have evolved to serve a different physiological function than AhR2alpha in

  13. Trypanosoma cruzi trans-sialidase initiates a program independent of the transcription factors RORγt and Ahr that leads to IL-17 production by activated B cells.

    PubMed

    Bermejo, Daniela A; Jackson, Shaun W; Gorosito-Serran, Melisa; Acosta-Rodriguez, Eva V; Amezcua-Vesely, Maria C; Sather, Blythe D; Singh, Akhilesh K; Khim, Socheath; Mucci, Juan; Liggitt, Denny; Campetella, Oscar; Oukka, Mohamed; Gruppi, Adriana; Rawlings, David J

    2013-05-01

    Here we identified B cells as a major source of rapid, innate-like production of interleukin 17 (IL-17) in vivo in response to infection with Trypanosoma cruzi. IL-17(+) B cells had a plasmablast phenotype, outnumbered cells of the TH17 subset of helper T cells and were required for an optimal response to this pathogen. With both mouse and human primary B cells, we found that exposure to parasite-derived trans-sialidase in vitro was sufficient to trigger modification of the cell-surface mucin CD45, which led to signaling dependent on the kinase Btk and production of IL-17A or IL-17F via a transcriptional program independent of the transcription factors RORγt and Ahr. Our combined data suggest that the generation of IL-17(+) B cells may be a previously unappreciated feature of innate immune responses required for pathogen control or IL-17-mediated autoimmunity.

  14. Activation of Protease Activated Receptor 2 by Exogenous Agonist Exacerbates Early Radiation Injury in Rat Intestine

    SciTech Connect

    Wang Junru; Boerma, Marjan; Kulkarni, Ashwini; Hollenberg, Morley D.; Hauer-Jensen, Martin

    2010-07-15

    Purpose: Protease-activated receptor-2 (PAR{sub 2}) is highly expressed throughout the gut and regulates the inflammatory, mitogenic, fibroproliferative, and nociceptive responses to injury. PAR{sub 2} is strikingly upregulated and exhibits increased activation in response to intestinal irradiation. We examined the mechanistic significance of radiation enteropathy development by assessing the effect of exogenous PAR{sub 2} activation. Methods and Materials: Rat small bowel was exposed to localized single-dose radiation (16.5 Gy). The PAR{sub 2} agonist (2-furoyl-LIGRLO-NH{sub 2}) or vehicle was injected intraperitoneally daily for 3 days before irradiation (before), for 7 days after irradiation (after), or both 3 days before and 7 days after irradiation (before-after). Early and delayed radiation enteropathy was assessed at 2 and 26 weeks after irradiation using quantitative histologic examination, morphometry, and immunohistochemical analysis. Results: The PAR{sub 2} agonist did not elicit changes in the unirradiated (shielded) intestine. In contrast, in the irradiated intestine procured 2 weeks after irradiation, administration of the PAR{sub 2} agonist was associated with more severe mucosal injury and increased intestinal wall thickness in all three treatment groups (p <.05) compared with the vehicle-treated controls. The PAR{sub 2} agonist also exacerbated the radiation injury score, serosal thickening, and mucosal inflammation (p <.05) in the before and before-after groups. The short-term exogenous activation of PAR{sub 2} did not affect radiation-induced intestinal injury at 26 weeks. Conclusion: The results of the present study support a role for PAR{sub 2} activation in the pathogenesis of early radiation-induced intestinal injury. Pharmacologic PAR{sub 2} antagonists might have the potential to reduce the intestinal side effects of radiotherapy and/or as countermeasures in radiologic accidents or terrorism scenarios.

  15. Skatole (3-Methylindole) Is a Partial Aryl Hydrocarbon Receptor Agonist and Induces CYP1A1/2 and CYP1B1 Expression in Primary Human Hepatocytes

    PubMed Central

    Balaguer, Patrick; Ekstrand, Bo; Daujat-Chavanieu, Martine; Gerbal-Chaloin, Sabine

    2016-01-01

    Skatole (3-methylindole) is a product of bacterial fermentation of tryptophan in the intestine. A significant amount of skatole can also be inhaled during cigarette smoking. Skatole is a pulmonary toxin that induces the expression of aryl hydrocarbon receptor (AhR) regulated genes, such as cytochrome P450 1A1 (CYP1A1), in human bronchial cells. The liver has a high metabolic capacity for skatole and is the first organ encountered by the absorbed skatole; however, the effect of skatole in the liver is unknown. Therefore, we investigated the impact of skatole on hepatic AhR activity and AhR-regulated gene expression. Using reporter gene assays, we showed that skatole activates AhR and that this is accompanied by an increase of CYP1A1, CYP1A2 and CYP1B1 expression in HepG2-C3 and primary human hepatocytes. Specific AhR antagonists and siRNA-mediated AhR silencing demonstrated that skatole-induced CYP1A1 expression is dependent on AhR activation. The effect of skatole was reduced by blocking intrinsic cytochrome P450 activity and indole-3-carbinole, a known skatole metabolite, was a more potent inducer than skatole. Finally, skatole could reduce TCDD-induced CYP1A1 expression, suggesting that skatole is a partial AhR agonist. In conclusion, our findings suggest that skatole and its metabolites affect liver homeostasis by modulating the AhR pathway. PMID:27138278

  16. Common Commercial and Consumer Products Contain Activators of the Aryl Hydrocarbon (Dioxin) Receptor

    PubMed Central

    Zhao, Bin; Bohonowych, Jessica E. S.; Timme-Laragy, Alicia; Jung, Dawoon; Affatato, Alessandra A.; Rice, Robert H.; Di Giulio, Richard T.; Denison, Michael S.

    2013-01-01

    Activation of the Ah receptor (AhR) by halogenated aromatic hydrocarbons (HAHs), such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin), can produce a wide variety of toxic and biological effects. While recent studies have shown that the AhR can bind and be activated by structurally diverse chemicals, how widespread of these AhR agonists are in environmental, biological and synthetic materials remains to be determined. Using AhR-based assays, we demonstrate the presence of potent AhR agonists in a variety of common commercial and consumer items. Solvent extracts of paper, rubber and plastic products contain chemicals that can bind to and stimulate AhR DNA binding and/or AhR-dependent gene expression in hepatic cytosol, cultured cell lines, human epidermis and zebrafish embryos. In contrast to TCDD and other persistent dioxin-like HAHs, activation of AhR-dependent gene expression by these extracts was transient, suggesting that the agonists are metabolically labile. Solvent extracts of rubber products produce AhR-dependent developmental toxicity in zebrafish in vivo, and inhibition of expression of the metabolic enzyme CYP1A, significantly increased their toxic potency. Although the identity of the responsible AhR-active chemicals and their toxicological impact remain to be determined, our data demonstrate that AhR active chemicals are widely distributed in everyday products. PMID:23441220

  17. Dehydroepiandrosterone Derivatives as Potent Antiandrogens with Marginal Agonist Activity

    DTIC Science & Technology

    2010-07-01

    or 9), although these compounds still showed anti-DHT effects (lanes 2 vs. 6, 8, or 10). Figure 4 . The effects of DHEA derivatives on PSA...2009 - 30 JUN 2010 4 . TITLE AND SUBTITLE Dehydroepiandrosterone Derivatives as Potent Antiandrogens 5a. CONTRACT NUMBER with Marginal Agonist...words) We hypothesized that dehydroepiandrosterone ( DHEA ) metabolites or their synthetic derivatives are able to bind to the androgen receptor with

  18. In vitro screening for aryl hydrocarbon receptor agonistic activity in 200 pesticides using a highly sensitive reporter cell line, DR-EcoScreen cells, and in vivo mouse liver cytochrome P450-1A induction by propanil, diuron and linuron.

    PubMed

    Takeuchi, Shinji; Iida, Mitsuru; Yabushita, Hisatoshi; Matsuda, Tadashi; Kojima, Hiroyuki

    2008-12-01

    The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that regulates genes involved in xenobiotic metabolism, cellular proliferation and differentiation. In this study, we have developed a highly sensitive AhR-mediated reporter cell line, DR-EcoScreen cells, which are mouse hepatoma Hepa1c1c7 cells stably transfected with a reporter plasmid containing seven copies of dioxin-responsive element. Using these DR-EcoScreen cells, we performed the reporter gene assay and characterized the AhR agonistic activities of 200 pesticides (29 organochlorines, 11 diphenyl ethers, 56 organophosphorus pesticides, 12 pyrethroids, 22 carbamates, 12 acid amides, 7 triazines, 6 ureas, and 45 others). Eleven of the 200 pesticides (acifluorfen-methyl, bifenox, chlorpyrifos, isoxathion, quinalphos, chlorpropham, diethofencarb, propanil, diuron, linuron, and prochloraz) showed AhR-mediated transcriptional activity. In particular, three herbicides (propanil, diuron, and linuron) have a common chemical structure and showed more potent agonistic activity than other pesticides. To investigate the in vivo effects, we examined the gene expression of AhR-inducible cytochrome P450 1As (CYP1As) in the liver of female C57BL/6 mice intraperitoneally injected with these three herbicides (300 mg kg(-1)) by quantitative RT-PCR, resulting in induction of significant high levels of CYP1A1 and CYP1A2 mRNAs. This indicates that propanil, diuron and linuron possess AhR-mediated transactivation effect in vivo as well as in vitro. Through the present study, we demonstrated that DR-EcoScreen cells are useful for sensitive, rapid and simple identification of AhR agonists among a large number of environmental chemicals.

  19. Aryl hydrocarbon receptor activation inhibits in vitro differentiation of human monocytes and Langerhans dendritic cells.

    PubMed

    Platzer, Barbara; Richter, Susanne; Kneidinger, Doris; Waltenberger, Darina; Woisetschläger, Maximilian; Strobl, Herbert

    2009-07-01

    The transcription factor aryl hydrocarbon receptor (AhR) represents a promising therapeutic target in allergy and autoimmunity. AhR signaling induced by the newly described ligand VAF347 inhibits allergic lung inflammation as well as suppresses pancreatic islet allograft rejection. These effects are likely mediated via alterations in dendritic cell (DC) function. Moreover, VAF347 induces tolerogenic DCs. Langerhans cells (LCs) are immediate targets of exogenous AhR ligands at epithelial surfaces; how they respond to AhR ligands remained undefined. We studied AhR expression and function in human LCs and myelopoietic cell subsets using a lineage differentiation and gene transduction model of human CD34(+) hematopoietic progenitors. We found that AhR is highly regulated during myeloid subset differentiation. LCs expressed highest AhR levels followed by monocytes. Conversely, neutrophil granulocytes lacked AhR expression. AhR ligands including VAF347 arrested the differentiation of monocytes and LCs at an early precursor cell stage, whereas progenitor cell expansion or granulopoiesis remained unimpaired. AhR expression was coregulated with the transcription factor PU.1 during myeloid subset differentiation. VAF347 inhibited PU.1 induction during initial monocytic differentiation, and ectopic PU.1 restored monocyte and LC generation in the presence of this compound. AhR ligands failed to interfere with cytokine receptor signaling during LC differentiation and failed to impair LC activation/maturation. VAF347-mediated antiproliferative effect on precursors undergoing LC lineage differentiation occurred in a clinically applicable serum-free culture model and was not accompanied by apoptosis induction. In conclusion, AhR agonist signaling interferes with transcriptional processes leading to monocyte/DC lineage commitment of human myeloid progenitor cells.

  20. Plant polyphenols differentially modulate inflammatory responses of human keratinocytes by interfering with activation of transcription factors NF{kappa}B and AhR and EGFR-ERK pathway

    SciTech Connect

    Potapovich, Alla I.; Lulli, Daniela; Fidanza, Paolo; Kostyuk, Vladimir A.; De Luca, Chiara; Pastore, Saveria; Korkina, Liudmila G.

    2011-09-01

    Molecular mechanisms underlying modulation of inflammatory responses in primary human keratinocytes by plant polyphenols (PPs), namely the glycosylated phenylpropanoid verbascoside, the stilbenoid resveratrol and its glycoside polydatin, and the flavonoid quercetin and its glycoside rutin were evaluated. As non-lethal stimuli, the prototypic ligand for epidermal growth factor receptor (EGFR) transforming growth factor alpha (TGFalpha), the combination of tumor necrosis factor (TNFalpha) and interferon (IFNgamma) (T/I), UVA + UVB irradiation, and bacterial lipopolysaccharide (LPS) were used. We demonstrated differential modulation of inflammatory responses in keratinocytes at signal transduction, gene transcription, and protein synthesis levels as a function of PP chemical structure, the pro-inflammatory trigger used, and PP interaction with intracellular detoxifying systems. The PPs remarkably inhibited constitutive, LPS- and T/I-induced but not TGFalpha-induced ERK phosphorylation. They also suppressed NFkappaB activation by LPS and T/I. Verbascoside and quercetin invariably impaired EGFR phosphorylation and UV-associated aryl hydrocarbon receptor (AhR)-mediated signaling, while rutin, polydatin and resveratrol did not affect EGFR phosphorylation and further activated AhR machinery in UV-exposed keratinocytes. In general, PPs down-regulated gene expression of pro-inflammatory cytokines/enzymes, except significant up-regulation of IL-8 observed under stimulation with TGFalpha. Both spontaneous and T/I-induced release of IL-8 and IP-10 was suppressed, although 50 {mu}M resveratrol and polydatin up-regulated IL-8. At this concentration, resveratrol activated both gene expression and de novo synthesis of IL-8 and AhR-mediated mechanisms were involved. We conclude that PPs differentially modulate the inflammatory response of human keratinocytes through distinct signal transduction pathways, including AhR and EGFR. - Graphical abstract: Display Omitted Highlights

  1. Dehydroepiandrosterone Derivatives as Potent Antiandrogens with Marginal Agonist Activity

    DTIC Science & Technology

    2013-07-01

    DATES COVERED 01 July 2012 – 30 June 2013 4 . TITLE AND SUBTITLE Dehydroepiandrosterone Derivatives as Potent Antiandrogens with Marginal Agonist...Page Introduction…………………………………………………………….………..….. 1 Body………………………………………………………………………………….. 1- 4 Key Research...In addition, we previously found that androstenediol (Adiol), a physiological metabolite from dehydroepiandrosterone ( DHEA ) and a precursor of

  2. AHR2-Mediated Transcriptomic Responses Underlying the Synergistic Cardiac Developmental Toxicity of PAHs

    PubMed Central

    Jayasundara, Nishad; Van Tiem Garner, Lindsey; Meyer, Joel N.; Erwin, Kyle N.; Di Giulio, Richard T.

    2015-01-01

    Polycyclic aromatic hydrocarbons (PAHs) induce developmental defects including cardiac deformities in fish. The aryl hydrocarbon receptor (AHR) mediates the toxicity of some PAHs. Exposure to a simple PAH mixture during embryo development consisting of an AHR agonist (benzo(a)pyrene-BaP) with fluoranthene (FL), an inhibitor of cytochrome p450 1(CYP1)—a gene induced by AHR activation—results in cardiac deformities. Exposure to BaP or FL alone at similar concentrations alters heart rates, but does not induce morphological deformities. Furthermore, AHR2 knockdown prevents the toxicity of BaP + FL mixture. Here, we used a zebrafish microarray analysis to identify heart-specific transcriptomic changes during early development that might underlie cardiotoxicity of BaP + FL. We used AHR2 morphant embryos to determine the role of this receptor in mediating toxicity. Control and knockdown embryos at 36 h post-fertilization were exposed to DMSO, 100 μg/l BaP, 500 μg/l FL, or 100 μg/l BaP + 500 μg/l FL, and heart tissues for RNA were extracted at 2, 6, 12, and 18 h-post-exposure (hpe), prior to the appearance of cardiac deformities. Data show AHR2-dependent BaP + FL effects on expression of genes involved in protein biosynthesis and neuronal development in addition to signaling molecules and their associated molecular pathways. Ca2+-cycling and muscle contraction genes were the most significantly differentially expressed category of transcripts when comparing BaP + FL-treated AHR2 morphant and control embryos. These differences were most prominent at 2 and 6 hpe. Therefore, we postulate that BaP + FL may affect cellular Ca2+ levels and subsequently cardiac muscle function, potentially underlying BaP + FL cardiotoxicity. PMID:25412620

  3. Alternative in vitro approach for assessing AHR-mediated CYP1A induction by dioxins in wild cormorant (Phalacrocorax carbo) population.

    PubMed

    Thuruthippallil, Leena Mol; Kubota, Akira; Kim, Eun-Young; Iwata, Hisato

    2013-06-18

    Our line of papers revealed that the common (great) cormorant (Phalacrocorax carbo) possesses two isoforms of the aryl hydrocarbon receptor (ccAHR1 and ccAHR2). This paper addresses in vitro tests of the ccAHR signaling pathways to solve two questions: (1) whether there are functional differences in the two ccAHR isoforms, and (2) whether a molecular perturbation, cytochrome P450 1A (ccCYP1A) induction, in the population-level can be predicted from the in vitro tests. The transactivation potencies mediated by ccAHR1 and ccAHR2 were measured in COS-7 cells treated with 15 selected dioxins and related compounds (DRCs), where ccAHR1 or ccAHR2 expression plasmid and ccCYP1A5 promoter/enhancer-linked luciferase reporter plasmid were transfected. For congeners that exhibited dose-dependent luciferase activities, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) relative potencies (REPs) and induction equivalency factors (IEFs) were estimated. ccAHR1-IEF profile was similar to WHO avian TCDD toxic equivalency factor (TEF) profile except for dioxin-like polychlorinated biphenyls that showed lower IEFs in ccAHR1-driven reporter assay. ccAHR2-IEF profile was different from WHO TEFs and ccAHR1-IEFs. Notably, 2,3,4,7,8-PeCDF was more potent than TCDD for ccAHR2-mediated response. Using ccAHR1- and ccAHR2-IEFs and hepatic DRC concentrations in the Lake Biwa cormorant population, total TCDD induction equivalents (IEQs) were calculated for each ccAHR-mediated response. Nonlinear regression analyses provided significant sigmoidal relationships of ccAHR1- and ccAHR2-derived IEQs with hepatic ccCYP1A5 mRNA levels, supporting the results of in vitro ccAHR-mediated TCDD dose-response curves. Collectively, our in vitro AHR reporter assay potentially could be an alternative to molecular epidemiology of the species of concern regarding CYP1A induction by AHR ligands.

  4. TRPA1 agonist activity of probenecid desensitizes channel responses: consequences for screening.

    PubMed

    McClenaghan, Conor; Zeng, Fanning; Verkuyl, Jan Martin

    2012-12-01

    The transient receptor potential channel subtype A member 1 (TRPA1) is a nonselective cation channel widely viewed as having therapeutic potential, particularly for pain-related indications. Realization of this potential will require potent, selective modulators; however, currently the pharmacology of TRPA1 is poorly defined. As TRPA1 is calcium permeable, calcium indicators offer a simple assay format for high-throughput screening. In this report, we show that probenecid, a uricosuric agent used experimentally in screening to increase loading of calcium-sensitive dyes, activates TRPA1. Prolonged probenecid incubation during the dye-loading process reduces agonist potency upon subsequent challenge. When Chinese Hamster Ovary (CHO)-hTRPA1 or STC-1 cells, which endogenously express TRPA1, were dye loaded in the presence of 2 mM probenecid TRPA1, agonists appeared less potent; EC(50) for allyl isothiocyante agonists in CHO-hTRPA1 was increased from 1.5±0.19 to 7.32±1.20 μM (P<0.01). No significant effect on antagonist potency was observed when using the agonist EC(80) concentration determined under the appropriate dye-loading conditions. We suggest an alternative protocol for calcium imaging using another blocker of anion transport, sulfinpyrazone. This blocker significantly augments indicator dye loading and the screening window, but is not a TRPA1 agonist and has no effect on agonist potency.

  5. Analysis of the agonist activity of fenoldopam (SKF 82526) at the vascular 5-HT2 receptor.

    PubMed Central

    Christie, M. I.; Harper, D.; Smith, G. W.

    1992-01-01

    1. The 5-HT2 receptor agonist activity of fenoldopam (SKF 82526) was characterized in the rabbit isolated aorta preparation. 2. Fenoldopam was an agonist at the vascular 5-HT2 receptor with lower affinity and efficacy than the naturally occurring agonist 5-hydroxytryptamine (5-HT). Fenoldopam had an affinity (pKA) of 5.84 +/- 0.04 and efficacy (tau) of 0.57 +/- 0.04, whereas 5-HT had a pKA of 6.65 +/- 0.12 and tau of 2.66 +/- 0.41. 3. The constrictor effects of fenoldopam and 5-HT were competitively antagonized by the 5-HT2 antagonist, ketanserin, with pKB values of 8.81 +/- 0.11 and 8.83 +/- 0.10 respectively. 4. Prior incubation with fenoldopam produced a concentration-related rightward shift of a subsequent 5-HT concentration-response curve. This inhibition was specific for 5-HT since constrictor responses to angiotensin II were unaffected. 5. This study indicates that the D1 receptor agonist, fenoldopam, acts as an agonist at the vascular 5-HT2 receptor, but with an affinity and efficacy less than that of the naturally occurring agonist, 5-HT. PMID:1361397

  6. Tetrachlorodibenzo-p-dioxin exposure alters radial arm maze performance and hippocampal morphology in female AhR mice.

    PubMed

    Powers, B E; Lin, T-M; Vanka, A; Peterson, R E; Juraska, J M; Schantz, S L

    2005-02-01

    Perinatal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been reported to alter spatial learning in rats tested on a radial arm maze (RAM). TCDD is believed to exert most of its effects through binding to the aryl hydrocarbon receptor (AhR). To determine whether the AhR mediates TCDD-induced alterations in spatial learning, we tested male and female AhR-knockout (AhR-/-), heterozygous (AhR+/-) and wild-type (AhR+/+) mice on the RAM. AhR+/- male and female mice were time mated, and treated dams were dosed with 5 microg TCDD/kg body weight on day 13 of gestation. When offspring reached adulthood, male and female AhR+/+, AhR+/- and AhR-/- mice from TCDD-exposed and unexposed litters were tested on the eight-arm RAM. After testing, we examined hippocampal morphology as visualized by the Timm's silver sulfide stain. TCDD-exposed female AhR+/- mice made more errors than their respective controls on the RAM and exhibited a decrease in the size of the intra- and infrapyramidal mossy fiber (IIP-MF) field of the hippocampus. None of the other TCDD-exposed groups differed from their respective control groups with regard to maze performance or hippocampal morphology. The reduction of IIP-MF field indicates a possible morphological basis for the learning deficit that was observed in the female AhR+/- mice. It is hypothesized that the effect of TCDD exposure is AhR dependent and that TCDD may alter GABAergic activity in the hippocampus of female mice during development.

  7. Dioxin-Dependent and Dioxin-Independent Gene Batteries: Comparison of Liver and Kidney in AHR-Null Mice

    PubMed Central

    Boutros, Paul C.; Bielefeld, Kirsten A.; Pohjanvirta, Raimo; Harper, Patricia A.

    2009-01-01

    The aryl hydrocarbon receptor (AHR) is a widely expressed ligand-dependent transcription factor that mediates cellular responses to dioxins and other planar aromatic hydrocarbons. Ahr-null mice are refractory to the toxic effects of dioxin exposure. Although some mechanistic aspects of AHR activity are well understood, the tissue specificity of AHR effects remains unclear, both during development and following administration of exogenous ligands. To address the latter issue, we defined and compared transcriptional responses to dioxin exposure in the liver and kidney of wild-type and Ahr-null adult C57BL/6J mice treated with either 2,3,7,8-tetrachlorodibenzo-p-dioxin or corn-oil vehicle. In both tissues, essentially all effects of dioxin on hepatic mRNA levels were mediated by the AHR. Although 297 genes were altered by dioxin exposure in the liver, only 17 were changed in the kidney, including a number of well-established AHR target genes. Ahr genotype had a large effect in both tissues, profoundly remodeling both the renal and hepatic transcriptomes. Surprisingly, a large number of genes were affected by Ahr genotype in both tissues, suggesting the presence of a basal AHR gene battery. Alterations of the renal transcriptome in Ahr-null animals were associated with perturbation of specific functional pathways and enrichment of specific DNA motifs. Our results demonstrate the importance of intertissue comparisons, highlight the basal role of the AHR in liver and kidney, and support a role in development or normal physiology. PMID:19759094

  8. Structure-Activity Relationship and Signaling of New Chimeric CXCR4 Agonists.

    PubMed

    Mona, Christine E; Besserer-Offroy, Élie; Cabana, Jérôme; Lefrançois, Marilou; Boulais, Philip E; Lefebvre, Marie-Reine; Leduc, Richard; Lavigne, Pierre; Heveker, Nikolaus; Marsault, Éric; Escher, Emanuel

    2016-08-25

    The CXCR4 receptor binds with meaningful affinities only CXCL12 and synthetic antagonists/inverse agonists. We recently described high affinity synthetic agonists for this chemokine receptor, obtained by grafting the CXCL12 N-terminus onto the inverse agonist T140. While those chimeric molecules behave as agonists for CXCR4, their binding and activation mode are unknown. The present SAR of those CXCL12-oligopeptide grafts reveals the key determinants involved in CXCR4 activation. Position 3 (Val) controls affinity, whereas position 7 (Tyr) acts as an efficacy switch. Chimeric molecules bearing aromatic residues in position 3 possess high binding affinities for CXCR4 and are Gαi full agonists with robust chemotactic properties. Fine-tuning of electron-poor aromatic rings in position 7 enhances receptor activation. To rationalize these results, a homology model of a receptor-ligand complex was built using the published crystal structures of CXCR4. Molecular dynamics simulations reveal further details accounting for the observed SAR for this series.

  9. Inhibitory effects of peroxisome proliferator-activated receptor γ agonists on collagen IV production in podocytes.

    PubMed

    Li, Yanjiao; Shen, Yachen; Li, Min; Su, Dongming; Xu, Weifeng; Liang, Xiubin; Li, Rongshan

    2015-07-01

    Peroxisome proliferator-activated receptor-γ (PPAR-γ) agonists have beneficial effects on the kidney diseases through preventing microalbuminuria and glomerulosclerosis. However, the mechanisms underlying these effects remain to be fully understood. In this study, we investigate the effects of PPAR-γ agonist, rosiglitazone (Rosi) and pioglitazone (Pio), on collagen IV production in mouse podocytes. The endogenous expression of PPAR-γ was found in the primary podocytes and can be upregulated by Rosi and Pio, respectively, detected by RT-PCR and Western blot. PPAR-γ agonist markedly blunted the increasing of collagen IV expression and extraction in podocytes induced by TGF-β. In contrast, adding PPAR-γ antagonist, GW9662, to podocytes largely prevented the inhibition of collagen IV expression from Pio treatment. Our data also showed that phosphorylation of Smad2/3 enhanced by TGF-β in a time-dependent manner was significantly attenuated by adding Pio. The promoter region of collagen IV gene contains one putative consensus sequence of Smad-binding element (SBE) by promoter analysis, Rosi and Pio significantly ameliorated TGF-β-induced SBE4-luciferase activity. In conclusion, PPAR-γ activation by its agonist, Rosi or Pio, in vitro directly inhibits collagen IV expression and synthesis in primary mouse podocytes. The suppression of collagen IV production was related to the inhibition of TGF-β-driven phosphorylation of Smad2/3 and decreased response activity of SBEs of collagen IV in PPAR-γ agonist-treated mouse podocytes. This represents a novel mechanistic support regarding PPAR-γ agonists as podocyte protective agents.

  10. Pungency of TRPV1 agonists is directly correlated with kinetics of receptor activation and lipophilicity.

    PubMed

    Ursu, Daniel; Knopp, Kelly; Beattie, Ruth E; Liu, Bin; Sher, Emanuele

    2010-09-01

    TRPV1 (transient receptor potential vanilloid 1) is a ligand-gated ion channel expressed predominantly in nociceptive primary afferents that plays a key role in pain processing. In vivo activation of TRPV1 receptors by natural agonists like capsaicin is associated with a sharp and burning pain, frequently described as pungency. To elucidate the mechanisms underlying pungency we investigated a series of TRPV1 agonists that included both pungent and non-pungent compounds covering a large range of potencies. Pungency of capsaicin, piperine, arvanil, olvanil, RTX (resiniferatoxin) and SDZ-249665 was evaluated in vivo, by determining the increase in the number of eye wipes caused by direct instillation of agonist solutions into the eye. Agonist-induced calcium fluxes were recorded using the FLIPR technique in a recombinant, TRPV1-expressing cell line. Current-clamp recordings were performed in rat DRG (dorsal root ganglia) neurons in order to assess the consequences of TRPV1 activation on neuronal excitability. Using the eye wipe assay the following rank of pungency was obtained: capsaicin>piperine>RTX>arvanil>olvanil>SDZ-249665. We found a strong correlation between kinetics of calcium flux, pungency and lipophilicity of TRPV1 agonists. Current-clamp recordings confirmed that the rate of receptor activation translates in the ability of agonists to generate action potentials in sensory neurons. We have demonstrated that the lipophilicity of the compounds is directly related to the kinetics of TRPV1 activation and that the latter influences their ability to trigger action potentials in sensory neurons and, ultimately, pungency.

  11. Promotion of adiponectin multimerization by emodin: a novel AMPK activator with PPARγ-agonist activity.

    PubMed

    Chen, Zhifen; Zhang, Lu; Yi, Junyang; Yang, Zhuanbo; Zhang, Zhijie; Li, Zhen

    2012-11-01

    Adiponectin is an important insulin-sensitizing adipokine with multiple beneficial effects on obesity-associated medical complications. It is secreted from adipocytes into circulation as high, medium, and low molecular weight forms (HMW, MMW, and LMW). Each oligomeric form of adiponectin exerts non-overlapping biological functions, with the HMW oligomer possessing the most potent insulin-sensitizing activity. In this study, we reported that emodin, a natural product and active ingredient of various Chinese herbs, activates AMPK in both 3T3-L1 adipocytes and 293T cells. Activation of AMPK by emodin promotes the assembly of HMW adiponectin and increases the ratio of HMW adiponectin to total adiponectin in 3T1-L1 adipocytes. Emodin might activate AMPK by an indirect mechanism similar to berberine. We also found that emodin activates PPARγ and promotes differentiation and adiponectin expression during differentiation of 3T3-L1 preadipocytes. Therefore, emodin is a novel AMPK activator with PPARγ-agonist activity. Our results demonstrate that the effects of emodin on adiponectin expression and multimerization are the ultimate effects resulting from both AMPK activation and PPARγ activation. The dual-activity makes emodin or the derivatives potential drug candidates for the treatment of type 2 diabetes and other obesity-related metabolic diseases.

  12. The most effective influence of 17-(3-ethoxypropyl) substituent on the binding affinity and the agonistic activity in KNT-127 derivatives, δ opioid receptor agonists.

    PubMed

    Nemoto, Toru; Ida, Yoshihiro; Iihara, Yusuke; Nakajima, Ryo; Hirayama, Shigeto; Iwai, Takashi; Fujii, Hideaki; Nagase, Hiroshi

    2013-12-15

    We investigated the structure-activity relationship of KNT-127 (opioid δ agonist) derivatives with various 17-substituents which are different in length and size. The 17-substituent in KNT-127 derivatives exerted a great influence on the affinity and agonistic activity for the δ receptor. While the compounds with electron-donating 17-substituents showed higher affinities for the δ receptor than those with electron-withdrawing groups, KNT-127 derivatives with 17-fluoroalkyl groups (the high electron-withdrawing groups) showed high selectivities for the δ receptor among evaluated compounds. In addition, the basicity of nitrogen as well as the structure of the 17-N substituent such as the length and configuration at an asymmetric carbon atom contributed to agonist properties for the δ receptor. Thus, the analog with a 17-(3-ethoxypropyl) group showed the best selectively and potent agonistic activity for the δ receptor among KNT-127 derivatives. These findings should be useful for designing novel δ selective agonists.

  13. Platelet-activating factor receptor agonists mediate xeroderma pigmentosum A photosensitivity.

    PubMed

    Yao, Yongxue; Harrison, Kathleen A; Al-Hassani, Mohammed; Murphy, Robert C; Rezania, Samin; Konger, Raymond L; Travers, Jeffrey B

    2012-03-16

    To date, oxidized glycerophosphocholines (Ox-GPCs) with platelet-activating factor (PAF) activity produced non-enzymatically have not been definitively demonstrated to mediate any known disease processes. Here we provide evidence that these Ox-GPCs play a pivotal role in the photosensitivity associated with the deficiency of the DNA repair protein xeroderma pigmentosum type A (XPA). It should be noted that XPA-deficient cells are known to have decreased antioxidant defenses. These studies demonstrate that treatment of human XPA-deficient fibroblasts with the pro-oxidative stressor ultraviolet B (UVB) radiation resulted in increased reactive oxygen species and PAF receptor (PAF-R) agonistic activity in comparison with gene-corrected cells. The UVB irradiation-generated PAF-R agonists were inhibited by antioxidants. UVB irradiation of XPA-deficient (Xpa-/-) mice also resulted in increased PAF-R agonistic activity and skin inflammation in comparison with control mice. The increased UVB irradiation-mediated skin inflammation and TNF-α production in Xpa-/- mice were blocked by systemic antioxidants and by PAF-R antagonists. Structural characterization of PAF-R-stimulating activity in UVB-irradiated XPA-deficient fibroblasts using mass spectrometry revealed increased levels of sn-2 short-chain Ox-GPCs along with native PAF. These studies support a critical role for PAF-R agonistic Ox-GPCs in the pathophysiology of XPA photosensitivity.

  14. Aryl‐hydrocarbon receptor activity modulates prolactin expression in the pituitary

    SciTech Connect

    Moran, Tyler B.; Brannick, Katherine E.; Raetzman, Lori T.

    2012-11-15

    Pituitary tumors account for 15% of intracranial neoplasms, however the extent to which environmental toxicants contribute to the proliferation and hormone expression of pituitary cells is unknown. Aryl-hydrocarbon receptor (AhR) interacting protein (AIP) loss of function mutations cause somatotrope and lactotrope adenomas in humans. AIP sequesters AhR and inhibits its transcriptional function. Because of the link between AIP and pituitary tumors, we hypothesize that exposure to dioxins, potent exogenous ligands for AhR that are persistent in the environment, may predispose to pituitary dysfunction through activation of AhR. In the present study, we examined the effect of AhR activation on proliferation and endogenous pituitary hormone expression in the GH3 rat somatolactotrope tumor cell line and the effect of loss of AhR action in knockout mice. GH3 cells respond to nM doses of the reversible AhR agonist β-naphthoflavone with a robust induction of Cyp1a1. Although mRNA levels of the anti-proliferative signaling cytokine TGFbeta1 are suppressed upon β-naphthoflavone treatment, we did not observe an alteration in cell proliferation. AhR activation with β-naphthoflavone suppresses Ahr expression and impairs expression of prolactin (PRL), but not growth hormone (GH) mRNA in GH3 cells. In mice, loss of Ahr similarly leads to a reduction in Prl mRNA at P3, while Gh is unaffected. Additionally, there is a significant reduction in pituitary hormones Lhb and Fshb in the absence of Ahr. Overall, these results demonstrate that AhR is important for pituitary hormone expression and suggest that environmental dioxins can exert endocrine disrupting effects at the pituitary. -- Highlights: ► AhR signaling suppresses Prl mRNA expression. ► AhR signaling does not influence pituitary proliferation in culture. ► AhR is necessary for Prl, Lhb and Fshb expression at postnatal day 3.

  15. In silico screening for agonists and blockers of the β2 adrenergic receptor: implications of inactive and activated state structures

    PubMed Central

    Costanzi, Stefano; Vilar, Santiago

    2011-01-01

    Ten crystal structures of the β2 adrenergic receptor (β2AR) have been published, reflecting different signaling states. Here, through controlled docking experiments, we examined the implications of using inactive or activated structures on the in silico screening for agonists and blockers of the receptor. Specifically, we targeted the crystal structures solved in complex with carazolol (2RH1), the neutral antagonist alprenalol (3NYA), the irreversible agonist FAUC50 (3PDS) and the full agonist BI-167017 (3P0G). Our results indicate that activated structures favor agonists over blockers while inactive structures favor blockers over agonists. This tendency is more marked for activated than for inactive structures. Additionally, agonists tend to receive more favorable docking scores when docked at activated rather than inactive structures, while blockers do the opposite. Hence, the difference between the docking scores attained with an activated and an inactive structure is an excellent means for the classification of ligands into agonists and blockers, as we determined through receiver operating characteristic (ROC) curves and linear discriminant analysis (LDA). With respect to virtual screening, all structures prioritized well agonists and blockers over non-binders. However, inactive structures worked better for blockers and activated structures worked better for agonists. Notably, the combination of individual docking experiments through receptor ensemble docking (RED) resulted in an excellent performance in the retrieval of both agonists and blockers. Finally, we demonstrated that the induced fit docking of agonists is a viable way of modifying an inactive crystal structure and bias it towards the in silico recognition of agonists rather than blockers. PMID:22170280

  16. Modulation Effect of Peroxisome Proliferator-Activated Receptor Agonists on Lipid Droplet Proteins in Liver

    PubMed Central

    Zhu, Yun-Xia; Zhang, Ming-Liang; Zhong, Yuan; Wang, Chen; Jia, Wei-Ping

    2016-01-01

    Peroxisome proliferator-activated receptor (PPAR) agonists are used for treating hyperglycemia and type 2 diabetes. However, the mechanism of action of these agonists is still under investigation. The lipid droplet-associated proteins FSP27/CIDEC and LSDP5, regulated directly by PPARγ and PPARα, are associated with hepatic steatosis and insulin sensitivity. Here, we evaluated the expression levels of FSP27/CIDEC and LSDP5 and the regulation of these proteins by consumption of a high-fat diet (HFD) or administration of PPAR agonists. Mice with diet-induced obesity were treated with the PPARγ or PPARα agonist, pioglitazone or fenofibrate, respectively. Liver tissues from db/db diabetic mice and human were also collected. Interestingly, FSP27/CIEDC was expressed in mouse and human livers and was upregulated in obese C57BL/6J mice. Fenofibrate treatment decreased hepatic triglyceride (TG) content and FSP27/CIDEC protein expression in mice fed an HFD diet. In mice, LSDP5 was not detected, even in the context of insulin resistance or treatment with PPAR agonists. However, LSDP5 was highly expressed in humans, with elevated expression observed in the fatty liver. We concluded that fenofibrate greatly decreased hepatic TG content and FSP27/CIDEC protein expression in mice fed an HFD, suggesting a potential regulatory role for fenofibrate in the amelioration of hepatic steatosis. PMID:26770990

  17. Potent complement C3a receptor agonists derived from oxazole amino acids: Structure-activity relationships.

    PubMed

    Singh, Ranee; Reed, Anthony N; Chu, Peifei; Scully, Conor C G; Yau, Mei-Kwan; Suen, Jacky Y; Durek, Thomas; Reid, Robert C; Fairlie, David P

    2015-12-01

    Potent ligands for the human complement C3a receptor (C3aR) were developed from the almost inactive tripeptide Leu-Ala-Arg corresponding to the three C-terminal residues of the endogenous peptide agonist C3a. The analogous Leu-Ser-Arg was modified by condensing the serine side chain with the leucine carbonyl with elimination of water to form leucine-oxazole-arginine. Subsequent elaboration with a variety of N-terminal amide capping groups produced agonists as potent as human C3a itself in stimulating Ca(2+) release from human macrophages. Structure-activity relationships are discussed.

  18. Dopamine Agonist Increases Risk Taking but Blunts Reward-Related Brain Activity

    PubMed Central

    Riba, Jordi; Krämer, Ulrike M.; Heldmann, Marcus; Richter, Sylvia; Münte, Thomas F.

    2008-01-01

    The use of D2/D3 dopaminergic agonists in Parkinson's disease (PD) may lead to pathological gambling. In a placebo-controlled double-blind study in healthy volunteers, we observed riskier choices in a lottery task after administration of the D3 receptor-preferring agonist pramipexole thus mimicking risk-taking behavior in PD. Moreover, we demonstrate decreased activation in the rostral basal ganglia and midbrain, key structures of the reward system, following unexpected high gains and therefore propose that pathological gambling in PD results from the need to seek higher rewards to overcome the blunted response in this system. PMID:18575579

  19. Chemical communication in scarab beetles: reciprocal behavioral agonist-antagonist activities of chiral pheromones.

    PubMed Central

    Leal, W S

    1996-01-01

    A novel mechanism of reciprocal behavioral agonist-antagonist activities of enantiomeric pheromones plays a pivotal role in overcoming the signal-to-noise problem derived from the use of a single-constituent pheromone system in scarab beetles. Female Anomala osakana produce (S, Z)-5-(+)-(1-decenyl)oxacyclopentan-2-one, which is highly attractive to males; the response is completely inhibited even by 5% of its antipode. These two enantiomers have reverse roles in the Popillia japonica sex pheromone system. Chiral GC-electroantennographic detector experiments suggest that A. osakana and P. japonica have both R and S receptors that are responsible for behavioral agonist and antagonist responses. PMID:8901541

  20. Induction of rotational behaviour by intranigral baclofen suggests possible GABA-agonist activity.

    PubMed

    Waddington, J L

    1977-10-15

    In rats, unilateral injections of the GABA-derivative baclofen into the zona reticulata of the substantia nigra produced a contralateral rotation that was translated to ipsilateral rotation under the influence of amphetamine. These results mimic those following unilateral elevation of GABA levels in the substantia nigra and suggest that baclofen may have some GABA agonist activity following intracerebral injection.

  1. Accessory Cell Mediated Activation of Porcine NK Cells by TLR7 and TLR8 Agonists

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The induction of innate immune responses by toll-like receptor (TLR) agonists is the subject of intense investigation in many different species. In large part, this reflects the potential of such compounds to be effective vaccine adjuvants. For that reason, we analyzed the activation of innate cells...

  2. NICOTINE EFFECTS ON THE ACTIVITY OF MICE EXPOSED PRENATALLY TO THE NICOTINIC AGONIST ANATOXIN-A.

    EPA Science Inventory

    Considerable research has shown long-lasting effects of early exposure in experimental animals to nicotine. Anatoxin-a is produced by cyanobacteria and has been shown to be a potent nicotinic agonist. This experiment evaluated the motor activity of adult mice, and their respons...

  3. Distinct activities of GABA agonists at synaptic- and extrasynaptic-type GABAA receptors

    PubMed Central

    Mortensen, Martin; Ebert, Bjarke; Wafford, Keith; Smart, Trevor G

    2010-01-01

    The activation characteristics of synaptic and extrasynaptic GABAA receptors are important for shaping the profile of phasic and tonic inhibition in the central nervous system, which will critically impact on the activity of neuronal networks. Here, we study in isolation the activity of three agonists, GABA, muscimol and 4,5,6,7-tetrahydoisoxazolo[5,4-c]pyridin-3(2H)-one (THIP), to further understand the activation profiles of α1β3γ2, α4β3γ2 and α4β3δ receptors that typify synaptic- and extrasynaptic-type receptors expressed in the hippocampus and thalamus. The agonists display an order of potency that is invariant between the three receptors, which is reliant mostly on the agonist dissociation constant. At δ subunit-containing extrasynaptic-type GABAA receptors, both THIP and muscimol additionally exhibited, to different degrees, superagonist behaviour. By comparing whole-cell and single channel currents induced by the agonists, we provide a molecular explanation for their different activation profiles. For THIP at high concentrations, the unusual superagonist behaviour on α4β3δ receptors is a consequence of its ability to increase the duration of longer channel openings and their frequency, resulting in longer burst durations. By contrast, for muscimol, moderate superagonist behaviour was caused by reduced desensitisation of the extrasynaptic-type receptors. The ability to specifically increase the efficacy of receptor activation, by selected exogenous agonists over that obtained with the natural transmitter, may prove to be of therapeutic benefit under circumstances when synaptic inhibition is compromised or dysfunctional. PMID:20176630

  4. Natural product agonists of peroxisome proliferator-activated receptor gamma (PPARγ): a review

    PubMed Central

    Wang, Limei; Waltenberger, Birgit; Pferschy-Wenzig, Eva-Maria; Blunder, Martina; Liu, Xin; Malainer, Clemens; Blazevic, Tina; Schwaiger, Stefan; Rollinger, Judith M.; Heiss, Elke H.; Schuster, Daniela; Kopp, Brigitte; Bauer, Rudolf; Stuppner, Hermann; Dirsch, Verena M.; Atanasov, Atanas G.

    2014-01-01

    Agonists of the nuclear receptor PPARγ are therapeutically used to combat hyperglycaemia associated with the metabolic syndrome and type 2 diabetes. In spite of being effective in normalization of blood glucose levels, the currently used PPARγ agonists from the thiazolidinedione type have serious side effects, making the discovery of novel ligands highly relevant. Natural products have proven historically to be a promising pool of structures for drug discovery, and a significant research effort has recently been undertaken to explore the PPARγ-activating potential of a wide range of natural products originating from traditionally used medicinal plants or dietary sources. The majority of identified compounds are selective PPARγ modulators (SPPARMs), transactivating the expression of PPARγ-dependent reporter genes as partial agonists. Those natural PPARγ ligands have different binding modes to the receptor in comparison to the full thiazolidinedione agonists, and on some occasions activate in addition PPARα (e.g. genistein, biochanin A, sargaquinoic acid, sargahydroquinoic acid, resveratrol, amorphastilbol) or the PPARγ-dimer partner retinoid X receptor (RXR; e.g. the neolignans magnolol and honokiol). A number of in vivo studies suggest that some of the natural product activators of PPARγ (e.g. honokiol, amorfrutin 1, amorfrutin B, amorphastilbol) improve metabolic parameters in diabetic animal models, partly with reduced side effects in comparison to full thiazolidinedione agonists. The bioactivity pattern as well as the dietary use of several of the identified active compounds and plant extracts warrants future research regarding their therapeutic potential and the possibility to modulate PPARγ activation by dietary interventions or food supplements. PMID:25083916

  5. Natural product agonists of peroxisome proliferator-activated receptor gamma (PPARγ): a review.

    PubMed

    Wang, Limei; Waltenberger, Birgit; Pferschy-Wenzig, Eva-Maria; Blunder, Martina; Liu, Xin; Malainer, Clemens; Blazevic, Tina; Schwaiger, Stefan; Rollinger, Judith M; Heiss, Elke H; Schuster, Daniela; Kopp, Brigitte; Bauer, Rudolf; Stuppner, Hermann; Dirsch, Verena M; Atanasov, Atanas G

    2014-11-01

    Agonists of the nuclear receptor PPARγ are therapeutically used to combat hyperglycaemia associated with the metabolic syndrome and type 2 diabetes. In spite of being effective in normalization of blood glucose levels, the currently used PPARγ agonists from the thiazolidinedione type have serious side effects, making the discovery of novel ligands highly relevant. Natural products have proven historically to be a promising pool of structures for drug discovery, and a significant research effort has recently been undertaken to explore the PPARγ-activating potential of a wide range of natural products originating from traditionally used medicinal plants or dietary sources. The majority of identified compounds are selective PPARγ modulators (SPPARMs), transactivating the expression of PPARγ-dependent reporter genes as partial agonists. Those natural PPARγ ligands have different binding modes to the receptor in comparison to the full thiazolidinedione agonists, and on some occasions activate in addition PPARα (e.g. genistein, biochanin A, sargaquinoic acid, sargahydroquinoic acid, resveratrol, amorphastilbol) or the PPARγ-dimer partner retinoid X receptor (RXR; e.g. the neolignans magnolol and honokiol). A number of in vivo studies suggest that some of the natural product activators of PPARγ (e.g. honokiol, amorfrutin 1, amorfrutin B, amorphastilbol) improve metabolic parameters in diabetic animal models, partly with reduced side effects in comparison to full thiazolidinedione agonists. The bioactivity pattern as well as the dietary use of several of the identified active compounds and plant extracts warrants future research regarding their therapeutic potential and the possibility to modulate PPARγ activation by dietary interventions or food supplements.

  6. K+ efflux agonists induce NLRP3 inflammasome activation independently of Ca2+ signaling1

    PubMed Central

    Katsnelson, Michael A.; Rucker, L. Graham; Russo, Hana M.; Dubyak, George R.

    2015-01-01

    Perturbation of intracellular ion homeostasis is a major cellular stress signal for activation of NLRP3 inflammasome signaling that results in caspase-1 mediated production of IL-1β and pyroptosis. However, the relative contributions of decreased cytosolic [K+] versus increased cytosolic [Ca2+] remain disputed and incompletely defined. We investigated roles for elevated cytosolic [Ca2+] in NLRP3 activation and downstream inflammasome signaling responses in primary murine dendritic cells and macrophages in response to two canonical NLRP3 agonists (ATP and nigericin) that facilitate primary K+ efflux by mechanistically distinct pathways or the lysosome-destabilizing agonist Leu-Leu-O-methyl ester (LLME). The study provides three major findings relevant to this unresolved area of NLRP3 regulation. First, increased cytosolic [Ca2+] was neither a necessary nor sufficient signal for the NLRP3 inflammasome cascade during activation by endogenous ATP-gated P2X7 receptor channels, the exogenous bacterial ionophore nigericin, or the lysosomotropic agent LLME. Second, agonists for three Ca2+-mobilizing G protein-coupled receptors (formyl peptide receptor/FPR; P2Y2 purinergic receptor/P2Y2R; calcium-sensing receptor/CaSR) expressed in murine dendritic cells were ineffective as activators of rapidly induced NLRP3 signaling when directly compared to the K+ efflux agonists. Third, the intracellular Ca2+ buffer, BAPTA, and the channel blocker, 2-aminoethoxydiphenyl borate (2-APB), widely used reagents for disruption of Ca2+-dependent signaling pathways, strongly suppressed nigericin-induced NLRP3 inflammasome signaling via mechanisms dissociated from their canonical or expected effects on Ca2+ homeostasis. The results indicate that the ability of K+ efflux agonists to activate NLRP3 inflammasome signaling can be dissociated from changes in cytosolic [Ca2+] as a necessary or sufficient signal. PMID:25762778

  7. In vivo characterization of an AHR-dependent long non-coding RNA required for proper Sox9b expression.

    PubMed

    Garcia, Gloria R; Goodale, Britton C; Wiley, Michelle W; La Du, Jane K; Hendrix, David A; Tanguay, Robert L

    2017-04-06

    Xenobiotic activation of the aryl hydrocarbon receptor (AHR) by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) prevents the proper formation of craniofacial cartilage and the heart in developing zebrafish. Downstream molecular targets responsible for AHR-dependent adverse effects remain largely unknown; however, in zebrafish sox9b has been identified as one of the most reduced transcripts in several target organs and is hypothesized to have a causal role in TCDD-induced toxicity. The reduction of sox9b expression in TCDD-exposed zebrafish embryos has been shown to contribute to heart and jaw malformation phenotypes. The mechanisms by which AHR2 (functional ortholog of mammalian AHR) activation leads to reduced sox9b expression levels and subsequent target organ toxicity are unknown. We have identified a novel long non-coding RNA (slincR) that is upregulated by strong AHR ligands and is located adjacent to the sox9b gene. We hypothesize that slincR is regulated by AHR2 and transcriptionally represses sox9b. The slincR transcript functions as an RNA macromolecule, and slincR expression is AHR2-dependent. Antisense knockdown of slincR results in an increase in sox9b expression during both normal development and AHR2 activation, which suggests a relief in repression. During development, slincR was expressed in tissues with sox9 essential functions, including the jaw/snout region, otic vesicle, eye, and brain. Reducing the levels of slincR resulted in altered neurological and/or locomotor behavioral responses. Our results place slincR as an intermediate between AHR2 activation and the reduction of sox9b mRNA in the AHR2 signaling pathway.

  8. Novel Oxazolidinone-Based Peroxisome Proliferator Activated Receptor Agonists: Molecular Modeling, Synthesis, and Biological Evaluation.

    PubMed

    Fresno, N; Macías-González, M; Torres-Zaguirre, A; Romero-Cuevas, M; Sanz-Camacho, P; Elguero, J; Pavón, F J; Rodríguez de Fonseca, F; Goya, P; Pérez-Fernández, R

    2015-08-27

    A series of new peroxisome proliferator activated receptors (PPARs) chiral ligands have been designed following the accepted three-module structure comprising a polar head, linker, and hydrophobic tail. The majority of the ligands incorporate the oxazolidinone moiety as a novel polar head, and the nature of the hydrophobic tail has also been varied. Docking studies using the crystal structure of an agonist bound to the ligand binding domain of the PPARα receptor have been performed as a tool for their design. Suitable synthetic procedures have been developed, and compounds with different stereochemistries have been prepared. Evaluation of basal and ligand-induced activity proved that several compounds showed agonist activity at the PPARα receptor, thus validating the oxazolidinone template for PPAR activity. In addition, two compounds, 2 and 4, showed dual PPARα/PPARγ agonism and interesting food intake reduction in rats.

  9. An Accessory Agonist Binding Site Promotes Activation of α4β2* Nicotinic Acetylcholine Receptors*

    PubMed Central

    Wang, Jingyi; Kuryatov, Alexander; Sriram, Aarati; Jin, Zhuang; Kamenecka, Theodore M.; Kenny, Paul J.; Lindstrom, Jon

    2015-01-01

    Neuronal nicotinic acetylcholine receptors containing α4, β2, and sometimes other subunits (α4β2* nAChRs) regulate addictive and other behavioral effects of nicotine. These nAChRs exist in several stoichiometries, typically with two high affinity acetylcholine (ACh) binding sites at the interface of α4 and β2 subunits and a fifth accessory subunit. A third low affinity ACh binding site is formed when this accessory subunit is α4 but not if it is β2. Agonists selective for the accessory ACh site, such as 3-[3-(3-pyridyl)-1,2,4-oxadiazol-5-yl]benzonitrile (NS9283), cannot alone activate a nAChR but can facilitate more efficient activation in combination with agonists at the canonical α4β2 sites. We therefore suggest categorizing agonists according to their site selectivity. NS9283 binds to the accessory ACh binding site; thus it is termed an accessory site-selective agonist. We expressed (α4β2)2 concatamers in Xenopus oocytes with free accessory subunits to obtain defined nAChR stoichiometries and α4/accessory subunit interfaces. We show that α2, α3, α4, and α6 accessory subunits can form binding sites for ACh and NS9283 at interfaces with α4 subunits, but β2 and β4 accessory subunits cannot. To permit selective blockage of the accessory site, α4 threonine 126 located on the minus side of α4 that contributes to the accessory site, but not the α4β2 sites, was mutated to cysteine. Alkylation of this cysteine with a thioreactive reagent blocked activity of ACh and NS9283 at the accessory site. Accessory agonist binding sites are promising drug targets. PMID:25869137

  10. Conformational variability of the glycine receptor M2 domain in response to activation by different agonists.

    PubMed

    Pless, Stephan A; Dibas, Mohammed I; Lester, Henry A; Lynch, Joseph W

    2007-12-07

    Models describing the structural changes mediating Cys loop receptor activation generally give little attention to the possibility that different agonists may promote activation via distinct M2 pore-lining domain structural rearrangements. We investigated this question by comparing the effects of different ligands on the conformation of the external portion of the homomeric alpha1 glycine receptor M2 domain. Conformational flexibility was assessed by tethering a rhodamine fluorophore to cysteines introduced at the 19' or 22' positions and monitoring fluorescence and current changes during channel activation. During glycine activation, fluorescence of the label attached to R19'C increased by approximately 20%, and the emission peak shifted to lower wavelengths, consistent with a more hydrophobic fluorophore environment. In contrast, ivermectin activated the receptors without producing a fluorescence change. Although taurine and beta-alanine were weak partial agonists at the alpha1R19'C glycine receptor, they induced large fluorescence changes. Propofol, which drastically enhanced these currents, did not induce a glycine-like blue shift in the spectral emission peak. The inhibitors strychnine and picrotoxin elicited fluorescence and current changes as expected for a competitive antagonist and an open channel blocker, respectively. Glycine and taurine (or beta-alanine) also produced an increase and a decrease, respectively, in the fluorescence of a label attached to the nearby L22'C residue. Thus, results from two separate labeled residues support the conclusion that the glycine receptor M2 domain responds with distinct conformational changes to activation by different agonists.

  11. Dehydroepiandrosterone Derivatives as Potent Antiandrogens With Marginal Agonist Activity

    DTIC Science & Technology

    2011-07-01

    of the molecules, which was only marginally suppressed by the steroid derivatives. Figure 4 . The effects of DHEA derivatives on expression of...DATE (DD-MM-YYYY) 2. REPORT TYPE 3. DATES COVERED (From - To) 4 . TITLE AND SUBTITLE 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM...physiological metabolite from dehydroepiandrosterone ( DHEA ) and a precursor of testosterone, has an intrinsic androgenic activity which was not

  12. Synthesis, activity, and docking study of phenylthiazole acids as potential agonists of PPARγ

    PubMed Central

    Ma, Liang; Wang, Taijin; Shi, Min; Ye, Haoyu

    2016-01-01

    Peroxisome proliferator-activated receptor gamma (PPARγ) is a ligand-mediated transcription factor playing key roles in glucose and lipid homeostasis, and PPARγ ligands possess therapeutic potential in these as well as other areas. In this study, a series of phenylthiazole acids have been synthesized and evaluated for agonistic activity by a convenient fluorescence polarization-based PPARγ ligand screening assay. Compound 4t, as a potential PPARγ agonist with half maximal effective concentration (EC50) 0.75±0.20 μM, exhibited in vitro potency comparable with a 0.83±0.14 μM of the positive control rosiglitazone. Molecular docking and molecular dynamics simulations indicated that phenylthiazole acid 4t interacted with the amino acid residues of the active site of the PPARγ complex in a stable manner, consistent with the result of the in vitro ligand assay. PMID:27313447

  13. Peroxisome proliferator-activated receptor agonists modulate neuropathic pain: a link to chemokines?

    PubMed Central

    Freitag, Caroline M.; Miller, Richard J.

    2014-01-01

    Chronic pain presents a widespread and intractable medical problem. While numerous pharmaceuticals are used to treat chronic pain, drugs that are safe for extended use and highly effective at treating the most severe pain do not yet exist. Chronic pain resulting from nervous system injury (neuropathic pain) is common in conditions ranging from multiple sclerosis to HIV-1 infection to type II diabetes. Inflammation caused by neuropathy is believed to contribute to the generation and maintenance of neuropathic pain. Chemokines are key inflammatory mediators, several of which (MCP-1, RANTES, MIP-1α, fractalkine, SDF-1 among others) have been linked to chronic, neuropathic pain in both human conditions and animal models. The important roles chemokines play in inflammation and pain make them an attractive therapeutic target. Peroxisome proliferator-activated receptors (PPARs) are a family of nuclear receptors known for their roles in metabolism. Recent research has revealed that PPARs also play a role in inflammatory gene repression. PPAR agonists have wide-ranging effects including inhibition of chemokine expression and pain behavior reduction in animal models. Experimental evidence suggests a connection between the pain ameliorating effects of PPAR agonists and suppression of inflammatory gene expression, including chemokines. In early clinical research, one PPARα agonist, palmitoylethanolamide (PEA), shows promise in relieving chronic pain. If this link can be better established, PPAR agonists may represent a new drug therapy for neuropathic pain. PMID:25191225

  14. Dehydroepiandrosterone Derivatives as Potent Antiandrogens with Marginal Agonist Activity

    DTIC Science & Technology

    2015-05-01

    figure not shown). Figure 4. The effects of DHEA derivatives on the expression of PSA (A), AR ( B ), bFGF (C), VEGF (D), IL-6 (E), and MMP-9 ( F ...induced bladder carcinogenesis. Mol Carcinogen 52(2): 94-102, 2013. PMID: 22086872 10. Li Y, Zheng Y, Izumi K, Ishiguro H, Ye B , Li F , Miyamoto H...Ishiguro H, Ye B , Li F , Miyamoto H: Androgen activates β-catenin signaling in bladder cancer cells. Endocr-Relat Cancer 20(3): 293-304, 2013. 11. Izumi K

  15. Dehydroepiandrosterone Derivatives as Potent Antiandrogens with Marginal Agonist Activity

    DTIC Science & Technology

    2014-07-01

    Summary 3. DATES COVERED 1 JUL 2013 - 30 JUN 2014 4 . TITLE AND SUBTITLE Dehydroepiandrosterone Derivatives as Potent Antiandrogens with... 4 Conclusion…………………………………………………………………………… 5 References……………………………………………………………………………. 6 Appendices...metabolite from dehydroepiandrosterone ( DHEA ) and a precursor of testosterone, has an intrinsic androgenic activity which was not completely antagonized by

  16. Agonist antibodies activating the Met receptor protect cardiomyoblasts from cobalt chloride-induced apoptosis and autophagy.

    PubMed

    Gallo, S; Gatti, S; Sala, V; Albano, R; Costelli, P; Casanova, E; Comoglio, P M; Crepaldi, T

    2014-04-17

    Met, the tyrosine kinase receptor for hepatocyte growth factor (HGF), mainly activates prosurvival pathways, including protection from apoptosis. In this work, we investigated the cardioprotective mechanisms of Met activation by agonist monoclonal antibodies (mAbs). Cobalt chloride (CoCl2), a chemical mimetic of hypoxia, was used to induce cardiac damage in H9c2 cardiomyoblasts, which resulted in reduction of cell viability by (i) caspase-dependent apoptosis and (ii) - surprisingly - autophagy. Blocking either apoptosis with the caspase inhibitor benzyloxycarbonyl-VAD-fluoromethylketone or autophagosome formation with 3-methyladenine prevented loss of cell viability, which suggests that both processes contribute to cardiomyoblast injury. Concomitant treatment with Met-activating antibodies or HGF prevented apoptosis and autophagy. Pro-autophagic Redd1, Bnip3 and phospho-AMPK proteins, which are known to promote autophagy through inactivation of the mTOR pathway, were induced by CoCl2. Mechanistically, Met agonist antibodies or HGF prevented the inhibition of mTOR and reduced the flux of autophagosome formation. Accordingly, their anti-autophagic function was completely blunted by Temsirolimus, a specific mTOR inhibitor. Targeted Met activation was successful also in the setting of low oxygen conditions, in which Met agonist antibodies or HGF demonstrated anti-apoptotic and anti-autophagic effects. Activation of the Met pathway is thus a promising novel therapeutic tool for ischaemic injury.

  17. Collybolide is a novel biased agonist of κ-opioid receptors with potent antipruritic activity

    PubMed Central

    Gupta, Achla; Gomes, Ivone; Bobeck, Erin N.; Fakira, Amanda K.; Massaro, Nicholas P.; Sharma, Indrajeet; Cavé, Adrien; Hamm, Heidi E.; Parello, Joseph

    2016-01-01

    Among the opioid receptors, the κ-opioid receptor (κOR) has been gaining considerable attention as a potential therapeutic target for the treatment of complex CNS disorders including depression, visceral pain, and cocaine addiction. With an interest in discovering novel ligands targeting κOR, we searched natural products for unusual scaffolds and identified collybolide (Colly), a nonnitrogenous sesquiterpene from the mushroom Collybia maculata. This compound has a furyl-δ-lactone core similar to that of Salvinorin A (Sal A), another natural product from the plant Salvia divinorum. Characterization of the molecular pharmacological properties reveals that Colly, like Sal A, is a highly potent and selective κOR agonist. However, the two compounds differ in certain signaling and behavioral properties. Colly exhibits 10- to 50-fold higher potency in activating the mitogen-activated protein kinase pathway compared with Sal A. Taken with the fact that the two compounds are equipotent for inhibiting adenylyl cyclase activity, these results suggest that Colly behaves as a biased agonist of κOR. Behavioral studies also support the biased agonistic activity of Colly in that it exhibits ∼10-fold higher potency in blocking non–histamine-mediated itch compared with Sal A, and this difference is not seen in pain attenuation by these two compounds. These results represent a rare example of functional selectivity by two natural products that act on the same receptor. The biased agonistic activity, along with an easily modifiable structure compared with Sal A, makes Colly an ideal candidate for the development of novel therapeutics targeting κOR with reduced side effects. PMID:27162327

  18. In vitro activity of RO363, a beta1-adrenoceptor selective agonist.

    PubMed Central

    Iakovidis, D.; Malta, E.; McPherson, G. A.; Raper, C.

    1980-01-01

    1 The beta-adrenoceptor stimulant effects of RO363 and (--)-isoprenaline have been compared in a variety of isolated tissue preparations. 2 RO363 is approximately half as potent as (--)-isoprenaline in tissues where actions are due to beta1-receptor activation (guinea-pig atrial and ileal preparations and ventricular strips from the rabbit, rat and guinea-pig. 3 In uterine and lung strip preparations from the guinea-pig, where responses are due to beta2-receptor stimulation. RO363 is 100 to 350 times less active than (--)-isoprenaline and has a low intrinsic activity. 4 In spontaneously contracted tracheal preparations from the guinea-pig, RO363 is a full agonist and is approximately half as potent as (--)-isoprenaline. These effects of RO363 are due to the activation of a population of beta1-receptors in the tissue since RO363 and (--)-isoprenaline have the same relative potencies in trachea, cardiac and ileal preparations. In addition the Kb values for practolol are similar in all these preparations when RO363 is used as the agonist. 5 The results show that RO363 is a potent and highly selective beta1-receptor agonist. PMID:6103722

  19. The emerging role of aryl hydrocarbon receptor in the activation and differentiation of Th17 cells.

    PubMed

    Baricza, Eszter; Tamási, Viola; Marton, Nikolett; Buzás, Edit I; Nagy, György

    2016-01-01

    The aryl hydrocarbon receptor (AHR) is a cytoplasmic transcription factor, which plays an essential role in the xenobiotic metabolism in a wide variety of cells. The AHR gene is evolutionarily conserved and it has a central role not only in the differentiation and maturation of many tissues, but also in the toxicological metabolism of the cell by the activation of metabolizing enzymes. Several lines of evidence support that both AHR agonists and antagonists have profound immunological effects; and recently, the AHR has been implicated in antibacterial host defense. According to recent studies, the AHR is essential for the differentiation and activation of T helper 17 (Th17) cells. It is well known that Th17 cells have a central role in the development of inflammation, which is crucial in the defense against pathogens. In addition, Th17 cells play a major role in the pathogenesis of several autoimmune diseases such as rheumatoid arthritis. Therefore, the AHR may provide connection between the environmental chemicals, the immune regulation, and autoimmunity. In the present review, we summarize the role of the AHR in the Th17 cell functions.

  20. A selective TrkB agonist with potent neurotrophic activities by 7,8-dihydroxyflavone

    PubMed Central

    Jang, Sung-Wuk; Liu, Xia; Yepes, Manuel; Shepherd, Kennie R.; Miller, Gary W.; Liu, Yang; Wilson, W. David; Xiao, Ge; Blanchi, Bruno; Sun, Yi E.; Ye, Keqiang

    2010-01-01

    Brain-derived neurotrophic factor (BDNF), a cognate ligand for the tyrosine kinase receptor B (TrkB) receptor, mediates neuronal survival, differentiation, synaptic plasticity, and neurogenesis. However, BDNF has a poor pharmacokinetic profile that limits its therapeutic potential. Here we report the identification of 7,8-dihydroxyflavone as a bioactive high-affinity TrkB agonist that provokes receptor dimerization and autophosphorylation and activation of downstream signaling. 7,8-Dihydroxyflavone protected wild-type, but not TrkB-deficient, neurons from apoptosis. Administration of 7,8-dihydroxyflavone to mice activated TrkB in the brain, inhibited kainic acid-induced toxicity, decreased infarct volumes in stroke in a TrkB-dependent manner, and was neuroprotective in an animal model of Parkinson disease. Thus, 7,8-dihydroxyflavone imitates BDNF and acts as a robust TrkB agonist, providing a powerful therapeutic tool for the treatment of various neurological diseases. PMID:20133810

  1. A Light-Controlled TLR4 Agonist and Selectable Activation of Cell Subpopulations

    PubMed Central

    Stutts, Lalisa

    2015-01-01

    Spatial and temporal aspects of immune cell signalling are key parameters in defining the magnitude of an immune response. Toll-like receptors (TLRs) on innate immune cells are important in early detection of pathogens and initiation of an immune response. Controlling the spatial and temporal signalling of TLRs would enable further study of immune synergies and assist in the development of new vaccines. Here, we show a light-based method for spatial control of TLR4 signalling. A TLR4 agonist, pyrimido[5,4-b]indole, was protected with a cage at a position critical for receptor binding. This afforded a photo-controllable agonist that was inactive while caged, yet effected NF-κB activity in cells following UV photo-controlled deprotection. We demonstrated spatial control of NF-κB activation within a population of cells by treating all cells with the caged TLR4 agonist and constraining light exposure, thereby activation, to a region of interest. PMID:26097006

  2. Number and locations of agonist binding sites required to activate homomeric Cys-loop receptors.

    PubMed

    Rayes, Diego; De Rosa, María José; Sine, Steven M; Bouzat, Cecilia

    2009-05-06

    Homo-pentameric Cys-loop receptors contain five identical agonist binding sites, each formed at a subunit interface. To determine the number and locations of binding sites required to generate a stable active state, we constructed a receptor subunit with a mutation that disables the agonist binding site and a reporter mutation that alters unitary conductance and coexpressed mutant and nonmutant subunits. Although receptors with a range of different subunit compositions are produced, patch-clamp recordings reveal that the amplitude of each single-channel opening event reports the number and, for certain subunit combinations, the locations of subunits with intact binding sites. We find that receptors with three binding sites at nonconsecutive subunit interfaces exhibit maximal mean channel open time, receptors with binding sites at three consecutive or two nonconsecutive interfaces exhibit intermediate open time, and receptors with binding sites at two consecutive or one interface exhibit brief open time. Macroscopic recordings after rapid application of agonist reveal that channel activation slows and the extent of desensitization decreases as the number of binding sites per receptor decreases. The overall results provide a framework for defining mechanisms of activation and drug modulation for homo-pentameric Cys-loop receptors.

  3. β2-Adrenergic receptor agonists activate CFTR in intestinal organoids and subjects with cystic fibrosis.

    PubMed

    Vijftigschild, Lodewijk A W; Berkers, Gitte; Dekkers, Johanna F; Zomer-van Ommen, Domenique D; Matthes, Elizabeth; Kruisselbrink, Evelien; Vonk, Annelotte; Hensen, Chantal E; Heida-Michel, Sabine; Geerdink, Margot; Janssens, Hettie M; van de Graaf, Eduard A; Bronsveld, Inez; de Winter-de Groot, Karin M; Majoor, Christof J; Heijerman, Harry G M; de Jonge, Hugo R; Hanrahan, John W; van der Ent, Cornelis K; Beekman, Jeffrey M

    2016-09-01

    We hypothesized that people with cystic fibrosis (CF) who express CFTR (cystic fibrosis transmembrane conductance regulator) gene mutations associated with residual function may benefit from G-protein coupled receptor (GPCR)-targeting drugs that can activate and enhance CFTR function.We used intestinal organoids to screen a GPCR-modulating compound library and identified β2-adrenergic receptor agonists as the most potent inducers of CFTR function.β2-Agonist-induced organoid swelling correlated with the CFTR genotype, and could be induced in homozygous CFTR-F508del organoids and highly differentiated primary CF airway epithelial cells after rescue of CFTR trafficking by small molecules. The in vivo response to treatment with an oral or inhaled β2-agonist (salbutamol) in CF patients with residual CFTR function was evaluated in a pilot study. 10 subjects with a R117H or A455E mutation were included and showed changes in the nasal potential difference measurement after treatment with oral salbutamol, including a significant improvement of the baseline potential difference of the nasal mucosa (+6.35 mV, p<0.05), suggesting that this treatment might be effective in vivo Furthermore, plasma that was collected after oral salbutamol treatment induced CFTR activation when administered ex vivo to organoids.This proof-of-concept study suggests that organoids can be used to identify drugs that activate CFTR function in vivo and to select route of administration.

  4. Thrombopoietin potentiates agonist-stimulated activation of p38 mitogen-activated protein kinase in human platelets.

    PubMed

    Ezumi, Y; Nishida, E; Uchiyama, T; Takayama, H

    1999-07-22

    Thrombopoietin (TPO) plays a crucial role in megakaryocyte differentiation and platelet production. c-Mpl, a receptor for TPO, is also expressed in terminally differentiated platelets. We investigated the effects of TPO on activation of p38 mitogen-activated protein kinase in human platelets. Thrombin, a thrombin receptor agonist peptide, a thromboxane A(2) analogue, collagen, crosslinking the glycoprotein VI, ADP, and epinephrine, but not phorbol 12, 13-dibutyrate activated p38. TPO did not activate p38 by itself, whereas TPO pretreatment potentiated the agonist-induced activation of p38. TPO did not promote phosphorylation of Hsp27 and cytosolic phospholipase A(2) by itself, but enhanced thrombin-induced phosphorylation of them. The specific p38 inhibitor SB203580 strongly inhibited such phosphorylation. Thus, TPO possesses the priming effect on p38 activation in human platelets and could affect platelet functions through the p38 pathway.

  5. GPR119 Agonist AS1269574 Activates TRPA1 Cation Channels to Stimulate GLP-1 Secretion.

    PubMed

    Chepurny, Oleg G; Holz, George G; Roe, Michael W; Leech, Colin A

    2016-06-01

    GPR119 is a G protein-coupled receptor expressed on intestinal L cells that synthesize and secrete the blood glucose-lowering hormone glucagon-like peptide-1 (GLP-1). GPR119 agonists stimulate the release of GLP-1 from L cells, and for this reason there is interest in their potential use as a new treatment for type 2 diabetes mellitus. AS1269574 is one such GPR119 agonist, and it is the prototype of a series of 2,4,6 trisubstituted pyrimidines that exert positive glucoregulatory actions in mice. Here we report the unexpected finding that AS1269574 stimulates GLP-1 release from the STC-1 intestinal cell line by directly promoting Ca(2+) influx through transient receptor potential ankyrin 1 (TRPA1) cation channels. These GPR119-independent actions of AS1269574 are inhibited by TRPA1 channel blockers (AP-18, A967079, HC030031) and are not secondary to intracellular Ca(2+) release or cAMP production. Patch clamp studies reveal that AS1269574 activates an outwardly rectifying membrane current with properties expected of TRPA1 channels. However, the TRPA1 channel-mediated action of AS1269574 to increase intracellular free calcium concentration is not replicated by GPR119 agonists (AR231453, oleoylethanolamide) unrelated in structure to AS1269574. Using human embryonic kidney-293 cells expressing recombinant rat TRPA1 channels but not GPR119, direct TRPA1 channel activating properties of AS1269574 are validated. Because we find that AS1269574 also acts in a conventional GPR119-mediated manner to stimulate proglucagon gene promoter activity in the GLUTag intestinal L cell line, new findings reported here reveal the surprising capacity of AS1269574 to act as a dual agonist at two molecular targets (GPR119/TRPA1) important to the control of L-cell function and type 2 diabetes mellitus drug discovery research.

  6. Design, synthesis, and functional activity of labeled CD1d glycolipid agonists.

    PubMed

    Jervis, Peter J; Polzella, Paolo; Wojno, Justyna; Jukes, John-Paul; Ghadbane, Hemza; Garcia Diaz, Yoel R; Besra, Gurdyal S; Cerundolo, Vincenzo; Cox, Liam R

    2013-04-17

    Invariant natural killer T cells (iNKT cells) are restricted by CD1d molecules and activated upon CD1d-mediated presentation of glycolipids to T cell receptors (TCRs) located on the surface of the cell. Because the cytokine response profile is governed by the structure of the glycolipid, we sought a method for labeling various glycolipids to study their in vivo behavior. The prototypical CD1d agonist, α-galactosyl ceramide (α-GalCer) 1, instigates a powerful immune response and the generation of a wide range of cytokines when it is presented to iNKT cell TCRs by CD1d molecules. Analysis of crystal structures of the TCR-α-GalCer-CD1d ternary complex identified the α-methylene unit in the fatty acid side chain, and more specifically the pro-S hydrogen at this position, as a site for incorporating a label. We postulated that modifying the glycolipid in this way would exert a minimal impact on the TCR-glycolipid-CD1d ternary complex, allowing the labeled molecule to function as a good mimic for the CD1d agonist under investigation. To test this hypothesis, the synthesis of a biotinylated version of the CD1d agonist threitol ceramide (ThrCer) was targeted. Both diastereoisomers, epimeric at the label tethering site, were prepared, and functional experiments confirmed the importance of substituting the pro-S, and not the pro-R, hydrogen with the label for optimal activity. Significantly, functional experiments revealed that biotinylated ThrCer (S)-10 displayed behavior comparable to that of ThrCer 5 itself and also confirmed that the biotin residue is available for streptavidin and antibiotin antibody recognition. A second CD1d agonist, namely α-GalCer C20:2 4, was modified in a similar way, this time with a fluorescent label. The labeled α-GalCer C20:2 analogue (11) again displayed functional behavior comparable to that of its unlabeled substrate, supporting the notion that the α-methylene unit in the fatty acid amide chain should be a suitable site for attaching

  7. Design, Synthesis, and Functional Activity of Labeled CD1d Glycolipid Agonists

    PubMed Central

    2013-01-01

    Invariant natural killer T cells (iNKT cells) are restricted by CD1d molecules and activated upon CD1d-mediated presentation of glycolipids to T cell receptors (TCRs) located on the surface of the cell. Because the cytokine response profile is governed by the structure of the glycolipid, we sought a method for labeling various glycolipids to study their in vivo behavior. The prototypical CD1d agonist, α-galactosyl ceramide (α-GalCer) 1, instigates a powerful immune response and the generation of a wide range of cytokines when it is presented to iNKT cell TCRs by CD1d molecules. Analysis of crystal structures of the TCR−α-GalCer–CD1d ternary complex identified the α-methylene unit in the fatty acid side chain, and more specifically the pro-S hydrogen at this position, as a site for incorporating a label. We postulated that modifying the glycolipid in this way would exert a minimal impact on the TCR–glycolipid–CD1d ternary complex, allowing the labeled molecule to function as a good mimic for the CD1d agonist under investigation. To test this hypothesis, the synthesis of a biotinylated version of the CD1d agonist threitol ceramide (ThrCer) was targeted. Both diastereoisomers, epimeric at the label tethering site, were prepared, and functional experiments confirmed the importance of substituting the pro-S, and not the pro-R, hydrogen with the label for optimal activity. Significantly, functional experiments revealed that biotinylated ThrCer (S)-10 displayed behavior comparable to that of ThrCer 5 itself and also confirmed that the biotin residue is available for streptavidin and antibiotin antibody recognition. A second CD1d agonist, namely α-GalCer C20:2 4, was modified in a similar way, this time with a fluorescent label. The labeled α-GalCer C20:2 analogue (11) again displayed functional behavior comparable to that of its unlabeled substrate, supporting the notion that the α-methylene unit in the fatty acid amide chain should be a suitable site for

  8. Coal tar induces AHR-dependent skin barrier repair in atopic dermatitis

    PubMed Central

    van den Bogaard, Ellen H.; Bergboer, Judith G.M.; Vonk-Bergers, Mieke; van Vlijmen-Willems, Ivonne M.J.J.; Hato, Stanleyson V.; van der Valk, Pieter G.M.; Schröder, Jens Michael; Joosten, Irma; Zeeuwen, Patrick L.J.M.; Schalkwijk, Joost

    2013-01-01

    Topical application of coal tar is one of the oldest therapies for atopic dermatitis (AD), a T helper 2 (Th2) lymphocyte–mediated skin disease associated with loss-of-function mutations in the skin barrier gene, filaggrin (FLG). Despite its longstanding clinical use and efficacy, the molecular mechanism of coal tar therapy is unknown. Using organotypic skin models with primary keratinocytes from AD patients and controls, we found that coal tar activated the aryl hydrocarbon receptor (AHR), resulting in induction of epidermal differentiation. AHR knockdown by siRNA completely abrogated this effect. Coal tar restored filaggrin expression in FLG-haploinsufficient keratinocytes to wild-type levels, and counteracted Th2 cytokine–mediated downregulation of skin barrier proteins. In AD patients, coal tar completely restored expression of major skin barrier proteins, including filaggrin. Using organotypic skin models stimulated with Th2 cytokines IL-4 and IL-13, we found coal tar to diminish spongiosis, apoptosis, and CCL26 expression, all AD hallmarks. Coal tar interfered with Th2 cytokine signaling via dephosphorylation of STAT6, most likely due to AHR-regulated activation of the NRF2 antioxidative stress pathway. The therapeutic effect of AHR activation herein described opens a new avenue to reconsider AHR as a pharmacological target and could lead to the development of mechanism-based drugs for AD. PMID:23348739

  9. Intrinsic Relative Activities of Opioid Agonists in Activating Gα proteins and Internalizing Receptor: Differences between Human and Mouse Receptors

    PubMed Central

    DiMattio, Kelly M.; Ehlert, Frederick J.; Liu-Chen, Lee-Yuan

    2015-01-01

    Several investigators recently identified biased opioid receptor (KOP receptor) agonists. However, no comprehensive study of the functional selectivity of available KOP receptor agonists at the human and mouse KOP receptors (hKOP receptor and mKOP receptor, respectively) has been published. Here we examined the ability of over 20 KOP receptor agonists to activate G proteins and to internalize the receptor. Clonal neuro-2a mouse neuroblastoma (N2a) cells stably transfected with the hKOP receptor or mKOP receptor were used. We employed agonist-induced [35S]GTPγS binding and KOP receptor internalization as measures of activation of G protein and β-arrestin pathways, respectively. The method of Ehlert and colleagues was used to quantify intrinsic relative activities at G protein activation (RAi−G) and receptor internalization (RAi−I) and the degree of functional selectivity between the two [Log RAi−G − Log RAi−I, RAi−G/RAi−I and bias factor]. The parameter, RAi, represents a relative estimate of agonist affinity for the active receptor state that elicits a given response. The endogenous ligand dynorphin A (1–17) was designated as the balanced ligand with a bias factor of 1. Interestingly, we found that there were species differences in functional selectivity. The most striking differences were for 12-epi-salvinorin A, U69,593, and ICI-199,441. 12-Epi-salvinorin A was highly internalization-biased at the mKOP receptor, but apparently G protein-biased at hKOP receptor. U69,593 was much more internalization-biased at mKOP receptor than hKOP receptor. ICI199,441 showed internalization-biased at the mKOP receptor and G protein-biased at the hKOP receptor. Possible mechanisms for the observed species differences are discussed. PMID:26057692

  10. Quantitative structure-activity studies of octopaminergic agonists and antagonists against nervous system of Locusta migratoria.

    PubMed

    Hirashima, A; Pan, C; Shinkai, K; Tomita, J; Kuwano, E; Taniguchi, E; Eto, M

    1998-07-01

    The quantitative structure activity relationship (QSAR) of octopaminergic agonists and antagonists against the thoracic nerve cord of the migratory locust, Locusta migratoria L., was analyzed using physicochemical parameters and regression analysis. The hydrophobic effect, dipole moment, and shape index were important in terms of Ki: the more hydrophobic, the greater dipole moment, and the smaller shape index of the molecules, the greater the activity. A receptor surface model (RSM) was generated using some subset of the most active structures. Three-dimensional energetics descriptors were calculated from RSM/ligand interaction and these three-dimensional descriptors were used in QSAR analysis. This data set was studied further using molecular shape analysis.

  11. The aryl hydrocarbon receptor (AhR) pathway as a regulatory pathway for cell adhesion and matrix metabolism

    PubMed Central

    Kung, Tiffany; Murphy, K.A.; White, L.A.

    2009-01-01

    The aryl hydrocarbon receptor (AhR) is an orphan receptor in the basic-helix-loop-helix PAS family of transcriptional regulators. Although the endogenous regulator of this pathway has not been identified, the AhR is known to bind and be activated by a variety of compounds ranging from environmental contaminants to flavanoids. The function of this receptor is still unclear; however, animal models indicate that the AhR is important for normal development. One hypothesis is that the AhR senses cellular stress and initiates the cellular response by altering gene expression and inhibiting cell cycle progression and that activation of the AhR by exogenous environmental chemicals results in the dysregulation of this normal function. In this review we will examine the role of the AhR in the regulation of genes and proteins involved in cell adhesion and matrix remodeling, and discuss the implications of these changes in development and disease. In addition, we will discuss evidence suggesting that the AhR pathway is responsive to changes in matrix composition as well as cell-cell and cell-matrix interactions. PMID:18940186

  12. Highly Selective Salicylketoxime-Based Estrogen Receptor β Agonists Display Antiproliferative Activities in a Glioma Model

    PubMed Central

    2016-01-01

    Estrogen receptor β (ERβ) selective agonists are considered potential therapeutic agents for a variety of pathological conditions, including several types of cancer. Their development is particularly challenging, since differences in the ligand binding cavities of the two ER subtypes α and β are minimal. We have carried out a rational design of new salicylketoxime derivatives which display unprecedentedly high levels of ERβ selectivity for this class of compounds, both in binding affinity and in cell-based functional assays. An endogenous gene expression assay was used to further characterize the pharmacological action of these compounds. Finally, these ERβ-selective agonists were found to inhibit proliferation of a glioma cell line in vitro. Most importantly, one of these compounds also proved to be active in an in vivo xenograft model of human glioma, thus demonstrating the high potential of this type of compounds against this devastating disease. PMID:25559213

  13. A Novel Non-Peptidic Agonist of the Ghrelin Receptor with Orexigenic Activity In vivo

    PubMed Central

    Pastor-Cavada, Elena; Pardo, Leticia M.; Kandil, Dalia; Torres-Fuentes, Cristina; Clarke, Sarah L.; Shaban, Hamdy; McGlacken, Gerard P.; Schellekens, Harriet

    2016-01-01

    Loss of appetite in the medically ill and ageing populations is a major health problem and a significant symptom in cachexia syndromes, which is the loss of muscle and fat mass. Ghrelin is a gut-derived hormone which can stimulate appetite. Herein we describe a novel, simple, non-peptidic, 2-pyridone which acts as a selective agonist for the ghrelin receptor (GHS-R1a). The small 2-pyridone demonstrated clear agonistic activity in both transfected human cells and mouse hypothalamic cells with endogenous GHS-R1a receptor expression. In vivo tests with the hit compound showed significant increased food intake following peripheral administration, which highlights the potent orexigenic effect of this novel GHS-R1a receptor ligand. PMID:27819353

  14. A Novel Non-Peptidic Agonist of the Ghrelin Receptor with Orexigenic Activity In vivo

    NASA Astrophysics Data System (ADS)

    Pastor-Cavada, Elena; Pardo, Leticia M.; Kandil, Dalia; Torres-Fuentes, Cristina; Clarke, Sarah L.; Shaban, Hamdy; McGlacken, Gerard P.; Schellekens, Harriet

    2016-11-01

    Loss of appetite in the medically ill and ageing populations is a major health problem and a significant symptom in cachexia syndromes, which is the loss of muscle and fat mass. Ghrelin is a gut-derived hormone which can stimulate appetite. Herein we describe a novel, simple, non-peptidic, 2-pyridone which acts as a selective agonist for the ghrelin receptor (GHS-R1a). The small 2-pyridone demonstrated clear agonistic activity in both transfected human cells and mouse hypothalamic cells with endogenous GHS-R1a receptor expression. In vivo tests with the hit compound showed significant increased food intake following peripheral administration, which highlights the potent orexigenic effect of this novel GHS-R1a receptor ligand.

  15. Anti-hyperglycemic activity of a TGR5 agonist isolated from Olea europaea.

    PubMed

    Sato, Hiroyuki; Genet, Cédric; Strehle, Axelle; Thomas, Charles; Lobstein, Annelise; Wagner, Alain; Mioskowski, Charles; Auwerx, Johan; Saladin, Régis

    2007-11-03

    Olive tree (Olea europeaea) leaves are well known for their effect on metabolism in particular as a traditional anti-diabetic and anti-hypertensive herbal drug. These properties are until now only attributed to oleuropein, the major secoiridoid of olive leaves. Here we describe the isolation and the identification of another constituent implicated in the anti-diabetic effect of this plant, i.e. oleanolic acid. We show that this triterpene is an agonist for TGR5, a member of G-protein coupled receptor activated by bile acids and which mediates some of their various cellular and physiological effect. Oleanolic acid lowers serum glucose and insulin levels in mice fed with a high fat diet and it enhances glucose tolerance. Our data suggest that both oleuropein and oleanolic acid are involved in the anti-diabetic effect of olive leaves and further emphasize the potential role of TGR5 agonists to improve metabolic disorders.

  16. Suppression of cytokine-mediated complement factor gene expression through selective activation of the Ah receptor with 3',4'-dimethoxy-α-naphthoflavone.

    PubMed

    Murray, Iain A; Flaveny, Colin A; Chiaro, Christopher R; Sharma, Arun K; Tanos, Rachel S; Schroeder, Jennifer C; Amin, Shantu G; Bisson, William H; Kolluri, Siva K; Perdew, Gary H

    2011-03-01

    We have characterized previously a class of aryl hydrocarbon receptor (AHR) ligand termed selective AHR modulators (SAhRMs). SAhRMs exhibit anti-inflammatory properties, including suppression of cytokine-mediated acute phase genes (e.g., Saa1), through dissociation of non-dioxin-response element (DRE) AHR activity from DRE-dependent xenobiotic gene expression. The partial AHR agonist α-naphthoflavone (αNF) mediates the suppressive, non-DRE dependent effects on SAA1 expression and partial DRE-mediated CYP1A1 induction. These observations suggest that αNF may be structurally modified to a derivative exhibiting only SAhRM activity. A screen of αNF derivatives identifies 3',4'-dimethoxy-αNF (DiMNF) as a candidate SAhRM. Competitive ligand binding validates DiMNF as an AHR ligand, and DRE-dependent reporter assays with quantitative mRNA analysis of AHR target genes reveal minimal agonist activity associated with AHR binding. Consistent with loss of agonist activity, DiMNF fails to promote AHR binding to DRE probes as determined through electromobility shift assay. Importantly, mRNA analysis indicates that DiMNF retains the suppressive capacity of αNF regarding cytokine-mediated SAA1 expression in Huh7 cells. Interestingly, predictive docking modeling suggests that DiMNF adopts a unique orientation within the AHR ligand binding pocket relative to αNF and may facilitate the rational design of additional SAhRMs. Microarray studies with a non-DRE binding but otherwise functional AHR mutant identified complement factor C3 as a potential SAhRM target. We confirmed this observation in Huh7 cells using 10 μM DiMNF, which significantly repressed C3 mRNA and protein. These data expand the classes of AHR ligands exerting DRE-independent anti-inflammatory SAhRM activity, suggesting SAhRMs may have application in the amelioration of inflammatory disorders.

  17. Thrombin generation by activated factor VII on platelet activated by different agonists. Extending the cell-based model of hemostasis

    PubMed Central

    Altman, Raul; Scazziota, Alejandra Silvia; Herrera, Maria de Lourdes; Gonzalez, Claudio

    2006-01-01

    Background Platelet activation is crucial in normal hemostasis. Using a clotting system free of external tissue factor, we investigated whether activated Factor VII in combination with platelet agonists increased thrombin generation (TG) in vitro. Methods and results TG was quantified by time parameters: lag time (LT) and time to peak (TTP), and by amount of TG: peak of TG (PTG) and area under thrombin formation curve after 35 minutes (AUC→35min) in plasma from 29 healthy volunteers using the calibrated automated thrombography (CAT) technique. TG parameters were measured at basal conditions and after platelet stimulation by sodium arachidonate (AA), ADP, and collagen (Col). In addition, the effects of recombinant activated FVII (rFVIIa) alone or combined with the other platelet agonists on TG parameters were investigated. We found that LT and TTP were significantly decreased (p < 0.05) and PTG and AUC→35min were significantly increased (p < 0.05) in platelet rich plasma activated with AA, ADP, Col, and rFVIIa compared to non-activated platelet rich plasma from normal subjects (p = 0.01). Furthermore platelet rich plasma activated by the combined effects of rFVIIa plus AA, ADP or Col had significantly reduced LT and TTP and increased AUC→35min (but not PTG) when compared to platelet rich plasma activated with agonists in the absence of rFVIIa. Conclusion Platelets activated by AA, ADP, Col or rFVIIa triggered TG. This effect was increased by combining rFVIIa with other agonists. Our intrinsic coagulation system produced a burst in TG independent of external tissue factor activity an apparent hemostatic effect with little thrombotic capacity. Thus we suggest a modification in the cell-based model of hemostasis. PMID:16630353

  18. Partial Agonist and Antagonist Activities of a Mutant Scorpion β-Toxin on Sodium Channels*

    PubMed Central

    Karbat, Izhar; Ilan, Nitza; Zhang, Joel Z.; Cohen, Lior; Kahn, Roy; Benveniste, Morris; Scheuer, Todd; Catterall, William A.; Gordon, Dalia; Gurevitz, Michael

    2010-01-01

    Scorpion β-toxin 4 from Centruroides suffusus suffusus (Css4) enhances the activation of voltage-gated sodium channels through a voltage sensor trapping mechanism by binding the activated state of the voltage sensor in domain II and stabilizing it in its activated conformation. Here we describe the antagonist and partial agonist properties of a mutant derivative of this toxin. Substitution of seven different amino acid residues for Glu15 in Css4 yielded toxin derivatives with both increased and decreased affinities for binding to neurotoxin receptor site 4 on sodium channels. Css4E15R is unique among this set of mutants in that it retained nearly normal binding affinity but lost its functional activity for modification of sodium channel gating in our standard electrophysiological assay for voltage sensor trapping. More detailed analysis of the functional effects of Css4E15R revealed weak voltage sensor trapping activity, which was very rapidly reversed upon repolarization and therefore was not observed in our standard assay of toxin effects. This partial agonist activity of Css4E15R is observed clearly in voltage sensor trapping assays with brief (5 ms) repolarization between the conditioning prepulse and the test pulse. The effects of Css4E15R are fit well by a three-step model of toxin action involving concentration-dependent toxin binding to its receptor site followed by depolarization-dependent activation of the voltage sensor and subsequent voltage sensor trapping. Because it is a partial agonist with much reduced efficacy for voltage sensor trapping, Css4E15R can antagonize the effects of wild-type Css4 on sodium channel activation and can prevent paralysis by Css4 when injected into mice. Our results define the first partial agonist and antagonist activities for scorpion toxins and open new avenues of research toward better understanding of the structure-function relationships for toxin action on sodium channel voltage sensors and toward potential toxin

  19. Partial agonist and antagonist activities of a mutant scorpion beta-toxin on sodium channels.

    PubMed

    Karbat, Izhar; Ilan, Nitza; Zhang, Joel Z; Cohen, Lior; Kahn, Roy; Benveniste, Morris; Scheuer, Todd; Catterall, William A; Gordon, Dalia; Gurevitz, Michael

    2010-10-01

    Scorpion β-toxin 4 from Centruroides suffusus suffusus (Css4) enhances the activation of voltage-gated sodium channels through a voltage sensor trapping mechanism by binding the activated state of the voltage sensor in domain II and stabilizing it in its activated conformation. Here we describe the antagonist and partial agonist properties of a mutant derivative of this toxin. Substitution of seven different amino acid residues for Glu(15) in Css4 yielded toxin derivatives with both increased and decreased affinities for binding to neurotoxin receptor site 4 on sodium channels. Css4(E15R) is unique among this set of mutants in that it retained nearly normal binding affinity but lost its functional activity for modification of sodium channel gating in our standard electrophysiological assay for voltage sensor trapping. More detailed analysis of the functional effects of Css4(E15R) revealed weak voltage sensor trapping activity, which was very rapidly reversed upon repolarization and therefore was not observed in our standard assay of toxin effects. This partial agonist activity of Css4(E15R) is observed clearly in voltage sensor trapping assays with brief (5 ms) repolarization between the conditioning prepulse and the test pulse. The effects of Css4(E15R) are fit well by a three-step model of toxin action involving concentration-dependent toxin binding to its receptor site followed by depolarization-dependent activation of the voltage sensor and subsequent voltage sensor trapping. Because it is a partial agonist with much reduced efficacy for voltage sensor trapping, Css4(E15R) can antagonize the effects of wild-type Css4 on sodium channel activation and can prevent paralysis by Css4 when injected into mice. Our results define the first partial agonist and antagonist activities for scorpion toxins and open new avenues of research toward better understanding of the structure-function relationships for toxin action on sodium channel voltage sensors and toward

  20. Cancer-promoting and Inhibiting Effects of Dietary Compounds: Role of the Aryl Hydrocarbon Receptor (AhR)

    PubMed Central

    Powell, Joann B.; Ghotbaddini, Maryam

    2014-01-01

    Polyaromatic hydrocarbons, heterocyclic aromatic amines and dioxin-like compounds are environmental carcinogens shown to initiate cancer in a number of tissue types including prostate and breast. These environmental carcinogens elicit their effects through interacting with the aryl hydrocarbon receptor (AhR), a ligand activated transcription factor. Naturally occurring compounds found in fruits and vegetables shown to have anti-carcinogenic effects also interact with the AhR. This review explores dietary and environmental exposure to chemical carcinogens and beneficial natural compounds whose effects are elicited by the AhR. PMID:25258701

  1. DDE and PCB 153 independently induce aryl hydrocarbon receptor (AhR) expression in peripheral blood mononuclear cells.

    PubMed

    Gaspar-Ramírez, Octavio; Pérez-Vázquez, Francisco J; Salgado-Bustamante, Mariana; González-Amaro, Roberto; Hernandez-Castro, Berenice; Pérez-Maldonado, Ivan N

    2015-01-01

    Recent studies have demonstrated that compounds inducing pro-inflammatory cytokines enhance AhR expression. The aim of this study was 2-fold: (1) to determine if two pro-inflammatory compounds, dichlorodiphenyldichloroethylene (DDE) and 2,2',4,4',5,5'-hexa-chlorobiphenyl (PCB 153), independently affect AhR gene expression in peripheral blood mononuclear cells (PBMC); and (2) if affected, to determine whether the mechanism involved was due to AhR activation or to a pro-inflammatory effect of the chemicals. PBMC isolated from healthy individuals were incubated in the presence of DDE (10 µg/ml) and PCB 153 (20 ng/ml) over time and AhR and CYP1A1 expression was assessed with a real-time PCR technique. The results indicated there was over-expression of the AhR mRNA in PBMC when the cells were treated with DDE and PCB 153. No changes in expression levels of CYP1A1 mRNA were found. Importantly, when the cells were exposed to DDE and PCB 153 in the presence of an antagonist of tumor necrosis factor (TNF)-α, the over-expression of AhR was abolished; as expected, the expression of CYP1A1 was unaffected. In conclusion, these studies demonstrated for the first time an increment of AhR expression "in vitro" in PBMC treated with two pro-inflammatory environmental pollutants, DDE and PCB153. Moreover, the over-expression of AhR was dependent of TNFα induced by DDE and PCB 153 and was independent of AhR activation.

  2. Airway Peroxidases Catalyze Nitration of the β2-Agonist Salbutamol and Decrease Its Pharmacological Activity

    PubMed Central

    Sallans, Larry; Macha, Stephen; Brown, Kari; McGraw, Dennis W.; Kovacic, Melinda Butsch; Britigan, Bradley E.

    2011-01-01

    β2-Agonists are the most effective bronchodilators for the rapid relief of asthma symptoms, but for unclear reasons, their effectiveness may be decreased during severe exacerbations. Because peroxidase activity and nitrogen oxides are increased in the asthmatic airway, we examined whether salbutamol, a clinically important β2-agonist, is subject to potentially inactivating nitration. When salbutamol was exposed to myeloperoxidase, eosinophil peroxidase or lactoperoxidase in the presence of hydrogen peroxide (H2O2) and nitrite (NO2−), both absorption spectroscopy and mass spectrometry indicated formation of a new metabolite with features expected for the nitrated drug. The new metabolites showed an absorption maximum at 410 nm and pKa of 6.6 of the phenolic hydroxyl group. In addition to nitrosalbutamol (m/z 285.14), a salbutamol-derived nitrophenol, formed by elimination of the formaldehyde group, was detected (m/z 255.13) by mass spectrometry. It is noteworthy that the latter metabolite was detected in exhaled breath condensates of asthma patients receiving salbutamol but not in unexposed control subjects, indicating the potential for β2-agonist nitration to occur in the inflamed airway in vivo. Salbutamol nitration was inhibited in vitro by ascorbate, thiocyanate, and the pharmacological agents methimazole and dapsone. The efficacy of inhibition depended on the nitrating system, with the lactoperoxidase/H2O2/NO2− being the most affected. Functionally, nitrated salbutamol showed decreased affinity for β2-adrenergic receptors and impaired cAMP synthesis in airway smooth muscle cells compared with the native drug. These results suggest that under inflammatory conditions associated with asthma, phenolic β2-agonists may be subject to peroxidase-catalyzed nitration that could potentially diminish their therapeutic efficacy. PMID:20974700

  3. Insights into the structural basis of endogenous agonist activation of family B G protein-coupled receptors.

    PubMed

    Dong, Maoqing; Gao, Fan; Pinon, Delia I; Miller, Laurence J

    2008-06-01

    Agonist drugs targeting the glucagon-like peptide-1 (GLP1) receptor represent important additions to the clinical management of patients with diabetes mellitus. In the current report, we have explored whether the recently described concept of a receptor-active endogenous agonist sequence within the amino terminus of the secretin receptor may also be applicable to the GLP1 receptor. If so, this could provide a lead for the development of additional small molecule agonists targeting this and other important family members. Indeed, the region of the GLP1 receptor analogous to that containing the active WDN within the secretin receptor was found to possess full agonist activity at the GLP1 receptor. The minimal fragment within this region that had full agonist activity was NRTFD. Despite having no primary sequence identity with the WDN, it was also active at the secretin receptor, where it had similar potency and efficacy to WDN, suggesting common structural features. Molecular modeling demonstrated that an intradomain salt bridge between the side chains of arginine and aspartate could yield similarities in structure with cyclic WDN. This directly supports the relevance of the endogenous agonist concept to the GLP1 receptor and provides new insights into the rational development and refinement of new types of drugs activating this important receptor.

  4. Cholinergic and glutamatergic agonists induce gamma frequency activity in dorsal subcoeruleus nucleus neurons

    PubMed Central

    Simon, Christen; Kezunovic, Nebojsa; Williams, D. Keith; Urbano, Francisco J.

    2011-01-01

    The dorsal subcoeruleus nucleus (SubCD) is involved in generating two signs of rapid eye movement (REM) sleep: muscle atonia and ponto-geniculo-occipital (PGO) waves. We tested the hypothesis that single cell and/or population responses of SubCD neurons are capable of generating gamma frequency activity in response to intracellular stimulation or receptor agonist activation. Whole cell patch clamp recordings (immersion chamber) and population responses (interface chamber) were conducted on 9- to 20-day-old rat brain stem slices. All SubCD neurons (n = 103) fired at gamma frequency when subjected to depolarizing steps. Two statistically distinct populations of neurons were observed, which were distinguished by their high (>80 Hz, n = 24) versus low (35–80 Hz, n = 16) initial firing frequencies. Both cell types exhibited subthreshold oscillations in the gamma range (n = 43), which may underlie the gamma band firing properties of these neurons. The subthreshold oscillations were blocked by the sodium channel blockers tetrodotoxin (TTX, n = 21) extracellularly and N-(2,6-dimethylphenylcarbamoylmethyl)triethylammonium bromide (QX-314) intracellularly (n = 5), indicating they were sodium channel dependent. Gamma frequency subthreshold oscillations were observed in response to the nonspecific cholinergic receptor agonist carbachol (CAR, n = 11, d = 1.08) and the glutamate receptor agonists N-methyl-d-aspartic acid (NMDA, n = 12, d = 1.09) and kainic acid (KA, n = 13, d = 0.96), indicating that cholinergic and glutamatergic inputs may be involved in the activation of these subthreshold currents. Gamma band activity also was observed in population responses following application of CAR (n = 4, P < 0.05), NMDA (n = 4, P < 0.05) and KA (n = 4, P < 0.05). Voltage-sensitive, sodium channel-dependent gamma band activity appears to be a part of the intrinsic membrane properties of SubCD neurons. PMID:21543743

  5. The characterization of a novel rigid nicotine analog with alpha7-selective nAChR agonist activity and modulation of agonist properties by boron inclusion.

    PubMed

    Papke, Roger L; Zheng, Guangrong; Horenstein, Nicole A; Dwoskin, Linda P; Crooks, Peter A

    2005-09-01

    The alpha7 nAChR subtype is of particular interest as a potential therapeutic target since it has been implicated as a mediator of both cognitive and neuroprotective activity. The rigid nicotine analog ACME and the N-cyanoborane conjugate ACME-B are selective partial agonists of rat alpha7 receptors expressed in Xenopus oocytes, with no significant activation of either alpha3beta4 or alpha4beta2 receptors. ACME-B is both more potent and efficacious than ACME. The efficacies of ACME-B and ACME are approximately 26% and 10% of the efficacy of ACh, respectively. Similar N-conjugation of S(-)nicotine with cyanoborane decreased efficacy for alpha3beta4 and alpha4beta2 receptors, as well as for alpha7 nAChR. Structural comparison of ACME with the benzylidene anabaseines, another class of previously identified alpha7-selective agonists, suggests that they share a similar structural motif that may be applicable to other alpha7-selective agonists.

  6. Structure-Activity Relationships of the Sustained Effects of Adenosine A2A Receptor Agonists Driven by Slow Dissociation Kinetics

    PubMed Central

    Hothersall, J. Daniel; Guo, Dong; Sarda, Sunil; Sheppard, Robert J.; Chen, Hongming; Keur, Wesley; Waring, Michael J.; IJzerman, Adriaan P.; Hill, Stephen J.; Dale, Ian L.

    2017-01-01

    The duration of action of adenosine A2A receptor (A2A) agonists is critical for their clinical efficacy, and we sought to better understand how this can be optimized. The in vitro temporal response profiles of a panel of A2A agonists were studied using cAMP assays in recombinantly (CHO) and endogenously (SH-SY5Y) expressing cells. Some agonists (e.g., 3cd; UK-432,097) but not others (e.g., 3ac; CGS-21680) demonstrated sustained wash-resistant agonism, where residual receptor activation continued after washout. The ability of an antagonist to reverse pre-established agonist responses was used as a surrogate read-out for agonist dissociation kinetics, and together with radioligand binding studies suggested a role for slow off-rate in driving sustained effects. One compound, 3ch, showed particularly marked sustained effects, with a reversal t1/2 > 6 hours and close to maximal effects that remained for at least 5 hours after washing. Based on the structure-activity relationship of these compounds, we suggest that lipophilic N6 and bulky C2 substituents can promote stable and long-lived binding events leading to sustained agonist responses, although a high compound logD is not necessary. This provides new insight into the binding interactions of these ligands and we anticipate that this information could facilitate the rational design of novel long-acting A2A agonists with improved clinical efficacy. PMID:27803241

  7. AIP mutations impair AhR signaling in pituitary adenoma patients fibroblasts and in GH3 cells.

    PubMed

    Lecoq, Anne-Lise; Viengchareun, Say; Hage, Mirella; Bouligand, Jérôme; Young, Jacques; Boutron, Audrey; Zizzari, Philippe; Lombès, Marc; Chanson, Philippe; Kamenický, Peter

    2016-05-01

    Germline mutations in the aryl hydrocarbon receptor-interacting protein (AIP) gene predispose humans to pituitary adenomas through unknown molecular mechanisms. The best-known interacting partner of AIP is the aryl hydrocarbon receptor (AhR), a transcription factor that mediates the effects of xenobiotics implicated in carcinogenesis. As 75% of AIP mutations disrupt the physical and/or functional interaction with AhR, we postulated that the tumorigenic potential of AIP mutations might result from altered AhR signaling. We evaluated the impact of AIP mutations on the AhR signaling pathway, first in fibroblasts from AIP-mutated patients with pituitary adenomas, by comparison with fibroblasts from healthy subjects, then in transfected pituitary GH3 cells. The AIP protein level in mutated fibroblasts was about half of that in cells from healthy subjects, but AhR expression was unaffected. Gene expression analyses showed significant modifications in the expression of the AhR target genes CYP1B1 and AHRR in AIP-mutated fibroblasts, both before and after stimulation with the endogenous AhR ligand kynurenine. Kynurenine increased Cyp1b1 expression to a greater extent in GH3 cells overexpressing wild type compared with cells expressing mutant AIP Knockdown of endogenous Aip in these cells attenuated Cyp1b1 induction by the AhR ligand. Both mutant AIP expression and knockdown of endogenous Aip affected the kynurenine-dependent GH secretion of GH3 cells. This study of human fibroblasts bearing endogenous heterozygous AIP mutations and transfected pituitary GH3 cells shows that AIP mutations affect the AIP protein level and alter AhR transcriptional activity in a gene- and tissue-dependent manner.

  8. Activation of Cyclic AMP Synthesis by Full and Partial Beta-Adrenergic Receptor Agonists in Chicken Skeletal Muscle Cells

    NASA Technical Reports Server (NTRS)

    Young, R. B.; Bridge, K. Y.

    2003-01-01

    Several beta-adrenergic receptor (bAR) agonists are known to cause hypertrophy of skeletal muscle tissue. Accordingly, five bAR agonists encompassing a range in activity from strong to weak were evaluated for their ability to stimulate CAMP accumulation in embryonic chicken skeletal muscle cells in culture. Two strong agonists (epinephrine and isoproterenol), one moderate agonist (albuterol), and two weak agonists known to cause hypertrophy in animals (clenbuterol and cimaterol) were studied. Dose response curves were determined over six orders of magnitude in concentration for each agonist, and values were determined for their maximum stimulation of CAMP synthesis rate (Bmax) and the agonist concentration at which 50% stimulation of CAMP synthesis (EC50) occurred. Bmax values decreased in the following order: isoproterenol, epinephrine, albuterol, cimaterol, clenbuterol. Cimaterol and clenbuterol at their Bmax concentrations were approximately 15-fold weaker than isoproterenol in stimulating the rate of CAMP synthesis. When cimaterol and clenbuterol were added to culture media at concentrations known to cause significant muscle hypertrophy in animals, there was no detectable effect on stimulation of CAMP synthesis. Finally, these same levels of cimaterol and clenbuterol did not antagonize the stimulation of CAMP by either epinephrine or isoproterenol.

  9. Activation of Cyclic AMP Synthesis by Full and Partial Beta-Adrenergic Receptor Agonists in Chicken Skeletal Muscle Cells

    NASA Technical Reports Server (NTRS)

    Young, R. B.; Bridge, K. Y.; Cureri, Peter A. (Technical Monitor)

    2002-01-01

    Several beta-adrenergic receptor (bAR) agonists are known to cause hypertrophy of skeletal muscle tissue. Accordingly, five bAR agonists encompassing a range in activity from strong to weak were evaluated for their ability to stimulate cAMP accumulation in embryonic chicken skeletal muscle cells in culture. Two strong agonists (epinephrine and isoproterenol), one moderate agonist (albuterol), and two weak agonists known to cause hypertrophy in animals (clenbuterol and cimaterol) were studied. Dose response curves were determined over six orders of magnitude in concentration for each agonist, and values were determined for their maximum stimulation of cAMP synthesis rate (Bmax) and the agonist concentration at which 50% stimulation of cAMP synthesis (EC50) occurred. Bmax values decreased in the following order: isoproterenol, epinephrine, albuterol, cimaterol, clenbuterol. Cimaterol and clenbuterol at their Bmax concentrations were approximately 15-fold weaker than isoproterenol in stimulating the rate of cAMP synthesis. When cimaterol and clenbuterol were added to culture media at concentrations known to cause significant muscle hypertrophy in animals, there was no detectable effect on stimulation of cAMP synthesis. Finally, these same levels of cimaterol and clenbuterol did not antagonize the stimulation of cAMP by either epinephrine or isoproterenol.

  10. Reduction of vitellogenin synthesis by an aryl hydrocarbon receptor agonist in the white sturgeon (Acipenser transmontamus).

    PubMed

    Palumbo, Amanda J; Denison, Michael S; Doroshov, Serge I; Tjeerdema, Ronald S

    2009-08-01

    Migrating white sturgeon (Acipenser transmontamus) may be subject to agricultural, municipal, and industrial wastewater effluents that likely contain different classes of endocrine-disrupting contaminants. Concern is mounting about the negative effects of environmental estrogens on fish reproduction; however, in environmental mixtures, the affects from estrogenic compounds may be suppressed by aryl hydrocarbon receptor (AhR) ligands. Indeed, reductions in 17beta-estradiol-induced (0.01 and 1 mg/kg) vitellogenin (VTG) levels were observed in white sturgeon coinjected with beta-naphthoflavone (BNF; 50 mg/kg), a model for contaminants that activate the AhR. Variation in the time of injection was used to attempt to correlate VTG inhibition to ethoxyresorufin-O-deethylase activity. No evidence was found to suggest that the inhibition of VTG is a direct result of enhanced estrogen metabolism by BNF-induced enzymes. Results of the present study are relevant for monitoring programs that measure VTG, because these results show that AhR-active environmental contaminants can repress VTG synthesis, which commonly is used as an indicator of estrogen-mimicking contaminants. Furthermore, suppression of natural estrogen signaling by AhR agonists may have significant effects on fish reproduction.

  11. Human and rodent aryl hydrocarbon receptor (AHR): from mediator of dioxin toxicity to physiologic AHR functions and therapeutic options.

    PubMed

    Bock, Karl Walter

    2017-04-01

    Metabolism of aryl hydrocarbons and toxicity of dioxins led to the discovery of the aryl hydrocarbon receptor (AHR). Tremendous advances have been made on multiplicity of AHR signaling and identification of endogenous ligands including the tryptophan metabolites FICZ and kynurenine. However, human AHR functions are still poorly understood due to marked species differences as well as cell-type- and cell context-dependent AHR functions. Observations in dioxin-poisoned individuals may provide hints to physiologic AHR functions in humans. Based on these observations three human AHR functions are discussed: (1) Chemical defence and homeostasis of endobiotics. The AHR variant Val381 in modern humans leads to reduced AHR affinity to aryl hydrocarbons in comparison with Neanderthals and primates expressing the Ala381 variant while affinity to indoles remains unimpaired. (2) Homeostasis of stem/progenitor cells. Dioxins dysregulate homeostasis in sebocyte stem cells. (3) Modulation of immunity. In addition to microbial defence, AHR may be involved in a 'disease tolerance defence pathway'. Further characterization of physiologic AHR functions may lead to therapeutic options.

  12. Hydroxy monounsaturated fatty acids as agonists for peroxisome proliferator-activated receptors.

    PubMed

    Yokoi, Hiroshi; Mizukami, Hajime; Nagatsu, Akito; Tanabe, Hiroki; Inoue, Makoto

    2010-01-01

    The physiological and pathological role of oxidized polyunsaturated fatty acids (PUFAs) has been extensively studied, whereas those of hydroxy monounsaturated fatty acids (MUFAs) are not well understood. This study demonstrated that 11-hydroxy-(9Z)-octadecenoic acid ((9Z)-11-HOE), which was isolated from adlay seeds (Coix lacryma-jobi L. var. ma-yuen STAF.), can activate peroxisome proliferator-activated receptor (PPAR)alpha, delta and gamma in luciferase reporter assays more efficiently than (9Z)-octadecenoic acid (oleic acid), and to the same degree as linoleic acid. (9Z)-11-HOE increased the mRNA levels of UCP2 and CD36 in C2C12 myotubes and THP- 1 cells, respectively, and these effects were blocked by the PPARdelta- and gamma-specific antagonists GSK0660 and T0070907, respectively. Evaluation of the structure.activity relationship between hydroxy MUFAs and PPAR activation revealed that (9E)-11-HOE, the geometrical isomer of (9Z)-11-HOE, activated PPARs more potently than (9Z)-11-HOE, and that PPAR activation by hydroxyl MUFAs was not markedly influenced by the position of the hydroxy group or the double bond, although PPARdelta seemed to possess ligand specificity different to that of PPARalpha or gamma . Additionally, the finding that 11-hydroxy octadecanoic acid, the hydrogenated product of (9E)-11- HOE, was also capable of activating PPARs to a similar extent as (9E)-11-HOE indicates that the double bond in hydroxy MUFAs is not essential for PPAR activation. In conclusion, (9Z)-11-HOE derived from alday seeds and hydroxy MUFAs with a chain length of 16 or 18 acted as PPAR agonists. Hydroxylation of MUFAs may change these compounds from silent PPAR ligands to active PPAR agonists.

  13. Agonist and antagonist muscle activation during maximal and submaximal isokinetic fatigue tests of the knee extensors.

    PubMed

    Hassani, A; Patikas, D; Bassa, E; Hatzikotoulas, K; Kellis, E; Kotzamanidis, C

    2006-12-01

    The purpose of this study was to examine the differences in electromyographic activity of agonist and antagonist knee musculature between a maximal and a submaximal isokinetic fatigue protocol. Fourteen healthy males (age: 24.3+/-2.5 years) performed 25 maximal (MIFP) and 60 submaximal (SIFP) isokinetic concentric efforts of the knee extensors at 60 degrees s(-1), across a 90 degrees range of motion. The two protocols were performed a week apart. The EMG activity of vastus medialis (VM), vastus lateralis (VL) and biceps femoris (BF) were recorded using surface electrodes. The peak torque (PT) and average EMG (aEMG) were expressed as percentages of pre-fatigue maximal value. One-way analysis of variance indicated a significant (p<0.05) decline of PT during the maximal (45.7%) and submaximal (46.8%) protocols. During the maximal test, the VM and VL aEMG initially increased and then decreased. In contrast, VM and VL aEMG continuously increased during submaximal testing (p<0.05). The antagonist (BF) aEMG remained constant during maximal test but it increased significantly and then declined during the submaximal testing. The above results indicate that agonist and antagonist activity depends on the intensity of the selected isokinetic fatigue test.

  14. Synthesis and SAR studies of benzyl ether derivatives as potent orally active S1P₁ agonists.

    PubMed

    Tsuji, Takashi; Suzuki, Keisuke; Nakamura, Tsuyoshi; Goto, Taiji; Sekiguchi, Yukiko; Ikeda, Takuya; Fukuda, Takeshi; Takemoto, Toshiyasu; Mizuno, Yumiko; Kimura, Takako; Kawase, Yumi; Nara, Futoshi; Kagari, Takashi; Shimozato, Takaichi; Yahara, Chizuko; Inaba, Shinichi; Honda, Tomohiro; Izumi, Takashi; Tamura, Masakazu; Nishi, Takahide

    2014-08-01

    We report herein the synthesis and structure-activity relationships (SAR) of a series of benzyl ether compounds as an S1P₁ receptor modulator. From our SAR studies, the installation of substituents onto the central benzene ring of 2a was revealed to potently influence the S1P₁ and S1P₃ agonistic activities, in particular, an ethyl group on the 2-position afforded satisfactory S1P₁/S1P₃ selectivity. These changes of the S1P₁ and S1P₃ agonistic activities caused by the alteration of substituents on the 2-position were reasonably explained by a docking study using an S1P₁ X-ray crystal structure and S1P₃ homology modeling. We found that compounds 2b and 2e had a potent in vivo immunosuppressive efficacy along with acceptable S1P₁/S1P₃ selectivity, and confirmed that these compounds had less in vivo bradycardia risk through the evaluation of heart rate change after oral administration of the compounds (30 mg/kg, p.o.) in rats.

  15. Dioxin induces Ahr-dependent robust DNA demethylation of the Cyp1a1 promoter via Tdg in the mouse liver

    NASA Astrophysics Data System (ADS)

    Amenya, Hesbon Z.; Tohyama, Chiharu; Ohsako, Seiichiroh

    2016-10-01

    The aryl hydrocarbon receptor (Ahr) is a highly conserved nuclear receptor that plays an important role in the manifestation of toxicity induced by polycyclic aromatic hydrocarbons. As a xenobiotic sensor, Ahr is involved in chemical biotransformation through activation of drug metabolizing enzymes. The activated Ahr cooperates with coactivator complexes to induce epigenetic modifications at target genes. Thus, it is conceivable that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a potent Ahr ligand, may elicit robust epigenetic changes in vivo at the Ahr target gene cytochrome P450 1a1 (Cyp1a1). A single dose of TCDD administered to adult mice induced Ahr-dependent CpG hypomethylation, changes in histone modifications, and thymine DNA glycosylase (Tdg) recruitment at the Cyp1a1 promoter in the liver within 24 hrs. These epigenetic changes persisted until 40 days post-TCDD treatment and there was Cyp1a1 mRNA hyperinduction upon repeat administration of TCDD at this time-point. Our demethylation assay using siRNA knockdown and an in vitro methylated plasmid showed that Ahr, Tdg, and the ten-eleven translocation methyldioxygenases Tet2 and Tet3 are required for the TCDD-induced DNA demethylation. These results provide novel evidence of Ahr-driven active DNA demethylation and epigenetic memory. The epigenetic alterations influence response to subsequent chemical exposure and imply an adaptive mechanism to xenobiotic stress.

  16. Dioxin induces Ahr-dependent robust DNA demethylation of the Cyp1a1 promoter via Tdg in the mouse liver

    PubMed Central

    Amenya, Hesbon Z.; Tohyama, Chiharu; Ohsako, Seiichiroh

    2016-01-01

    The aryl hydrocarbon receptor (Ahr) is a highly conserved nuclear receptor that plays an important role in the manifestation of toxicity induced by polycyclic aromatic hydrocarbons. As a xenobiotic sensor, Ahr is involved in chemical biotransformation through activation of drug metabolizing enzymes. The activated Ahr cooperates with coactivator complexes to induce epigenetic modifications at target genes. Thus, it is conceivable that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a potent Ahr ligand, may elicit robust epigenetic changes in vivo at the Ahr target gene cytochrome P450 1a1 (Cyp1a1). A single dose of TCDD administered to adult mice induced Ahr-dependent CpG hypomethylation, changes in histone modifications, and thymine DNA glycosylase (Tdg) recruitment at the Cyp1a1 promoter in the liver within 24 hrs. These epigenetic changes persisted until 40 days post-TCDD treatment and there was Cyp1a1 mRNA hyperinduction upon repeat administration of TCDD at this time-point. Our demethylation assay using siRNA knockdown and an in vitro methylated plasmid showed that Ahr, Tdg, and the ten-eleven translocation methyldioxygenases Tet2 and Tet3 are required for the TCDD-induced DNA demethylation. These results provide novel evidence of Ahr-driven active DNA demethylation and epigenetic memory. The epigenetic alterations influence response to subsequent chemical exposure and imply an adaptive mechanism to xenobiotic stress. PMID:27713569

  17. Opposing effects of corepressor and coactivators in determining the dose-response curve of agonists, and residual agonist activity of antagonists, for glucocorticoid receptor-regulated gene expression.

    PubMed

    Szapary, D; Huang, Y; Simons, S S

    1999-12-01

    A distinguishing, but unexplained, characteristic of steroid hormone action is the dose-response curve for the regulation of gene expression. We have previously reported that the dose-response curve for glucocorticoid induction of a transfected reporter gene in CV-1 and HeLa cells is repositioned in the presence of increasing concentrations of glucocorticoid receptors (GRs). This behavior is now shown to be independent of the reporter, promoter, or enhancer, consistent with our proposal that a transacting factor(s) was being titrated by added receptors. Candidate factors have been identified by the observation that changes in glucocorticoid induction parameters in CV-1 cells could be reproduced by varying the cellular levels of coactivators [transcriptional intermediary factor 2 (TIF2), steroid receptor coactivator 1 (SRC-1), and amplified in breast cancer 1 (AIB1)], comodulator [CREB-binding protein (CBP)], or corepressor [silencing mediator for retinoid and thyroid-hormone receptors (SMRT)] without concomitant increases in GR. Significantly, the effects of TIF2 and SMRT were mutually antagonistic. Similarly, additional SMRT could reverse the action of increased levels of GRs in HeLa cells, thus indicating that the effects of cofactors on transcription may be general for GR in a variety of cells. These data further indicate that GRs are yet an additional target of the corepressor SMRT. At the same time, these cofactors were found to be capable of regulating the level of residual agonist activity displayed by antiglucocorticoids. Finally, these observations suggest that a novel role for cofactors is to participate in processes that determine the dose-response curve, and partial agonist activity, of GR-steroid complexes. This new activity of cofactors is disconnected from their ability to increase or decrease GR transactivation. An equilibrium model is proposed in which the ratio of coactivator-corepressor bound to either receptor-agonist or -antagonist complexes

  18. Streamlining the design of promising clinical trials: in-vitro testing of antithrombotic regimens and multiple agonists of platelet activation.

    PubMed

    Schneider, David J; Sobel, Burton E

    2009-03-01

    Platelets are activated in vivo by multiple agonists; however, platelet function testing in vitro has been performed predominantly with only one or two agonists of platelet activation. Greater insight into anticipated effects of antithrombotic regimens should enhance the design of successful clinical trials. To test this concept, we assessed platelet activation induced by multiple agonists and two antithrombotic regimens, unfractionated heparin (UFH) and eptifibatide compared with bivalirudin and cangrelor. Blood samples from 10 patients with coronary artery disease were spiked with pharmacologic concentrations achieved in vivo of either UFH (1.2 U/ml) and eptifibatide (1.7 microg/ml), or with bivalirudin (8 microg/ml) and cangrelor (500 nmol/l). Platelet function was assessed with the use of flow cytometry. Agonists included thrombin (50 nmol/l), adenosine diphosphate (1 micromol/l), the collagen-mimetic convulxin (5 ng/ml), and platelet-activating factor (10 nmol/l). When platelet activation was identified by the surface expression of P-selectin in response to multiple agonists, the combination of bivalirudin and cangrelor suppressed activation more than UFH and eptifibatide. When platelet activation was identified by the activation of glycoprotein IIb-IIIa (PAC-1 binding), the combination of bivalirudin and cangrelor was more effective in suppressing activation in response to thrombin and adenosine diphosphate, whereas UFH and eptifibatide more effectively prevented binding of PAC-1 when platelets were activated with the collagen-mimetic convulxin. In conclusion, bivalirudin and cangrelor suppressed platelet activation in response to diverse agonists in vitro more than UFH and eptifibatide. These results and this approach to selection of promising interventions should be helpful in streamlining the design of clinical trials.

  19. A Novel Class of Small Molecule Agonists with Preference for Human over Mouse TLR4 Activation

    PubMed Central

    Heeke, Darren S.; Rao, Eileen; Maynard, Sean K.; Hornigold, David; McCrae, Christopher; Fraser, Neil; Tovchigrechko, Andrey; Yu, Li; Williams, Nicola; King, Sarah; Cooper, Martin E.; Hajjar, Adeline M.; Woo, Jennifer C.

    2016-01-01

    The best-characterized Toll-like receptor 4 (TLR4) ligands are lipopolysaccharide (LPS) and its chemically modified and detoxified variant, monophosphoryl lipid A (MPL). Although both molecules are active for human TLR4, they demonstrate a potency preference for mouse TLR4 based on data from transfected cell lines and primary cells of both species. After a high throughput screening process of small molecule libraries, we have discovered a new class of TLR4 agonist with a species preference profile differing from MPL. Products of the 4-component Ugi synthesis reaction were demonstrated to potently trigger human TLR4-transfected HEK cells but not mouse TLR4, although inclusion of the human MD2 with mTLR4 was able to partially recover activity. Co-expression of CD14 was not required for optimal activity of Ugi compounds on transfected cells, as it is for LPS. The species preference profile for the panel of Ugi compounds was found to be strongly active for human and cynomolgus monkey primary cells, with reduced but still substantial activity for most Ugi compounds on guinea pig cells. Mouse, rat, rabbit, ferret, and cotton rat cells displayed little or no activity when exposed to Ugi compounds. However, engineering the human versions of TLR4 and MD2 to be expressed in mTLR4/MD2 deficient mice allowed for robust activity by Ugi compounds both in vitro and in vivo. These findings extend the range of compounds available for development as agonists of TLR4 and identify novel molecules which reverse the TLR4 triggering preference of MPL for mouse TLR4 over human TLR4. Such compounds may be amenable to formulation as more potent human-specific TLR4L-based adjuvants than typical MPL-based adjuvants. PMID:27736941

  20. A Novel Class of Small Molecule Agonists with Preference for Human over Mouse TLR4 Activation.

    PubMed

    Marshall, Jason D; Heeke, Darren S; Rao, Eileen; Maynard, Sean K; Hornigold, David; McCrae, Christopher; Fraser, Neil; Tovchigrechko, Andrey; Yu, Li; Williams, Nicola; King, Sarah; Cooper, Martin E; Hajjar, Adeline M; Woo, Jennifer C

    2016-01-01

    The best-characterized Toll-like receptor 4 (TLR4) ligands are lipopolysaccharide (LPS) and its chemically modified and detoxified variant, monophosphoryl lipid A (MPL). Although both molecules are active for human TLR4, they demonstrate a potency preference for mouse TLR4 based on data from transfected cell lines and primary cells of both species. After a high throughput screening process of small molecule libraries, we have discovered a new class of TLR4 agonist with a species preference profile differing from MPL. Products of the 4-component Ugi synthesis reaction were demonstrated to potently trigger human TLR4-transfected HEK cells but not mouse TLR4, although inclusion of the human MD2 with mTLR4 was able to partially recover activity. Co-expression of CD14 was not required for optimal activity of Ugi compounds on transfected cells, as it is for LPS. The species preference profile for the panel of Ugi compounds was found to be strongly active for human and cynomolgus monkey primary cells, with reduced but still substantial activity for most Ugi compounds on guinea pig cells. Mouse, rat, rabbit, ferret, and cotton rat cells displayed little or no activity when exposed to Ugi compounds. However, engineering the human versions of TLR4 and MD2 to be expressed in mTLR4/MD2 deficient mice allowed for robust activity by Ugi compounds both in vitro and in vivo. These findings extend the range of compounds available for development as agonists of TLR4 and identify novel molecules which reverse the TLR4 triggering preference of MPL for mouse TLR4 over human TLR4. Such compounds may be amenable to formulation as more potent human-specific TLR4L-based adjuvants than typical MPL-based adjuvants.

  1. Small-molecule nociceptin receptor agonist ameliorates mast cell activation and pain in sickle mice

    PubMed Central

    Vang, Derek; Paul, Jinny A.; Nguyen, Julia; Tran, Huy; Vincent, Lucile; Yasuda, Dennis; Zaveri, Nurulain T.; Gupta, Kalpna

    2015-01-01

    Treatment of pain with morphine and its congeners in sickle cell anemia is suboptimal, warranting the need for analgesics devoid of side effects, addiction and tolerance liability. Small-molecule nociceptin opioid receptor ligands show analgesic efficacy in acute and chronic pain models. We show that AT-200, a high affinity nociceptin opioid receptor agonist with low efficacy at the mu opioid receptor, ameliorated chronic and hypoxia/reoxygenation-induced mechanical, thermal and deep tissue/musculoskeletal hyperalgesia in HbSS-BERK sickle mice. The antinociceptive effect of AT-200 was antagonized by SB-612111, a nociceptin opioid receptor antagonist, but not naloxone, a non-selective mu opioid receptor antagonist. Daily 7-day treatment with AT-200 did not develop tolerance and showed a sustained anti-nociceptive effect, which improved over time and led to reduced plasma serum amyloid protein, neuropeptides, inflammatory cytokines and mast cell activation in the periphery. These data suggest that AT-200 ameliorates pain in sickle mice via the nociceptin opioid receptor by reducing inflammation and mast cell activation without causing tolerance. Thus, nociceptin opioid receptor agonists are promising drugs for treating pain in sickle cell anemia. PMID:26294734

  2. Small-molecule nociceptin receptor agonist ameliorates mast cell activation and pain in sickle mice.

    PubMed

    Vang, Derek; Paul, Jinny A; Nguyen, Julia; Tran, Huy; Vincent, Lucile; Yasuda, Dennis; Zaveri, Nurulain T; Gupta, Kalpna

    2015-12-01

    Treatment of pain with morphine and its congeners in sickle cell anemia is suboptimal, warranting the need for analgesics devoid of side effects, addiction and tolerance liability. Small-molecule nociceptin opioid receptor ligands show analgesic efficacy in acute and chronic pain models. We show that AT-200, a high affinity nociceptin opioid receptor agonist with low efficacy at the mu opioid receptor, ameliorated chronic and hypoxia/reoxygenation-induced mechanical, thermal and deep tissue/musculoskeletal hyperalgesia in HbSS-BERK sickle mice. The antinociceptive effect of AT-200 was antagonized by SB-612111, a nociceptin opioid receptor antagonist, but not naloxone, a non-selective mu opioid receptor antagonist. Daily 7-day treatment with AT-200 did not develop tolerance and showed a sustained anti-nociceptive effect, which improved over time and led to reduced plasma serum amyloid protein, neuropeptides, inflammatory cytokines and mast cell activation in the periphery. These data suggest that AT-200 ameliorates pain in sickle mice via the nociceptin opioid receptor by reducing inflammation and mast cell activation without causing tolerance. Thus, nociceptin opioid receptor agonists are promising drugs for treating pain in sickle cell anemia.

  3. Medium Chain Fatty Acids Are Selective Peroxisome Proliferator Activated Receptor (PPAR) γ Activators and Pan-PPAR Partial Agonists

    PubMed Central

    Ayers, Steven D.; Lin, Jean Z.; Cvoro, Aleksandra; Silveira, Rodrigo L.; Martínez, Leandro; Souza, Paulo C. T.; Saidemberg, Daniel; Deng, Tuo; Amato, Angela Angelica; Togashi, Marie; Hsueh, Willa A.; Phillips, Kevin; Palma, Mário Sérgio; Neves, Francisco A. R.; Skaf, Munir S.; Webb, Paul; Polikarpov, Igor

    2012-01-01

    Thiazolidinediones (TZDs) act through peroxisome proliferator activated receptor (PPAR) γ to increase insulin sensitivity in type 2 diabetes (T2DM), but deleterious effects of these ligands mean that selective modulators with improved clinical profiles are needed. We obtained a crystal structure of PPARγ ligand binding domain (LBD) and found that the ligand binding pocket (LBP) is occupied by bacterial medium chain fatty acids (MCFAs). We verified that MCFAs (C8–C10) bind the PPARγ LBD in vitro and showed that they are low-potency partial agonists that display assay-specific actions relative to TZDs; they act as very weak partial agonists in transfections with PPARγ LBD, stronger partial agonists with full length PPARγ and exhibit full blockade of PPARγ phosphorylation by cyclin-dependent kinase 5 (cdk5), linked to reversal of adipose tissue insulin resistance. MCFAs that bind PPARγ also antagonize TZD-dependent adipogenesis in vitro. X-ray structure B-factor analysis and molecular dynamics (MD) simulations suggest that MCFAs weakly stabilize C-terminal activation helix (H) 12 relative to TZDs and this effect is highly dependent on chain length. By contrast, MCFAs preferentially stabilize the H2-H3/β-sheet region and the helix (H) 11-H12 loop relative to TZDs and we propose that MCFA assay-specific actions are linked to their unique binding mode and suggest that it may be possible to identify selective PPARγ modulators with useful clinical profiles among natural products. PMID:22649490

  4. Medium chain fatty acids are selective peroxisome proliferator activated receptor (PPAR) γ activators and pan-PPAR partial agonists.

    PubMed

    Liberato, Marcelo Vizoná; Nascimento, Alessandro S; Ayers, Steven D; Lin, Jean Z; Cvoro, Aleksandra; Silveira, Rodrigo L; Martínez, Leandro; Souza, Paulo C T; Saidemberg, Daniel; Deng, Tuo; Amato, Angela Angelica; Togashi, Marie; Hsueh, Willa A; Phillips, Kevin; Palma, Mário Sérgio; Neves, Francisco A R; Skaf, Munir S; Webb, Paul; Polikarpov, Igor

    2012-01-01

    Thiazolidinediones (TZDs) act through peroxisome proliferator activated receptor (PPAR) γ to increase insulin sensitivity in type 2 diabetes (T2DM), but deleterious effects of these ligands mean that selective modulators with improved clinical profiles are needed. We obtained a crystal structure of PPARγ ligand binding domain (LBD) and found that the ligand binding pocket (LBP) is occupied by bacterial medium chain fatty acids (MCFAs). We verified that MCFAs (C8-C10) bind the PPARγ LBD in vitro and showed that they are low-potency partial agonists that display assay-specific actions relative to TZDs; they act as very weak partial agonists in transfections with PPARγ LBD, stronger partial agonists with full length PPARγ and exhibit full blockade of PPARγ phosphorylation by cyclin-dependent kinase 5 (cdk5), linked to reversal of adipose tissue insulin resistance. MCFAs that bind PPARγ also antagonize TZD-dependent adipogenesis in vitro. X-ray structure B-factor analysis and molecular dynamics (MD) simulations suggest that MCFAs weakly stabilize C-terminal activation helix (H) 12 relative to TZDs and this effect is highly dependent on chain length. By contrast, MCFAs preferentially stabilize the H2-H3/β-sheet region and the helix (H) 11-H12 loop relative to TZDs and we propose that MCFA assay-specific actions are linked to their unique binding mode and suggest that it may be possible to identify selective PPARγ modulators with useful clinical profiles among natural products.

  5. Agonist-Biased Signaling via Proteinase Activated Receptor-2: Differential Activation of Calcium and Mitogen-Activated Protein Kinase Pathways

    PubMed Central

    Ramachandran, Rithwik; Mihara, Koichiro; Mathur, Maneesh; Rochdi, Moulay Driss; Bouvier, Michel; DeFea, Kathryn

    2009-01-01

    We evaluated the ability of different trypsin-revealed tethered ligand (TL) sequences of rat proteinase-activated receptor 2 (rPAR2) and the corresponding soluble TL-derived agonist peptides to trigger agonist-biased signaling. To do so, we mutated the proteolytically revealed TL sequence of rPAR2 and examined the impact on stimulating intracellular calcium transients and mitogen-activated protein (MAP) kinase. The TL receptor mutants, rPAR2-Leu37Ser38, rPAR2-Ala37–38, and rPAR2-Ala39–42 were compared with the trypsin-revealed wild-type rPAR2 TL sequence, S37LIGRL42—. Upon trypsin activation, all constructs stimulated MAP kinase signaling, but only the wt-rPAR2 and rPAR2-Ala39–42 triggered calcium signaling. Furthermore, the TL-derived synthetic peptide SLAAAA-NH2 failed to cause PAR2-mediated calcium signaling but did activate MAP kinase, whereas SLIGRL-NH2 triggered both calcium and MAP kinase signaling by all receptors. The peptides AAIGRL-NH2 and LSIGRL-NH2 triggered neither calcium nor MAP kinase signals. Neither rPAR2-Ala37–38 nor rPAR2-Leu37Ser38 constructs recruited β-arrestins-1 or -2 in response to trypsin stimulation, whereas both β-arrestins were recruited to these mutants by SLIGRL-NH2. The lack of trypsin-triggered β-arrestin interactions correlated with impaired trypsin-activated TL-mutant receptor internalization. Trypsin-stimulated MAP kinase activation by the TL-mutated receptors was not blocked by inhibitors of Gαi (pertussis toxin), Gαq [N-cyclohexyl-1-(2,4-dichlorophenyl)-1,4-dihydro-6-methylindeno[1,2-c]pyrazole-3-carboxamide (GP2A)], Src kinase [4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]-pyrimidine (PP1)], or the epidermal growth factor (EGF) receptor [4-(3′-chloroanilino)-6,7-dimethoxy-quinazoline (AG1478)], but was inhibited by the Rho-kinase inhibitor (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide, 2HCl (Y27362). The data indicate that the proteolytically revealed TL sequence(s) and the mode

  6. Inhibition of AHR transcription by NF1C is affected by a single-nucleotide polymorphism, and is involved in suppression of human uterine endometrial cancer.

    PubMed

    Li, D; Takao, T; Tsunematsu, R; Morokuma, S; Fukushima, K; Kobayashi, H; Saito, T; Furue, M; Wake, N; Asanoma, K

    2013-10-10

    Involvement of the aryl hydrocarbon receptor (AHR) in carcinogenesis has been suggested in many studies. Upregulation of AHR has been reported in some cancer species, and an association between single-nucleotide polymorphisms (SNPs) of AHR and cancer risk or cancer development has also been reported. This evidence suggests the involvement of some specific SNPs in AHR transcriptional regulation in the process of carcinogenesis or cancer development, but there have been no studies to elucidate the mechanism involved. In this study, we identified the transcription factor Nuclear Factor 1-C (NF1C) as a candidate to regulate AHR transcription in a polymorphism-dependent manner. SNP rs10249788 was included in a consensus binding site for NF1C. Our results suggested that NF1C preferred the C allele to the T allele at rs10249788 for binding. Forced expression of NF1C suppressed the activity of the AHR promoter with C at rs10249788 stronger than that with T. Moreover, expression analysis of human uterine endometrial cancer (HEC) specimens showed greater upregulation of AHR and downregulation of NF1C than those of normal endometrium specimens. Sequence analysis showed HEC patients at advanced stages tended to possess T/T alleles more frequently than healthy women. We also demonstrated that NF1C suppressed proliferation, motility and invasion of HEC cells. This function was at least partially mediated by AHR. This study is the first to report that a polymorphism on the AHR regulatory region affected transcriptional regulation of the AHR gene in vitro. Because NF1C is a tumor suppressor, our new insights into AHR deregulation and its polymorphisms could reveal novel mechanisms of genetic susceptibility to cancer.

  7. Mimetics of caloric restriction include agonists of lipid-activated nuclear receptors.

    PubMed

    Corton, J Christopher; Apte, Udayan; Anderson, Steven P; Limaye, Pallavi; Yoon, Lawrence; Latendresse, John; Dunn, Corrie; Everitt, Jeffrey I; Voss, Kenneth A; Swanson, Cynthia; Kimbrough, Carie; Wong, Jean S; Gill, Sarjeet S; Chandraratna, Roshantha A S; Kwak, Mi-Kyoung; Kensler, Thomas W; Stulnig, Thomas M; Steffensen, Knut R; Gustafsson, Jan-Ake; Mehendale, Harihara M

    2004-10-29

    The obesity epidemic in industrialized countries is associated with increases in cardiovascular disease (CVD) and certain types of cancer. In animal models, caloric restriction (CR) suppresses these diseases as well as chemical-induced tissue damage. These beneficial effects of CR overlap with those altered by agonists of nuclear receptors (NR) under control of the fasting-responsive transcriptional co-activator, peroxisome proliferator-activated co-activator 1alpha (PGC-1alpha). In a screen for compounds that mimic CR effects in the liver, we found statistically significant overlaps between the CR transcript profile in wild-type mice and the profiles altered by agonists of lipid-activated NR, including peroxisome proliferator-activated receptor alpha (PPARalpha), liver X receptor, and their obligate heterodimer partner, retinoid X receptor. The overlapping genes included those involved in CVD (lipid metabolism and inflammation) and cancer (cell fate). Based on this overlap, we hypothesized that some effects of CR are mediated by PPARalpha. As determined by transcript profiling, 19% of all gene expression changes in wild-type mice were dependent on PPARalpha, including Cyp4a10 and Cyp4a14, involved in fatty acid omega-oxidation, acute phase response genes, and epidermal growth factor receptor but not increases in PGC-1alpha. CR protected the livers of wild-type mice from damage induced by thioacetamide, a liver toxicant and hepatocarcinogen. CR protection was lost in PPARalpha-null mice due to inadequate tissue repair. These results demonstrate that PPARalpha mediates some of the effects of CR and indicate that a pharmacological approach to mimicking many of the beneficial effects of CR may be possible.

  8. Novel Aryl Hydrocarbon Receptor Agonist Suppresses Migration and Invasion of Breast Cancer Cells

    PubMed Central

    Mohafez, Omar; Hairul-Islam, Villianur Ibrahim; Alzahrani, Abdullah; Bani Ismail, Mohammad; Thirugnanasambantham, Krishnaraj

    2016-01-01

    Background Despite the remarkable progress to fight against breast cancer, metastasis remains the dominant cause of treatment failure and recurrence. Therefore, control of invasiveness potential of breast cancer cells is crucial. Accumulating evidences suggest Aryl hydrocarbon receptor (Ahr), a helix-loop-helix transcription factor, as a promising target to control migration and invasion in breast cancer cells. Thus, an Ahr-based exploration was performed to identify a new Ahr agonist with inhibitory potentials on cancer cell motility. Methods For prediction of potential interactions between Ahr and candidate molecules, bioinformatics analysis was carried out. The interaction of the selected ligand with Ahr and its effects on migration and invasion were examined in vitro using the MDA-MB-231 and T47D cell lines. The silencing RNAs were transfected into cells by electroporation. Expressions of microRNAs (miRNAs) and coding genes were quantified by real-time PCR, and the protein levels were detected by western blot. Results The in silico and in vitro results identified Flavipin as a novel Ahr agonist. It induces formation of Ahr/Ahr nuclear translocator (Arnt) heterodimer to promote the expression of cytochrome P450 family 1 subfamily A member 1 (Cyp1a1). Migration and invasion of MDA-MB-231 and T47D cells were inhibited with Flavipin treatment in an Ahr-dependent fashion. Interestingly, Flavipin suppressed the pro-metastatic factor SRY-related HMG-box4 (Sox4) by inducing miR-212/132 cluster. Moreover, Flavipin inhibited growth and adhesion of both cell lines by suppressing gene expressions of B-cell lymphoma 2 (Bcl2) and integrinα4 (ITGA4). Conclusion Taken together, the results introduce Flavipin as a novel Ahr agonist, and provide first evidences on its inhibitory effects on cancer cell motility, suggesting Flavipin as a candidate to control cell invasiveness in breast cancer patients. PMID:27907195

  9. A new class of peroxisome proliferator-activated receptor agonists with a novel binding epitope shows antidiabetic effects.

    PubMed

    Ostberg, Tove; Svensson, Stefan; Selén, Göran; Uppenberg, Jonas; Thor, Markus; Sundbom, Maj; Sydow-Bäckman, Mona; Gustavsson, Anna-Lena; Jendeberg, Lena

    2004-09-24

    The peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors belonging to the NR1 subfamily of nuclear receptors. The PPARs play key roles in the control of glucose and lipid homeostasis, and the synthetic isoform-specific PPAR agonists are used clinically to improve insulin sensitivity and to lower serum triglyceride levels. All of the previously reported PPAR agonists form the same characteristic interactions with the receptor, which have been postulated to be important for the induction of agonistic activity. Here we describe a new class of PPARalpha/gamma modulators, the 5-substituted 2-benzoylaminobenzoic acids (2-BABAs). As shown by x-ray crystallography, the representative compounds BVT.13, BVT.762, and BVT.763, utilize a novel binding epitope and lack the agonist-characteristic interactions. Despite this, some compounds within the 2-BABA family are potent agonists in a cell-based reporter gene assay. Furthermore, BVT.13 displays antidiabetic effects in ob/ob mice. We concluded that the 2-BABA binding mode can be used to design isoform-specific PPAR modulators with biological activity in vivo.

  10. Muscle pain induces task-dependent changes in cervical agonist/antagonist activity.

    PubMed

    Falla, D; Farina, D; Dahl, M Kanstrup; Graven-Nielsen, T

    2007-02-01

    This study examined the effect of experimental neck muscle pain on the EMG-force relationship of cervical agonist and antagonist muscles. Surface EMG signals were detected from the sternomastoid, splenius capitis, and upper trapezius muscles bilaterally from 14 healthy subjects during cervical flexion and extension contractions of linearly increasing force from 0 to 60% of the maximum voluntary contraction (MVC). Measurements were performed before and after injection of 0.5 ml hypertonic and isotonic saline into either the sternomastoid or splenius capitis in two experimental sessions. EMG average rectified value (ARV) of the sternomastoid, splenius capitis, and upper trapezius muscles and the muscle fiber conduction velocity (CV) of the sternomastoid muscle were estimated at 5% MVC force increments. During cervical flexion with injection of hypertonic saline in sternomastoid, ARV of sternomastoid was lower on the side of pain in the force range 25-60% MVC (P < 0.05) and was associated with a bilateral reduction of splenius capitis and upper trapezius ARV (P < 0.01). During cervical extension, injection of hypertonic saline in splenius capitis resulted in lower estimates of splenius capitis ARV on the painful side from 45 to 60% MVC (P < 0.05), which was associated with a bilateral increase in upper trapezius ARV estimates from 50 to 60% MVC (P < 0.001). However, no significant change was identified for estimates of sternomastoid ARV. Experimentally induced neck muscle pain resulted in task-dependent changes in cervical agonist/antagonist activity without modifications in muscle fiber CV.

  11. Tethered agonists: a new mechanism underlying adhesion G protein-coupled receptor activation.

    PubMed

    Schöneberg, Torsten; Liebscher, Ines; Luo, Rong; Monk, Kelly R; Piao, Xianhua

    2015-06-01

    The family of adhesion G protein-coupled receptors (aGPCRs) comprises 33 members in the human genome, which are subdivided into nine subclasses. Many aGPCRs undergo an autoproteolytic process via their GPCR Autoproteolysis-INducing (GAIN) domain during protein maturation to generate an N- and a C-terminal fragments, NTF and CTF, respectively. The NTF and CTF are non-covalently reassociated on the plasma membrane to form a single receptor unit. How aGPCRs are activated upon ligand binding remains one of the leading questions in the field of aGPCR research. Recent work from our labs and others shows that ligand binding can remove the NTF from the plasma membrane-bound CTF, exposing a tethered agonist which potently activates downstream signaling.

  12. Sinomenine induces the generation of intestinal Treg cells and attenuates arthritis via activation of aryl hydrocarbon receptor.

    PubMed

    Tong, Bei; Yuan, Xusheng; Dou, Yannong; Wu, Xin; Wang, Yuhui; Xia, Yufeng; Dai, Yue

    2016-10-01

    Sinomenine (SIN), an anti-arthritis drug, has previously been proven to exert immunomodulatory activity in rats by inducing intestinal regulatory T-cells (Treg cells). Here, we assessed the effect of SIN on the generation and function of Treg cells in autoimmune arthritis, and the underlying mechanisms in view of aryl hydrocarbon receptor (AhR). The proportions of Treg cells and IL-17-producing T-cells (Th17 cells) differentiated from naive T-cells were analyzed by flow cytometric analysis. The AhR agonistic effect of SIN was tested by analyzing the activation of downstream signaling pathways and target genes. The dependence of intestinal Treg cell induction and arthritis alleviation by SIN on AhR activation was confirmed in a mouse collagen-induced arthritis (CIA) model. SIN promoted the differentiation and function of intestinal Treg cells in vitro. It induced the expression and activity of AhR target gene, promoted AhR/Hsp90 dissociation and AhR nuclear translocation, induced XRE reporter activity, and facilitated AhR/XRE binding in vitro, displaying the potential to be an agonist of AhR. In CIA mice, SIN induced the generation of intestinal Treg cells, and facilitated the immunosuppressive function of these Treg cells as shown by an adoptive transfer test. In addition, the induction of intestinal Treg cells and the anti-arthritic effect of SIN in CIA mice could be largely diminished by the AhR antagonist resveratrol. SIN attenuates arthritis by promoting the generation and function of Treg cells in an AhR-dependent manner.

  13. Structural basis for constitutive activity and agonist-induced activation of the enteroendocrine fat sensor GPR119

    PubMed Central

    Engelstoft, M S; Norn, C; Hauge, M; Holliday, N D; Elster, L; Lehmann, J; Jones, R M; Frimurer, T M; Schwartz, T W

    2014-01-01

    Background and Purpose GPR119 is a Gαs-coupled 7TM receptor activated by endogenous lipids such as oleoylethanolamide (OEA) and by the dietary triglyceride metabolite 2-monoacylglycerol. GPR119 stimulates enteroendocrine hormone and insulin secretion. But despite massive drug discovery efforts in the field, very little is known about the basic molecular pharmacology of GPR119. Experimental Approach GPR119 receptor signalling was studied in transfected cells. Mutational mapping (30 mutations in 23 positions) was performed on residues required for ligand-independent and agonist-induced GPR119 activation (AR231453 and OEA). Novel Rosetta-based receptor modelling was applied, using a composite template approach with segments from different X-ray structures and fully flexible ligand docking. Key Results The increased signalling induced by increasing the cell surface expression of GPR119 in the absence of agonist and the inhibitory effect of two synthetic inverse agonists demonstrated that GRP119 signals with a high degree of constitutive activity through the Gαs pathway. The mutational maps for AR231453 and OEA were very similar and, surprisingly, also similar to the mutational map for residues affecting the constitutive signalling – albeit with key differences. Surprisingly, almost all residues in extracellular loop-2b were important for the constitutive activity. The molecular modelling and docking demonstrated that AR231453 binds in a ‘vertical’ pocket in between mutational hits reaching from the centre of the receptor out to extracellular loop-2b. Conclusions and Implications The high constitutive activity of GPR119 should be taken into account in future drug discovery efforts, which can now be guided by the detailed knowledge of the physiochemical properties of the extended ligand-binding pocket. PMID:25117266

  14. The Toll-Like Receptor Agonist Imiquimod Is Active against Prions

    PubMed Central

    Beringue, Vincent; Soubigou, Flavie; Pang, Yanhong; Desban, Nathalie; Massacrier, Catherine; Morel, Yannis; Paturel, Carine; Contesse, Marie-Astrid; Bouaziz, Serge; Sanyal, Suparna; Galons, Hervé; Blondel, Marc; Voisset, Cécile

    2013-01-01

    Using a yeast-based assay, a previously unsuspected antiprion activity was found for imiquimod (IQ), a potent Toll-like receptor 7 (TLR7) agonist already used for clinical applications. The antiprion activity of IQ was first detected against yeast prions [PSI+] and [URE3], and then against mammalian prion both ex vivo in a cell-based assay and in vivo in a transgenic mouse model for prion diseases. In order to facilitate structure-activity relationship studies, we conducted a new synthetic pathway which provides a more efficient means of producing new IQ chemical derivatives, the activity of which was tested against both yeast and mammalian prions. The comparable antiprion activity of IQ and its chemical derivatives in the above life forms further emphasizes the conservation of prion controlling mechanisms throughout evolution. Interestingly, this study also demonstrated that the antiprion activity of IQ and IQ-derived compounds is independent from their ability to stimulate TLRs. Furthermore, we found that IQ and its active chemical derivatives inhibit the protein folding activity of the ribosome (PFAR) in vitro. PMID:23977222

  15. Ablating the aryl hydrocarbon receptor (AhR) in CD11c+ cells perturbs intestinal epithelium development and intestinal immunity

    PubMed Central

    Chng, Song Hui; Kundu, Parag; Dominguez-Brauer, Carmen; Teo, Wei Ling; Kawajiri, Kaname; Fujii-Kuriyama, Yoshiaki; Mak, Tak Wah; Pettersson, Sven

    2016-01-01

    Diet and microbiome derived indole derivatives are known to activate the ligand induced transcription factor, the Aryl hydrocarbon Receptor (AhR). While the current understanding of AhR biology has confirmed its role in mucosal lymphocytes, its function in intestinal antigen presenting cells (APCs) is poorly understood. Here, we report that Cre-mediated deletion of AhR in CD11c-expressing cells in C57/BL6 mice is associated with altered intestinal epithelial morphogenesis in vivo. Moreover, when co-cultured with AhR-deficient DCs ex vivo, intestinal organoids showed reduced SRY (sex determining region Y)-box 9 and increased Mucin 2 expression, which correlates with reduced Paneth cells and increased goblet cell differentiation, similar to the data obtained in vivo. Further, characterization of intestinal APC subsets, devoid of AhR, revealed an expression pattern associated with aberrant intrinsic Wnt pathway regulation. At a functional level, the loss of AhR in APCs resulted in a dysfunctional epithelial barrier, associated with a more aggressive chemically induced colitis compared to wild type animals. Our results are consistent with a model whereby the AhR signalling pathway may participate in the regulation of innate immunity through intestinal epithelium development and mucosal immunity. PMID:27068235

  16. Ablating the aryl hydrocarbon receptor (AhR) in CD11c+ cells perturbs intestinal epithelium development and intestinal immunity.

    PubMed

    Chng, Song Hui; Kundu, Parag; Dominguez-Brauer, Carmen; Teo, Wei Ling; Kawajiri, Kaname; Fujii-Kuriyama, Yoshiaki; Mak, Tak Wah; Pettersson, Sven

    2016-04-12

    Diet and microbiome derived indole derivatives are known to activate the ligand induced transcription factor, the Aryl hydrocarbon Receptor (AhR). While the current understanding of AhR biology has confirmed its role in mucosal lymphocytes, its function in intestinal antigen presenting cells (APCs) is poorly understood. Here, we report that Cre-mediated deletion of AhR in CD11c-expressing cells in C57/BL6 mice is associated with altered intestinal epithelial morphogenesis in vivo. Moreover, when co-cultured with AhR-deficient DCs ex vivo, intestinal organoids showed reduced SRY (sex determining region Y)-box 9 and increased Mucin 2 expression, which correlates with reduced Paneth cells and increased goblet cell differentiation, similar to the data obtained in vivo. Further, characterization of intestinal APC subsets, devoid of AhR, revealed an expression pattern associated with aberrant intrinsic Wnt pathway regulation. At a functional level, the loss of AhR in APCs resulted in a dysfunctional epithelial barrier, associated with a more aggressive chemically induced colitis compared to wild type animals. Our results are consistent with a model whereby the AhR signalling pathway may participate in the regulation of innate immunity through intestinal epithelium development and mucosal immunity.

  17. Peroxisome Proliferator–Activated Receptor-γ Agonists Prevent In Vivo Remodeling of Human Artery Induced by Alloreactive T Cells

    PubMed Central

    Tobiasova, Zuzana; Zhang, Lufeng; Yi, Tai; Qin, Linfeng; Manes, Thomas D.; Kulkarni, Sanjay; Lorber, Marc I.; Rodriguez, Frederick C.; Choi, Je-Min; Tellides, George; Pober, Jordan S.; Kawikova, Ivana; Bothwell, Alfred L.M.

    2012-01-01

    Background Ligands activating the transcription factor peroxisome proliferator–activated receptor-γ (PPARγ) have antiinflammatory effects. Vascular rejection induced by allogeneic T cells can be responsible for acute and chronic graft loss. Studies in rodents suggest that PPARγ agonists may inhibit graft vascular rejection, but human T-cell responses to allogeneic vascular cells differ from those in rodents, and the effects of PPARγ in human transplantation are unknown. Methods and Results We tested the effects of PPARγ agonists on human vascular graft rejection using a model in which human artery is interposed into the abdominal aorta of immunodeficient mice, followed by adoptive transfer of allogeneic (to the artery donor) human peripheral blood mononuclear cells. Interferon-γ–dependent rejection ensues within 4 weeks, characterized by intimal thickening, T-cell infiltrates, and vascular cell activation, a response resembling clinical intimal arteritis. The PPARγ agonists 15-deoxy-prostaglandin-J2, ciglitazone, and pioglitazone reduced intimal expansion, intimal infiltration of CD45RO+ memory T cells, and plasma levels of inflammatory cytokines. The PPARγ antagonist GW9662 reversed the protective effects of PPARγ agonists, confirming the involvement of PPARγ-mediated pathways. In vitro, pioglitazone inhibited both alloantigen-induced proliferation and superantigen-induced transendothelial migration of memory T cells, indicating the potential mechanisms of PPARγ effects. Conclusion Our results suggest that PPARγ agonists inhibit allogeneic human memory T cell responses and may be useful for the treatment of vascular graft rejection. PMID:21690493

  18. The Novel Toll-Like Receptor 2 Agonist SUP3 Enhances Antigen Presentation and T Cell Activation by Dendritic Cells

    PubMed Central

    Guo, Xueheng; Wu, Ning; Shang, Yingli; Liu, Xin; Wu, Tao; Zhou, Yifan; Liu, Xin; Huang, Jiaoyan; Liao, Xuebin; Wu, Li

    2017-01-01

    Dendritic cells (DCs) are highly specialized antigen-presenting cells that play crucial roles in innate and adaptive immunity. Previous studies suggested that Toll-like receptor (TLR) agonists could be used as potential adjuvants, as activation of TLRs can boost DC-induced immune responses. TLR2 agonists have been shown to enhance DC-mediated immune responses. However, classical TLR2 agonists such as Pam3CSK4 are not stable enough in vivo, which limits their clinical applications. In this study, a novel structurally stable TLR2 agonist named SUP3 was designed. Functional analysis showed that SUP3 induced much stronger antitumor response than Pam3CSK4 by promoting cytotoxic T lymphocytes activation in vivo. This effect was achieved through the following mechanisms: SUP3 strongly enhanced the ability of antigen cross-presentation by DCs and subsequent T cell activation. SUP3 upregulated the expression of costimulatory molecules on DCs and increased antigen deposition in draining lymph nodes. More interestingly, SUP3 induced less amount of pro-inflammatory cytokine production in vivo compared to other TLR agonists such as lipopolysaccharide. Taken together, SUP3 could serve as a novel promising immune adjuvant in vaccine development and immune modulations. PMID:28270814

  19. Catalposide is a natural agonistic ligand of peroxisome proliferator-activated receptor-{alpha}

    SciTech Connect

    Lee, Ji Hae; Jun, Hee-jin; Hoang, Minh-Hien; Jia, Yaoyao; Han, Xiang Hua; Lee, Dong-Ho; Lee, Hak-Ju; Hwang, Bang Yeon; Lee, Sung-Joon

    2012-06-15

    Highlights: Black-Right-Pointing-Pointer Catalposide is a novel ligand for PPAR{alpha}. Black-Right-Pointing-Pointer Cell stimulated with catalposide improved fatty acid uptake, regulated target genes in fatty acid {beta}-oxidation and synthesis. Black-Right-Pointing-Pointer Catalposdie reduces hepatic triacylglycerides. Black-Right-Pointing-Pointer Theses demonstrate catalposide could ameliorate hyperlipidemia and hepatic steatosis. -- Abstract: Peroxisome proliferator-activated receptor-alpha (PPAR{alpha}) is a nuclear receptor that regulates the expression of genes related to cellular lipid uptake and oxidation. Thus, PPAR{alpha} agonists may be important in the treatment of hypertriglyceridemia and hepatic steatosis. In this study, we demonstrated that catalposide is a novel natural PPAR{alpha} agonist, identified from reporter gene assay-based activity screening with approximately 900 natural plant and seaweed extracts. Results of time-resolved fluorescence resonance energy transfer analyses suggested that the compound interacted directly with the ligand-binding domain of PPAR{alpha}. Cultured hepatocytes stimulated with catalposide exhibited significantly reduced cellular triglyceride concentrations, by 21%, while cellular uptake of fatty acids was increased, by 70% (P < 0.05). Quantitative PCR analysis revealed that the increase in cellular fatty acid uptake was due to upregulation of fatty acid transporter protein-4 (+19% vs. the control) in cells stimulated with catalposide. Additionally, expression of genes related to fatty acid oxidation and high-density lipoprotein metabolism were upregulated, while that of genes related to fatty acid synthesis were suppressed. In conclusion, catalposide is hypolipidemic by activation of PPAR{alpha} via a ligand-mediated mechanism that modulates the expression of in lipid metabolism genes in hepatocytes.

  20. Specific activation of the TLR1-TLR2 heterodimer by small-molecule agonists

    PubMed Central

    Cheng, Kui; Gao, Meng; Godfroy, James I.; Brown, Peter N.; Kastelowitz, Noah; Yin, Hang

    2015-01-01

    Toll-like receptor (TLR) agonists activate both the innate and the adaptive immune systems. These TLR agonists have been exploited as potent vaccine adjuvants and antitumor agents. We describe the identification and characterization of a small molecule, N-methyl-4-nitro-2-(4-(4-(trifluoromethyl)phenyl)-1H-imidazol-1-yl)aniline (CU-T12-9), that directly targets TLR1/2 to initiate downstream signaling. CU-T12-9 specifically induces TLR1/2 activation, which can be blocked by either the anti-hTLR1 or the anti-hTLR2 antibody, but not the anti-hTLR6 antibody. Using a variety of different biophysical assays, we have demonstrated the binding mode of CU-T12-9. By binding to both TLR1 and TLR2, CU-T12-9 facilitates the TLR1/2 heterodimeric complex formation, which in turn activates the downstream signaling. Fluorescence anisotropy assays revealed competitive binding to the TLR1/2 complex between CU-T12-9 and Pam3CSK4 with a half-maximal inhibitory concentration (IC50) of 54.4 nM. Finally, we showed that CU-T12-9 signals through nuclear factor κB (NF-κB) and invokes an elevation of the downstream effectors tumor necrosis factor–α (TNF-α), interleukin-10 (IL-10), and inducible nitric oxide synthase (iNOS). Thus, our studies not only provide compelling new insights into the regulation of TLR1/2 signaling transduction but also may facilitate future therapeutic developments. PMID:26101787

  1. A behavioural and biochemical study in rats of 5-hydroxytryptamine receptor agonists and antagonists, with observations on structure-activity requirements for the agonists

    PubMed Central

    Green, A.R.; Hall, J.E.; Rees, A.R.

    1981-01-01

    1 The effect of the putative 5-hydroxytryptamine (5-HT) receptor antagonists, methysergide, methergoline, mianserin, cyproheptadine, cinanserin (all at 10 mg/kg), methiothepin (5 mg/kg) and (-)-propranolol (20 mg/kg) on the behavioural responses to tranylcypromine (10 mg/kg) followed 30 min later by L-tryptophan (100 mg/kg) was examined. 2 Methysergide, methergoline, methiothepin and (-)-propranolol inhibited head weaving, forepaw treading and hind-limb abduction. Methysergide and methergoline increased reactivity. In contrast, cypropheptadine, cinanserin and mianserin had no effects on the behaviour. 3 Similar findings were obtained when the behaviours were elicited by administration of tranylcypromine (10 mg/kg) followed by the putative 5-HT receptor agonist, 5-methoxy-N,N-dimethyltryptamine (5-MeODMT) (2 mg/kg). 4 When the behaviours were elicited by the putative 5-HT receptor agonist, quipazine (50 mg/kg), all the drugs effectively inhibited head weaving and forepaw treading. 5 When the dose of cypropheptadine was doubled to 20 mg/kg an inhibition of the tranylcypromine/L-tryptophan induced behaviours was seen. 6 Methiothepin produced a marked inhibition of apomorphine-induced locomotor activity whilst all the others enhanced this response, suggesting that only methiothepin inhibits the 5-HT behaviours by dopamine antagonism and that the increased reactivity seen following tranylcypromine/L-tryptophan after pretreatment with methysergide or methergoline might be due to enhanced dopamine function. 7 Pretreatment with p-chlorophenylalanine resulted in enhanced behavioural responses to both 5-MeODMT and quipazine. 8 Both methergoline and methiothepin decreased the rate of 5-HT synthesis in whole brain but not spinal cord and methergoline decreased spinal cord 5-HIAA concentration. None of the other drugs had any significant effects on the concentration of 5-HT, 5-HIAA or 5-HT synthesis rate in brain or spinal cord. 9 Experiments with compounds structurally related

  2. [Pathophysiological relevance of peroxisome proliferators activated receptors (PPAR) to joint diseases - the pro and con of agonists].

    PubMed

    Jouzeau, Jean-Yves; Moulin, David; Koufany, Meriem; Sebillaud, Sylvie; Bianchi, Arnaud; Netter, Patrick

    2008-01-01

    Peroxisome proliferators activated receptors (PPAR) are ligand-inducible nuclear transacting factors comprising three subtypes, PPARalpha, PPARbeta/delta and PPARgamma, which play a key role in lipids and glucose homeostasis. All PPAR subtypes have been identified in joint or inflammatory cells and their activation resulted in a transcriptional repression of pro-inflammatory cytokines (IL-1, TNFalpha), early inflammatory genes (NOS(2), COX-2, mPGES-1) or matrix metalloproteases (MMP-1, MMP-13), at least for the gamma subtype. PPAR full agonists were also shown to stimulate IL-1 receptor antagonist (IL-1Ra) production by cytokine-stimulated articular cells in a subtype-dependent manner. These anti-inflammatory and anti-catabolic properties were confirmed in animal models of joint diseases where PPAR agonists reduced synovial inflammation while preventing cartilage destruction or inflammatory bone loss, although many effects required much higher doses than needed to restore insulin sensitivity or to lower circulating lipid levels. However, these promising effects of PPAR full agonists were hampered by their ability to reduce the growth factor-dependent synthesis of extracellular matrix components or to induce chondrocyte apoptosis, by the possible contribution of immunosuppressive properties to their anti-arthritic effects, by the increased adipocyte differentiation secondary to prolonged stimulation of PPARgamma, and by a variable contribution of PPAR subtypes depending on the system. Clinical data are scarce in rheumatoid arthritis (RA) patients whereas thousands of patients worldwilde, treated with PPAR agonists for type 2 diabetes or dyslipidemia, are paradoxically prone to suffer from osteoarthritis (OA). Whereas high dosage of full agonists may expose RA patients to cardiovascular adverse effects, the proof of concept that PPAR agonists have therapeutical relevance to OA may benefit from an epidemiological follow-up of joint lesions in diabetic or

  3. Functionality of aryl hydrocarbon receptors (AhR1 and AhR2) of white sturgeon (Acipenser transmontanus) and implications for the risk assessment of dioxin-like compounds.

    PubMed

    Doering, Jon A; Farmahin, Reza; Wiseman, Steve; Kennedy, Sean W; Giesy, John P; Hecker, Markus

    2014-07-15

    Worldwide, populations of sturgeons are endangered, and it is hypothesized that anthropogenic chemicals, including dioxin-like compounds (DLCs), might be contributing to the observed declines in populations. DLCs elicit their toxic action through activation of the aryl hydrocarbon receptor (AhR), which is believed to regulate most, if not all, adverse effects associated with exposure to these chemicals. Currently, risk assessment of DLCs in fishes uses toxic equivalency factors (TEFs) developed for the World Health Organization (WHO) that are based on studies of embryo-lethality with salmonids. However, there is a lack of knowledge of the sensitivity of sturgeons to DLCs, and it is uncertain whether TEFs developed by the WHO are protective of these fishes. Sturgeons are evolutionarily distinct from salmonids, and the AhRs of sturgeons differ from those of salmonids. Therefore, this study investigated the sensitivity of white sturgeon (Acipenser transmontanus) to DLCs in vitro via the use of luciferase reporter gene assays using COS-7 cells transfected with AhR1 or AhR2 of white sturgeon. Specifically, activation and relative potencies (RePs) of 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD), 2,3,4,7,8-pentachloro-dibenzofuran, 2,3,7,8-tetrachloro-dibenzofuran, 3,3',4,4',5-pentachlorobiphenyl, 3,3',4,4'-tetrachlorobiphenyl, and 2,3,3',4,4'-pentachlorobiphenyl were determined for each AhR. It was demonstrated that white sturgeon expresses AhR1s and AhR2s that are both activated by DLCs with EC50 values for 2,3,7,8-TCDD that are lower than those of any other AhR of vertebrates tested to date. Both AhRs of white sturgeon had RePs for polychlorinated dibenzofurans more similar to TEFs for birds, while RePs for polychlorinated biphenyls were most similar to TEFs for fishes. Measured concentrations of select DLCs in tissues of white sturgeon from British Columbia, Canada, were used to calculate toxic equivalents (TEQs) by use of TEFs for fishes used by the WHO and TCDD

  4. Genetic and epigenetic regulation of AHR gene expression in MCF-7 breast cancer cells: role of the proximal promoter GC-rich region

    PubMed Central

    Englert, Neal A.; Turesky, Robert J.; Han, Weiguo; Bessette, Erin E.; Spivack, Simon D.; Caggana, Michele; Spink, David C.; Spink, Barbara C.

    2014-01-01

    The aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor, contributes to carcinogenesis through its role in the regulation of cytochrome P450 1 (CYP1)-catalyzed metabolism of carcinogens. Here, we investigated genetic and epigenetic mechanisms that affect AhR expression. Analyses of the human AHR proximal promoter in MCF-7 human breast cancer cells using luciferase assays and electrophoretic mobility shift assays revealed multiple specificity protein (Sp) 1 binding sequences that are transcriptional activators in vitro. The regulation of AhR expression was evaluated in long-term estrogen exposed (LTEE) MCF-7 cells, which showed increased AhR expression, enhanced CYP1 inducibility, and increased capacity to form DNA adducts when exposed to the dietary carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine. The increased AhR expression in LTEE cells was found not to result from increased mRNA stability, differential RNA processing, or decreased DNA methylation. Analysis of the AHR proximal promoter region using chromatin immunoprecipitation confirmed that enhanced expression of AhR in LTEE cells involves changes in histone modifications, notably decreased trimethylation of histone 3, lysine 27. Upon further examination of the GC-rich Sp1-binding region, we confirmed that it contains a polymorphic (GGGGC)n repeat. In a population of newborns from New York State, the allele frequency of (GGGGC)n was n = 4>5≫6, 2. Circular dichroism spectroscopy revealed the ability of sequences of this GC-rich region to form guanine-quadruplex structures in vitro. These studies revealed multiple levels at which AhR expression may be controlled, and offer additional insights into mechanisms regulating AhR expression that can ultimately impact carcinogenesis. PMID:22728919

  5. Genetic and epigenetic regulation of AHR gene expression in MCF-7 breast cancer cells: role of the proximal promoter GC-rich region.

    PubMed

    Englert, Neal A; Turesky, Robert J; Han, Weiguo; Bessette, Erin E; Spivack, Simon D; Caggana, Michele; Spink, David C; Spink, Barbara C

    2012-09-01

    The aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor, contributes to carcinogenesis through its role in the regulation of cytochrome P450 1 (CYP1)-catalyzed metabolism of carcinogens. Here, we investigated genetic and epigenetic mechanisms that affect AhR expression. Analyses of the human AHR proximal promoter in MCF-7 human breast cancer cells using luciferase assays and electrophoretic mobility shift assays revealed multiple specificity protein (Sp) 1 binding sequences that are transcriptional activators in vitro. The regulation of AhR expression was evaluated in long-term estrogen exposed (LTEE) MCF-7 cells, which showed increased AhR expression, enhanced CYP1 inducibility, and increased capacity to form DNA adducts when exposed to the dietary carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine. The increased AhR expression in LTEE cells was found not to result from increased mRNA stability, differential RNA processing, or decreased DNA methylation. Analysis of the AHR proximal promoter region using chromatin immunoprecipitation confirmed that enhanced expression of AhR in LTEE cells involves changes in histone modifications, notably decreased trimethylation of histone 3, lysine 27. Upon further examination of the GC-rich Sp1-binding region, we confirmed that it contains a polymorphic (GGGGC)(n) repeat. In a population of newborns from New York State, the allele frequency of (GGGGC)(n) was n = 4 > 5 ≫ 6, 2. Circular dichroism spectroscopy revealed the ability of sequences of this GC-rich region to form guanine-quadruplex structures in vitro. These studies revealed multiple levels at which AhR expression may be controlled, and offer additional insights into mechanisms regulating AhR expression that can ultimately impact carcinogenesis.

  6. Transgenic silkworms expressing human insulin receptors for evaluation of therapeutically active insulin receptor agonists.

    PubMed

    Matsumoto, Yasuhiko; Ishii, Masaki; Ishii, Kenichi; Miyaguchi, Wataru; Horie, Ryo; Inagaki, Yoshinori; Hamamoto, Hiroshi; Tatematsu, Ken-ichiro; Uchino, Keiro; Tamura, Toshiki; Sezutsu, Hideki; Sekimizu, Kazuhisa

    2014-12-12

    We established a transgenic silkworm strain expressing the human insulin receptor (hIR) using the GAL4/UAS system. Administration of human insulin to transgenic silkworms expressing hIR decreased hemolymph sugar levels and facilitated Akt phosphorylation in the fat body. The decrease in hemolymph sugar levels induced by injection of human insulin in the transgenic silkworms expressing hIR was blocked by co-injection of wortmannin, a phosphoinositide 3-kinase inhibitor. Administration of bovine insulin, an hIR ligand, also effectively decreased sugar levels in the transgenic silkworms. These findings indicate that functional hIRs that respond to human insulin were successfully induced in the transgenic silkworms. We propose that the humanized silkworm expressing hIR is useful for in vivo evaluation of the therapeutic activities of insulin receptor agonists.

  7. The Thrombopoietin Receptor: Structural Basis of Traffic and Activation by Ligand, Mutations, Agonists, and Mutated Calreticulin

    PubMed Central

    Varghese, Leila N.; Defour, Jean-Philippe; Pecquet, Christian; Constantinescu, Stefan N.

    2017-01-01

    A well-functioning hematopoietic system requires a certain robustness and flexibility to maintain appropriate quantities of functional mature blood cells, such as red blood cells and platelets. This review focuses on the cytokine receptor that plays a significant role in thrombopoiesis: the receptor for thrombopoietin (TPO-R; also known as MPL). Here, we survey the work to date to understand how this receptor functions at a molecular level throughout its lifecycle, from traffic to the cell surface, dimerization and binding cognate cytokine via its extracellular domain, through to its subsequent activation of associated Janus kinases and initiation of downstream signaling pathways, as well as the regulation of these processes. Atomic level resolution structures of TPO-R have remained elusive. The identification of disease-causing mutations in the receptor has, however, offered some insight into structure and function relationships, as has artificial means of receptor activation, through TPO mimetics, transmembrane-targeting receptor agonists, and engineering in dimerization domains. More recently, a novel activation mechanism was identified whereby mutated forms of calreticulin form complexes with TPO-R via its extracellular N-glycosylated domain. Such complexes traffic pathologically in the cell and persistently activate JAK2, downstream signal transducers and activators of transcription (STATs), and other pathways. This pathologic TPO-R activation is associated with a large fraction of human myeloproliferative neoplasms.

  8. Type I interferons and microbial metabolites of tryptophan modulate astrocyte activity and CNS inflammation via the aryl hydrocarbon receptor

    PubMed Central

    Rothhammer, Veit; Mascanfroni, Ivan D.; Bunse, Lukas; Takenaka, Maisa C.; Kenison, Jessica E.; Mayo, Lior; Chao, Chun-Cheih; Patel, Bonny; Yan, Raymond; Blain, Manon; Alvarez, Jorge I.; Kébir, Hania; Anandasabapathy, Niroshana; Izquierdo, Guillermo; Jung, Steffen; Obholzer, Nikolaus; Pochet, Nathalie; Clish, Clary B.; Prinz, Marco; Prat, Alexandre; Antel, Jack; Quintana, Francisco J.

    2016-01-01

    Astrocytes play important roles in the central nervous system (CNS) during health and disease. Through genome-wide analyses we detected a transcriptional response to type I interferons (IFN-I) in astrocytes during experimental CNS autoimmunity and also in CNS lesions from multiple sclerosis (MS) patients. IFN-I signaling in astrocytes reduces inflammation and experimental autoimmune encephalomyelitis (EAE) disease scores via the ligand-activated transcription factor aryl hydrocarbon receptor (AhR) and suppressor of cytokine signaling 2 (SOCS2). The anti-inflammatory effects of nasally administered IFN-β are partly mediated by AhR. Dietary tryptophan is metabolized by the gut microbiota into AhR agonists that act on astrocytes to limit CNS inflammation. EAE scores were increased following ampicillin treatment during the recovery phase, and CNS inflammation was reduced in antibiotic-treated mice by supplementation with the tryptophan metabolites indole, indoxyl-3-sulfate (I3S), indole-3-propionic acid (IPA) and indole-3-aldehyde (IAld), or the bacterial enzyme tryptophanase. In individuals with MS, the circulating levels of AhR agonists were decreased. These findings suggest that IFN-I produced in the CNS act in combination with metabolites derived from dietary tryptophan by the gut flora to activate AhR signaling in astrocytes and suppress CNS inflammation. PMID:27158906

  9. Selective Cannabinoid Receptor-1 Agonists Regulate Mast Cell Activation in an Oxazolone-Induced Atopic Dermatitis Model

    PubMed Central

    Nam, Gaewon; Jeong, Se Kyoo; Park, Bu Man; Lee, Sin Hee; Kim, Hyun Jong; Hong, Seung-Phil; Kim, Beomjoon

    2016-01-01

    Background Many inflammatory mediators, including various cytokines (e.g. interleukins and tumor necrosis factor [TNF]), inflammatory proteases, and histamine are released following mast cell activation. However, the endogenous modulators for mast cell activation and the underlying mechanism have yet to be elucidated. Endogenous cannabinoids such as palmitoylethanolamide (PEA) and N-arachidonoylethanolamine (anandamide or AEA), were found in peripheral tissues and have been proposed to possess autacoid activity, implying that cannabinoids may downregulate mast cell activation and local inflammation. Objective In order to investigate the effect of cannabinoid receptor-1 (CB1R) agonists on mast cell activation, AEA-derived compounds were newly synthesized and evaluated for their effect on mast cell activation. Methods The effects of selected compounds on FcεRI-induced histamine and β-hexosaminidase release were evaluated in a rat basophilic leukemia cell line (RBL-2H3). To further investigate the inhibitory effects of CB1R agonist in vivo, an oxazolone-induced atopic dermatitis mouse model was exploited. Results We found that CB1R inhibited the release of inflammatory mediators without causing cytotoxicity in RBL-2H3 cells and that CB1R agonists markedly and dose-dependently suppressed mast cell proliferation indicating that CB1R plays an important role in modulating antigen-dependent immunoglobulin E (IgE)-mediated mast cell activation. We also found that topical application of CB1R agonists suppressed the recruitment of mast cells into the skin and reduced the level of blood histamine. Conclusion Our results indicate that CB1R agonists down-regulate mast cell activation and may be used for relieving inflammatory symptoms mediated by mast cell activation, such as atopic dermatitis, psoriasis, and contact dermatitis. PMID:26848215

  10. TRIF Is a Critical Negative Regulator of TLR Agonist Mediated Activation of Dendritic Cells In Vivo

    PubMed Central

    Appledorn, Daniel M.; Aylsworth, Charles F.; Godbehere, Sarah; Liu, Chyong-Jy Joyce; Quiroga, Dionisia; Amalfitano, Andrea

    2011-01-01

    Despite recent advances in developing and licensing adjuvants, there is a great need for more potent formulations to enhance immunogenicity of vaccines. An Eimeria tenella derived antigen (rEA) augments immune responses against several pathogens in animal models and recently was confirmed to be safe for human use. In this study, we have analyzed the molecular mechanisms underlying rEA activity in mice, and confirmed that rEA activates multiple immune cell types, including DCs, macrophages, NK, B, and T cells. The rEA adjuvant also elicits the induction of pleiotropic pro-inflammatory cytokines, responses that completely depend upon the presence of the TLR adaptor protein MyD88. Surprisingly, we also found that the TRIF adaptor protein acts as a potent negative regulator of TLR agonist-triggered immune responses. For example, IL12 production and the induction of co-stimulatory molecule expression by DCs and IFNγ production by NK cells in vivo were significantly increased in rEA-treated TRIF-KO mice. Importantly, however, TRIF suppressive effects were not restricted to rEA-mediated responses, but were apparent in LPS- or ODN2006-activated DCs as well. Taken together, our findings confirm that rEA is a potent adjuvant, triggering robust activation of the innate immune system, in a manner that is augmented by MyD88 and inhibited by TRIF; thereby unveiling the potential complexities of modulating TLR activity to augment vaccine efficacy. PMID:21760953

  11. TLR4/MD-2 activation by a synthetic agonist with no similarity to LPS

    PubMed Central

    Wang, Ying; Su, Lijing; Morin, Matthew D.; Jones, Brian T.; Whitby, Landon R.; Surakattula, Murali M. R. P.; Huang, Hua; Shi, Hexin; Choi, Jin Huk; Wang, Kuan-wen; Moresco, Eva Marie Y.; Berger, Michael; Zhan, Xiaoming; Zhang, Hong; Boger, Dale L.; Beutler, Bruce

    2016-01-01

    Structurally disparate molecules reportedly engage and activate Toll-like receptor (TLR) 4 and other TLRs, yet the interactions that mediate binding and activation by dissimilar ligands remain unknown. We describe Neoseptins, chemically synthesized peptidomimetics that bear no structural similarity to the established TLR4 ligand, lipopolysaccharide (LPS), but productively engage the mouse TLR4 (mTLR4)/myeloid differentiation factor 2 (MD-2) complex. Neoseptin-3 activates mTLR4/MD-2 independently of CD14 and triggers canonical myeloid differentiation primary response gene 88 (MyD88)- and Toll-interleukin 1 receptor (TIR) domain-containing adaptor inducing IFN-beta (TRIF)-dependent signaling. The crystal structure mTLR4/MD-2/Neoseptin-3 at 2.57-Å resolution reveals that Neoseptin-3 binds as an asymmetrical dimer within the hydrophobic pocket of MD-2, inducing an active receptor complex similar to that induced by lipid A. However, Neoseptin-3 and lipid A form dissimilar molecular contacts to achieve receptor activation; hence strong TLR4/MD-2 agonists need not mimic LPS. PMID:26831104

  12. TLR4/MD-2 activation by a synthetic agonist with no similarity to LPS.

    PubMed

    Wang, Ying; Su, Lijing; Morin, Matthew D; Jones, Brian T; Whitby, Landon R; Surakattula, Murali M R P; Huang, Hua; Shi, Hexin; Choi, Jin Huk; Wang, Kuan-wen; Moresco, Eva Marie Y; Berger, Michael; Zhan, Xiaoming; Zhang, Hong; Boger, Dale L; Beutler, Bruce

    2016-02-16

    Structurally disparate molecules reportedly engage and activate Toll-like receptor (TLR) 4 and other TLRs, yet the interactions that mediate binding and activation by dissimilar ligands remain unknown. We describe Neoseptins, chemically synthesized peptidomimetics that bear no structural similarity to the established TLR4 ligand, lipopolysaccharide (LPS), but productively engage the mouse TLR4 (mTLR4)/myeloid differentiation factor 2 (MD-2) complex. Neoseptin-3 activates mTLR4/MD-2 independently of CD14 and triggers canonical myeloid differentiation primary response gene 88 (MyD88)- and Toll-interleukin 1 receptor (TIR) domain-containing adaptor inducing IFN-beta (TRIF)-dependent signaling. The crystal structure mTLR4/MD-2/Neoseptin-3 at 2.57-Å resolution reveals that Neoseptin-3 binds as an asymmetrical dimer within the hydrophobic pocket of MD-2, inducing an active receptor complex similar to that induced by lipid A. However, Neoseptin-3 and lipid A form dissimilar molecular contacts to achieve receptor activation; hence strong TLR4/MD-2 agonists need not mimic LPS.

  13. Intersection of AHR and Wnt Signaling in Development, Health, and Disease

    PubMed Central

    Schneider, Andrew J.; Branam, Amanda M.; Peterson, Richard E.

    2014-01-01

    The AHR (aryl hydrocarbon receptor) and Wnt (wingless-related MMTV integration site) signaling pathways have been conserved throughout evolution. Appropriately regulated signaling through each pathway is necessary for normal development and health, while dysregulation can lead to developmental defects and disease. Though both pathways have been vigorously studied, there is relatively little research exploring the possibility of crosstalk between these pathways. In this review, we provide a brief background on (1) the roles of both AHR and Wnt signaling in development and disease, and (2) the molecular mechanisms that characterize activation of each pathway. We also discuss the need for careful and complete experimental evaluation of each pathway and describe existing research that explores the intersection of AHR and Wnt signaling. Lastly, to illustrate in detail the intersection of AHR and Wnt signaling, we summarize our recent findings which show that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced disruption of Wnt signaling impairs fetal prostate development. PMID:25286307

  14. Identification of PPARgamma Partial Agonists of Natural Origin (II): In Silico Prediction in Natural Extracts with Known Antidiabetic Activity

    PubMed Central

    Guasch, Laura; Sala, Esther; Mulero, Miquel; Valls, Cristina; Salvadó, Maria Josepa; Pujadas, Gerard; Garcia-Vallvé, Santiago

    2013-01-01

    Background Natural extracts have played an important role in the prevention and treatment of diseases and are important sources for drug discovery. However, to be effectively used in these processes, natural extracts must be characterized through the identification of their active compounds and their modes of action. Methodology/Principal Findings From an initial set of 29,779 natural products that are annotated with their natural source and using a previously developed virtual screening procedure (carefully validated experimentally), we have predicted as potential peroxisome proliferators-activated receptor gamma (PPARγ) partial agonists 12 molecules from 11 extracts known to have antidiabetic activity. Six of these molecules are similar to molecules with described antidiabetic activity but whose mechanism of action is unknown. Therefore, it is plausible that these 12 molecules could be the bioactive molecules responsible, at least in part, for the antidiabetic activity of the extracts containing them. In addition, we have also identified as potential PPARγ partial agonists 10 molecules from 16 plants with undescribed antidiabetic activity but that are related (i.e., they are from the same genus) to plants with known antidiabetic properties. None of the 22 molecules that we predict as PPARγ partial agonists show chemical similarity with a group of 211 known PPARγ partial agonists obtained from the literature. Conclusions/Significance Our results provide a new hypothesis about the active molecules of natural extracts with antidiabetic properties and their mode of action. We also suggest plants with undescribed antidiabetic activity that may contain PPARγ partial agonists. These plants represent a new source of potential antidiabetic extracts. Consequently, our work opens the door to the discovery of new antidiabetic extracts and molecules that can be of use, for instance, in the design of new antidiabetic drugs or functional foods focused towards the

  15. The Role of AhR in Autoimmune Regulation and Its Potential as a Therapeutic Target against CD4 T Cell Mediated Inflammatory Disorder

    PubMed Central

    Zhu, Conghui; Xie, Qunhui; Zhao, Bin

    2014-01-01

    AhR has recently emerged as a critical physiological regulator of immune responses affecting both innate and adaptive systems. Since the AhR signaling pathway represents an important link between environmental stimulators and immune-mediated inflammatory disorder, it has become the object of great interest among researchers recently. The current review discusses new insights into the mechanisms of action of a select group of inflammatory autoimmune diseases and the ligand-activated AhR signaling pathway. Representative ligands of AhR, both exogenous and endogenous, are also reviewed relative to their potential use as tools for understanding the role of AhR and as potential therapeutics for the treatment of various inflammatory autoimmune diseases, with a focus on CD4 helper T cells, which play important roles both in self-immune tolerance and in inflammatory autoimmune diseases. Evidence indicating the potential use of these ligands in regulating inflammation in various diseases is highlighted, and potential mechanisms of action causing immune system effects mediated by AhR signaling are also discussed. The current review will contribute to a better understanding of the role of AhR and its signaling pathway in CD4 helper T cell mediated inflammatory disorder. Considering the established importance of AhR in immune regulation and its potential as a therapeutic target, we also think that both further investigation into the molecular mechanisms of immune regulation that are mediated by the ligand-specific AhR signaling pathway, and integrated research and development of new therapeutic drug candidates targeting the AhR signaling pathway should be pursued urgently. PMID:24905409

  16. The role of AhR in autoimmune regulation and its potential as a therapeutic target against CD4 T cell mediated inflammatory disorder.

    PubMed

    Zhu, Conghui; Xie, Qunhui; Zhao, Bin

    2014-06-05

    AhR has recently emerged as a critical physiological regulator of immune responses affecting both innate and adaptive systems. Since the AhR signaling pathway represents an important link between environmental stimulators and immune-mediated inflammatory disorder, it has become the object of great interest among researchers recently. The current review discusses new insights into the mechanisms of action of a select group of inflammatory autoimmune diseases and the ligand-activated AhR signaling pathway. Representative ligands of AhR, both exogenous and endogenous, are also reviewed relative to their potential use as tools for understanding the role of AhR and as potential therapeutics for the treatment of various inflammatory autoimmune diseases, with a focus on CD4 helper T cells, which play important roles both in self-immune tolerance and in inflammatory autoimmune diseases. Evidence indicating the potential use of these ligands in regulating inflammation in various diseases is highlighted, and potential mechanisms of action causing immune system effects mediated by AhR signaling are also discussed. The current review will contribute to a better understanding of the role of AhR and its signaling pathway in CD4 helper T cell mediated inflammatory disorder. Considering the established importance of AhR in immune regulation and its potential as a therapeutic target, we also think that both further investigation into the molecular mechanisms of immune regulation that are mediated by the ligand-specific AhR signaling pathway, and integrated research and development of new therapeutic drug candidates targeting the AhR signaling pathway should be pursued urgently.

  17. Predicting the sensitivity of fishes to dioxin-like compounds: possible role of the aryl hydrocarbon receptor (AhR) ligand binding domain.

    PubMed

    Doering, Jon A; Giesy, John P; Wiseman, Steve; Hecker, Markus

    2013-03-01

    Dioxin-like compounds are chronically toxic to most vertebrates. However, dramatic differences in sensitivity to these chemicals exist both within and among vertebrate classes. A recent study found that in birds, critical amino acid residues in the aryl hydrocarbon receptor (AhR) ligand binding domain are predictive of sensitivity to dioxin-like compounds in a range of species. It is currently unclear whether similar predictive relationships exist for fishes, a group of animals at risk of exposure to dioxin-like compounds. Effects of dioxin-like compounds are mediated through the AhR in fishes and birds. However, AhR dynamics are more complex among fishes. Fishes possess AhRs that can be grouped within at least three distinct clades (AhR1, AhR2, AhR3) with each clade possibly containing multiple isoforms. AhR2 has been shown to be the active form in most teleosts, with AhR1 not binding dioxin-like compounds. The role of AhR3 in dioxin-like toxicity has not been established to date and this clade is only known to be expressed in some cartilaginous fishes. Furthermore, multiple mechanisms of sensitivity to dioxin-like compounds that are not relevant in birds could exist among fishes. Although, at this time, deficiencies exist for the development of such a predictive relationship for application to fishes, successfully establishing such relationships would offer a substantial improvement in assessment of risks of dioxin-like compounds for this class of vertebrates. Elucidation of such relationships would provide a mechanistic foundation for extrapolation among species to allow the identification of the most sensitive fishes, with the ultimate goal of the prediction of risk posed to endangered species that are not easily studied.

  18. Effects of structural modifications of N-CPM-normorphine derivatives on agonist and antagonist activities in isolated organs.

    PubMed

    Riba, P; Tóth, Z; Hosztafi, S; Friedmann, T; Fürst, S

    2003-01-01

    The agonistic and antagonistic properties of N-cyclopropylmethyl (N-CPM) morphine derivatives were observed in mouse vas deferens (MVD), longitudinal muscle of guinea pig ileum (GPI) and rabbit vas deferens (LVD). In MVD the K(e) values of the titled compounds (N-CPM-morphine, N-CPM-isomorphine, N-CPM-dihydromorphine, N-CPM-dihydroisomorpPhine, N-CPM-dihydromorphone and naltrexone) were measured for mu-, kappa- and delta-receptors using normorphine, ethylketocyclazocine (EKC) and D-Pen2-D-Pen5-enkephaline (DPDPE) as selective agonists on the receptors, respectively. For mu-receptors of MVD the tested compounds showed similar affinity. For kappa-receptors the non-iso-6-OH derivatives possessed much less affinity than the iso-derivatives. Similar difference could be observed for delta-receptors. The agonistic activities of these compounds in MVD were observed to be between 0-20% of the inhibition of muscle contractions. In GPI the compounds except naltrexone possessed strong agonistic activities effectively antagonized by nor-binaltorphimine (nor-BNI) (K(e) of nor-BNI was 0.23 nM) suggesting that they were strong kappa-receptor agonists. We investigated these agents in LVD too, which contains kappa-receptors, but they did not produce any agonist potencies. It raises the possibility that the kappa-receptor subtypes of LVD and MVD are different from the kappa-receptor subtype of GPI or the vasa deferentia contain much fewer kappa-receptors than GPI and the intrinsic activities of these compounds are too small to reach the 50% inhibition of the contractions.

  19. Are styrene oligomers in coastal sediments of an industrial area aryl hydrocarbon-receptor agonists?

    PubMed

    Hong, Seongjin; Lee, Junghyun; Lee, Changkeun; Yoon, Seo Joon; Jeon, Seungyeon; Kwon, Bong-Oh; Lee, Jong-Hyeon; Giesy, John P; Khim, Jong Seong

    2016-06-01

    Effect-directed analysis (EDA) was performed to identify the major aryl hydrocarbon receptor (AhR) agonists in sediments collected from a highly industrialized area (Lake Shihwa, Korea). Great AhR-mediated potencies were found in fractions containing aromatic compounds with log Kow values of 5-8, and relatively great concentrations of styrene oligomers (SOs) and polycyclic aromatic hydrocarbons (PAHs) were detected in those fractions. Until now, there was little information on occurrences and toxic relative potencies (RePs) of SOs in coastal environments. In the present study; i) distributions and compositions, ii) AhR binding affinities, and iii) contributions of SOs to total AhR-mediated potencies were determined in coastal sediments. Elevated concentrations of 10 SOs were detected in sediments of inland creeks ranging from 61 to 740 ng g(-1) dry mass (dm), while lesser concentrations were found in inner (mean = 33 ng g(-1) dm) and outer regions (mean = 25 ng g(-1) dm) of the lake. Concentrations of PAHs in sediments were comparable to those of SOs. 2,4-diphenyl-1-butene (SD3) was the predominant SO analogue in sediments. SOs and PAHs were accumulated in sediments near sources, and could not be transported to remote regions due to their hydrophobicity. RePs of 3 SOs could be derived, which were 1000- to 10,000-fold less than that of one representative potent AhR active PAH, benzo[a]pyrene. Although concentrations of SOs in sediments were comparable to those of PAHs, the collective contribution of SOs to total AhR-mediated potencies were rather small (<1%), primarily due to their smaller RePs. Overall, the present study provides information on distributions and AhR binding affinities for SOs as baseline data for degradation products of polystyrene plastic in the coastal environment.

  20. Subunit rotation models activation of serotonin 5-HT3AB receptors by agonists

    NASA Astrophysics Data System (ADS)

    Maksay, Gábor; Simonyi, Miklós; Bikádi, Zsolt

    2004-10-01

    The N-terminal extracellular regions of heterooligomeric 3AB-type human 5-hydroxytryptamine receptors (5-HT 3ABR) were modelled based on the crystal structure of snail acetylcholine binding protein AChBP. Stepwise rotation of subunit A by 5° was performed between -10° and 15° to mimic agonist binding and receptor activation. Anticlockwise rotation reduced the size of the binding cavity in interface AB and reorganised the network of hydrogen bonds along the interface. AB subunit dimers with different rotations were applied for docking of ligands with different efficacies: 5-HT, m-chlorophenylbiguanide, SR 57227, quinolinyl piperazine and lerisetron derivatives. All ligands were docked into the dimer with -10° rotation representing ligand-free, open binding cavities similarly, without pharmacological discrimination. Their ammonium ions were in hydrogen bonding distance to the backbone carbonyl of W183. Anticlockwise rotation and contraction of the binding cavity led to distinctive docking interactions of agonists with E129 and cation-π interactions of their ammonium ions. Side chains of several further amino acids participating in docking (Y143, Y153, Y234 and E236) are in agreement with the effects of point mutations in the binding loops. Our model postulates that 5-HT binds to W183 in a hydrophobic cleft as well as to E236 in a hydrophilic vestibule. Then it elicits anticlockwise rotation to draw in loop C via π-cation-π interactions of␣its ammonium ion with W183 and Y234. Finally, closure of the binding cavity might end in rebinding of 5-HT to E129 in the hydrophilic vestibule.

  1. Identification of novel peroxisome proliferator-activated receptor-gamma (PPARγ) agonists using molecular modeling method

    NASA Astrophysics Data System (ADS)

    Gee, Veronica M. W.; Wong, Fiona S. L.; Ramachandran, Lalitha; Sethi, Gautam; Kumar, Alan Prem; Yap, Chun Wei

    2014-11-01

    Peroxisome proliferator-activated receptor-gamma (PPARγ) plays a critical role in lipid and glucose homeostasis. It is the target of many drug discovery studies, because of its role in various disease states including diabetes and cancer. Thiazolidinediones, a synthetic class of agents that work by activation of PPARγ, have been used extensively as insulin-sensitizers for the management of type 2 diabetes. In this study, a combination of QSAR and docking methods were utilised to perform virtual screening of more than 25 million compounds in the ZINC library. The QSAR model was developed using 1,517 compounds and it identified 42,378 potential PPARγ agonists from the ZINC library, and 10,000 of these were selected for docking with PPARγ based on their diversity. Several steps were used to refine the docking results, and finally 30 potentially highly active ligands were identified. Four compounds were subsequently tested for their in vitro activity, and one compound was found to have a K i values of <5 μM.

  2. Agonist activation of arachidonate-regulated Ca2+-selective (ARC) channels in murine parotid and pancreatic acinar cells.

    PubMed

    Mignen, Olivier; Thompson, Jill L; Yule, David I; Shuttleworth, Trevor J

    2005-05-01

    ARC channels (arachidonate-regulated Ca(2+)-selective channels) are a novel type of highly Ca(2+)-selective channel that are specifically activated by low concentrations of agonist-induced arachidonic acid. This activation occurs in the absence of any depletion of internal Ca(2+) stores (i.e. they are 'non-capacitative'). Previous studies in HEK293 cells have shown that these channels provide the predominant pathway for the entry of Ca(2+) seen at low agonist concentrations where oscillatory [Ca(2+)](i) signals are typically produced. In contrast, activation of the more widely studied store-operated Ca(2+) channels (e.g. CRAC channels) is only seen at higher agonist concentrations where sustained 'plateau-type'[Ca(2+)](i) responses are observed. We have now demonstrated the presence of ARC channels in both parotid and pancreatic acinar cells and shown that, again, they are specifically activated by the low concentrations of appropriate agonists (carbachol in the parotid, and both carbachol and cholecystokinin in the pancreas) that are associated with oscillatory [Ca(2+)](i) signals in these cells. Uncoupling the receptor-mediated activation of cytosolic phospholipase A(2) (cPLA(2)) with isotetrandrine reduces the activation of the ARC channels by carbachol and, correspondingly, markedly inhibits the [Ca(2+)](i) signals induced by low carbachol concentrations, whilst those signals seen at high agonist concentrations are essentially unaffected. Interestingly, in the pancreatic acinar cells, activation by cholecystokinin induces a current through the ARC channels that is only approximately 60% of that seen with carbachol. This is consistent with previous reports indicating that carbachol-induced [Ca(2+)](i) signals in these cells are much more dependent on Ca(2+) entry than are the cholecystokinin-induced responses.

  3. Structure−Activity Study of Dihydrocinnamic Acids and Discovery of the Potent FFA1 (GPR40) Agonist TUG-469

    PubMed Central

    2010-01-01

    The free fatty acid 1 receptor (FFA1 or GPR40), which is highly expressed on pancreatic β-cells and amplifies glucose-stimulated insulin secretion, has emerged as an attractive target for the treatment of type 2 diabetes. Several FFA1 agonists containing the para-substituted dihydrocinnamic acid moiety are known. We here present a structure−activity relationship study of this compound family suggesting that the central methyleneoxy linker is preferable for the smaller compounds, whereas the central methyleneamine linker gives higher potency to the larger compounds. The study resulted in the discovery of the potent and selective full FFA1 agonist TUG-469 (29). PMID:24900217

  4. Novel Agonist Bioisosteres and Common Structure-Activity Relationships for The Orphan G Protein-Coupled Receptor GPR139

    PubMed Central

    Shehata, Mohamed A.; Nøhr, Anne C.; Lissa, Delphine; Bisig, Christoph; Isberg, Vignir; Andersen, Kirsten B.; Harpsøe, Kasper; Björkling, Fredrik; Bräuner-Osborne, Hans; Gloriam, David E.

    2016-01-01

    GPR139 is an orphan class A G protein-coupled receptor found mainly in the central nervous system. It has its highest expression levels in the hypothalamus and striatum, regions regulating metabolism and locomotion, respectively, and has therefore been suggested as a potential target for obesity and Parkinson’s disease. The two aromatic amino acids L-Trp and L-Phe have been proposed as putative endogenous agonists, and three structurally related benzohydrazide, glycine benzamide, and benzotriazine surrogate agonist series have been published. Herein, we assayed 158 new analogues selected from a pharmacophore model, and identified 12 new GPR139 agonists, containing previously untested bioisosteres. Furthermore, we present the first combined structure-activity relationships, and a refined pharmacophore model to serve as a rationale for future ligand identification and optimization. PMID:27830715

  5. Differential pathway coupling efficiency of the activated insulin receptor drives signaling selectivity by xmeta, an allosteric partial agonist antibody

    Technology Transfer Automated Retrieval System (TEKTRAN)

    XMetA, an anti-insulin receptor (IR) monoclonal antibody, is an allosteric partial agonist of the IR. We have previously reported that XMetA activates the “metabolic-biased” Akt kinase signaling pathway while having little or no effect on the “mitogenic” MAPK signaling pathwayof ERK 1/2. To inves...

  6. Genome Editing of the CYP1A1 Locus in iPSCs as a Platform to Map AHR Expression throughout Human Development

    PubMed Central

    Smith, Brenden W.; Stanford, Elizabeth A.; Sherr, David H.; Murphy, George J.

    2016-01-01

    The aryl hydrocarbon receptor (AHR) is a ligand activated transcription factor that increases the expression of detoxifying enzymes upon ligand stimulation. Recent studies now suggest that novel endogenous roles of the AHR exist throughout development. In an effort to create an optimized model system for the study of AHR signaling in several cellular lineages, we have employed a CRISPR/CAS9 genome editing strategy in induced pluripotent stem cells (iPSCs) to incorporate a reporter cassette at the transcription start site of one of its canonical targets, cytochrome P450 1A1 (CYP1A1). This cell line faithfully reports on CYP1A1 expression, with luciferase levels as its functional readout, when treated with an endogenous AHR ligand (FICZ) at escalating doses. iPSC-derived fibroblast-like cells respond to acute exposure to environmental and endogenous AHR ligands, and iPSC-derived hepatocytes increase CYP1A1 in a similar manner to primary hepatocytes. This cell line is an important innovation that can be used to map AHR activity in discrete cellular subsets throughout developmental ontogeny. As further endogenous ligands are proposed, this line can be used to screen for safety and efficacy and can report on the ability of small molecules to regulate critical cellular processes by modulating the activity of the AHR. PMID:27148368

  7. In Silico Identification of an Aryl Hydrocarbon Receptor Antagonist with Biological Activity In Vitro and In Vivo

    PubMed Central

    Parks, Ashley J.; Pollastri, Michael P.; Hahn, Mark E.; Stanford, Elizabeth A.; Novikov, Olga; Franks, Diana G.; Haigh, Sarah E.; Narasimhan, Supraja; Ashton, Trent D.; Hopper, Timothy G.; Kozakov, Dmytro; Beglov, Dimitri; Vajda, Sandor; Schlezinger, Jennifer J.

    2014-01-01

    The aryl hydrocarbon receptor (AHR) is critically involved in several physiologic processes, including cancer progression and multiple immune system activities. We, and others, have hypothesized that AHR modulators represent an important new class of targeted therapeutics. Here, ligand shape–based virtual modeling techniques were used to identify novel AHR ligands on the basis of previously identified chemotypes. Four structurally unique compounds were identified. One lead compound, 2-((2-(5-bromofuran-2-yl)-4-oxo-4H-chromen-3-yl)oxy)acetamide (CB7993113), was further tested for its ability to block three AHR-dependent biologic activities: triple-negative breast cancer cell invasion or migration in vitro and AHR ligand–induced bone marrow toxicity in vivo. CB7993113 directly bound both murine and human AHR and inhibited polycyclic aromatic hydrocarbon (PAH)– and TCDD-induced reporter activity by 75% and 90% respectively. A novel homology model, comprehensive agonist and inhibitor titration experiments, and AHR localization studies were consistent with competitive antagonism and blockade of nuclear translocation as the primary mechanism of action. CB7993113 (IC50 3.3 × 10−7 M) effectively reduced invasion of human breast cancer cells in three-dimensional cultures and blocked tumor cell migration in two-dimensional cultures without significantly affecting cell viability or proliferation. Finally, CB7993113 effectively inhibited the bone marrow ablative effects of 7,12-dimethylbenz[a]anthracene in vivo, demonstrating drug absorption and tissue distribution leading to pharmacological efficacy. These experiments suggest that AHR antagonists such as CB7993113 may represent a new class of targeted therapeutics for immunomodulation and/or cancer therapy. PMID:25159092

  8. Acquired resistance to Ah receptor agonists in a population of Atlantic killifish (Fundulus heteroclitus) inhabiting a marine superfund site: in vivo and in vitro studies on the inducibility of xenobiotic metabolizing enzymes.

    PubMed

    Bello, S M; Franks, D G; Stegeman, J J; Hahn, M E

    2001-03-01

    New Bedford Harbor (NBH), MA, is a federal Superfund site that is heavily contaminated with polychlorinated biphenyls (PCBs) and other halogenated aromatic hydrocarbons (HAHs), including some potent aryl hydrocarbon receptor (AhR) agonists. A population of Atlantic killifish (Fundulus heteroclitus) continues to inhabit this site, despite accumulating extraordinarily high concentrations of PCBs (272 microg/g dry weight). To determine if NBH killifish have developed resistance to HAHs that act through the AhR, we examined the inducibility of cytochrome P4501A1 (CYP1A1), UDP glucuronosyl transferase (UGT), and glutathione S-transferase (GST) in fish from NBH and a reference site, Scorton Creek (SC, Cape Cod, MA; PCB concentrations 0.177 microg/g dry weight). 2,3,7,8-Tetrachlorodibenzofuran (TCDF) induced CYP1A1 mRNA, protein, and activity in SC fish in all tissues examined (liver, heart, gut, gill, kidney, spleen, and gonad). In contrast, NBH fish expressed low levels of CYP1A1 and showed no induction of CYP1A1 mRNA, protein, or activity by TCDF, or induction that was lower in magnitude or required higher doses of inducer. p-Nitrophenol UGT activity was not induced by TCDF in either population, while GST activity with 1-chloro-2,4-dinitrobenzene as substrate was induced only in NBH fish in one experiment. Inducibility of CYP1A1 by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or beta-naphthoflavone (BNF) was measured in primary hepatocyte cultures prepared from SC and NBH fish. TCDD induced CYP1A1 activity (ethoxyresorufin O-deethylase) to the same degree in hepatocytes from both populations, demonstrating the functionality of the AhR signaling pathway in NBH fish. However, hepatocytes from NBH fish were 14-fold less sensitive to TCDD than were those from SC fish. The nonhalogenated AhR agonist BNF also induced CYP1A1 in cells from both populations, although with only a 3-fold difference in sensitivity (NBH < SC). These results indicate that chronic exposure to high

  9. Paradoxical relationship between RAVE (relative activity versus endocytosis) values of several opioid receptor agonists and their liability to cause dependence

    PubMed Central

    Wang, Yu-hua; Sun, Jian-feng; Tao, Yi-min; Xu, Xue-jun; Chi, Zhi-qiang; Liu, Jing-gen

    2010-01-01

    Aim: To examine the relationship between the RAVE (relative activity versus endocytosis) values of opiate agonists and their dependence liability by studying several potent analgesics with special profiles in the development of physical and psychological dependence. Methods: The effects of (−)-cis-(3R,4S,2′R) ohmefentanyl (F9202), (+)-cis-(3R,4S,2′S) ohmefentanyl (F9204), dihydroetorphine (DHE) and morphine on [35S]GTPγS binding, forskolin-stimulated cAMP accumulation, and receptor internalization were studied in CHO cells stably expressing HA-tagged μ-opioid receptors (CHO-HA-MOR). cAMP overshoot in response to the withdrawal of these compound treatments was also tested. Results: All four agonists exhibited the same rank order of activity in stimulation of [35S]GTPγS binding, inhibition of adenylyl cyclase (AC) and induction of receptor internalization: DHE>F9204>F9202>morphine. Based on these findings and the previous in vivo analgesic data obtained from our and other laboratories, the RAVE values of the four agonists were calculated. The rank order of RAVE values was morphine>F9202>F9204>DHE. For the induction of cAMP overshoot, the rank order was F9202≥morphine>F9204≥DHE. Conclusion: Taken in combination with previous findings of these compounds' liability to develop dependence, the present study suggests that the agonist with the highest RAVE value seems to have a relatively greater liability to develop psychological dependence relative to the agonist with the lowest RAVE value. However, the RAVE values of these agonists are not correlated with their probability of developing physical dependence or inducing cAMP overshoot, a cellular hallmark of dependence. PMID:20228826

  10. Structure-activity relationship study around guanabenz identifies two derivatives retaining antiprion activity but having lost α2-adrenergic receptor agonistic activity.

    PubMed

    Nguyen, Phu Hai; Hammoud, Hassan; Halliez, Sophie; Pang, Yanhong; Evrard, Justine; Schmitt, Martine; Oumata, Nassima; Bourguignon, Jean-Jacques; Sanyal, Suparna; Beringue, Vincent; Blondel, Marc; Bihel, Frédéric; Voisset, Cécile

    2014-10-15

    Guanabenz (GA) is an orally active α2-adrenergic agonist that has been used for many years for the treatment of hypertension. We recently described that GA is also active against both yeast and mammalian prions in an α2-adrenergic receptor-independent manner. These data suggest that this side-activity of GA could be explored for the treatment of prion-based diseases and other amyloid-based disorders. In this perspective, the potent antihypertensive activity of GA happens to be an annoying side-effect that could limit its use. In order to get rid of GA agonist activity at α2-adrenergic receptors, we performed a structure-activity relationship study around GA based on changes of the chlorine positions on the benzene moiety and then on the modifications of the guanidine group. Hence, we identified the two derivatives 6 and 7 that still possess a potent antiprion activity but were totally devoid of any agonist activity at α2-adrenergic receptors. Similarly to GA, 6 and 7 were also able to inhibit the protein folding activity of the ribosome (PFAR) which has been suggested to be involved in prion appearance/maintenance. Therefore, these two GA derivatives are worth being considered as drug candidates.

  11. Pioglitazone, a Peroxisome Proliferator-Activated Receptor γ Agonist, Suppresses Rat Prostate Carcinogenesis

    PubMed Central

    Suzuki, Shugo; Mori, Yukiko; Nagano, Aya; Naiki-Ito, Aya; Kato, Hiroyuki; Nagayasu, Yuko; Kobayashi, Mizuho; Kuno, Toshiya; Takahashi, Satoru

    2016-01-01

    Pioglitazone (PGZ), a peroxisome proliferator-activated receptor γ agonist, which is known as a type 2 diabetes drug, inhibits cell proliferation in various cancer cell lines, including prostate carcinomas. This study focused on the effect of PGZ on prostate carcinogenesis using a transgenic rat for an adenocarcinoma of prostate (TRAP) model. Adenocarcinoma lesions as a percentage of overall lesions in the ventral prostate were significantly reduced by PGZ treatment in a dose-dependent manner. The number of adenocarcinomas per given area in the ventral prostate was also significantly reduced by PGZ treatment. The Ki67 labeling index in the ventral prostate was also significantly reduced by PGZ. Decreased cyclin D1 expression in addition to the inactivation of both p38 mitogen-activated protein kinase (MAPK) and nuclear factor (NF)κB were detected in PGZ-treated TRAP rat groups. In LNCaP, a human androgen-dependent prostate cancer cell line, PGZ also inhibited cyclin D1 expression and the activation of both p38 MAPK and NFκB. The suppression of cultured cell growth was mainly regulated by the NFκB pathway as detected using specific inhibitors in both LNCaP and PC3, a human androgen-independent prostate cancer cell line. These data suggest that PGZ possesses a chemopreventive potential for prostate cancer. PMID:27973395

  12. AHR2 morpholino knockdown reduces the toxicity of total particulate matter to zebrafish embryos.

    PubMed

    Massarsky, Andrey; Bone, Audrey J; Dong, Wu; Hinton, David E; Prasad, G L; Di Giulio, Richard T

    2016-10-15

    The zebrafish embryo has been proposed as a 'bridge model' to study the effects of cigarette smoke on early development. Previous studies showed that exposure to total particulate matter (TPM) led to adverse effects in developing zebrafish, and suggested that the antioxidant and aryl hydrocarbon receptor (AHR) pathways play important roles. This study investigated the roles of these two pathways in mediating TPM toxicity. The study consisted of four experiments. In experiment I, zebrafish embryos were exposed from 6h post fertilization (hpf) until 96hpf to TPM0.5 and TPM1.0 (corresponding to 0.5 and 1.0μg/mL equi-nicotine units) in the presence or absence of an antioxidant (N-acetyl cysteine/NAC) or a pro-oxidant (buthionine sulfoximine/BSO). In experiment II, TPM exposures were performed in embryos that were microinjected with nuclear factor erythroid 2-related factor 2 (Nrf2), AHR2, cytochrome P450 1A (CYP1A), or CYP1B1 morpholinos, and deformities were assessed. In experiment III, embryos were exposed to TPM, and embryos/larvae were collected at 24, 48, 72, and 96hpf to assess several genes associated with the antioxidant and AHR pathways. Lastly, experiment IV assessed the activity and protein levels of CYP1A and CYP1B1 after exposure to TPM. We demonstrate that the incidence of TPM-induced deformities was generally not affected by NAC/BSO treatments or Nrf2 knockdown. In contrast, AHR2 knockdown reduced, while CYP1A or CYP1B1 knockdowns elevated the incidence of some deformities. Moreover, as shown by gene expression the AHR pathway, but not the antioxidant pathway, was induced in response to TPM exposure, providing further evidence for its importance in mediating TPM toxicity.

  13. Identification of Stage-Specific Gene Modulation during Early Thymocyte Development by Whole-Genome Profiling Analysis after Aryl Hydrocarbon Receptor Activation

    PubMed Central

    Mills, Jeffrey H.; Lai, Zhi-Wei; Singh, Kameshwar P.; Middleton, Frank A.; Gasiewicz, Thomas A.; Silverstone, Allen E.

    2010-01-01

    The aryl hydrocarbon receptor (AHR) is a basic helix-loop-helix transcription factor, implicated as an important modulator of the immune system and of early thymocyte development. We have shown previously that AHR activation by the environmental contaminant and potent AHR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) leads to a significant decline in the percentage of S-phase cells in the CD3−CD4−CD8− triple-negative stage (TN) 3 and TN4 T-cell committed thymocytes 9 to 12 h after exposure. In the more immature TN1- or TN2-stage cells, no effect on cell cycle was observed. To identify early molecular targets, which could provide insight into how the AHR acts as a modulator of thymocyte development and cell cycle regulation, we performed gene-profiling experiments using RNA isolated from four intrathymic progenitor populations in which the AHR was activated for 6 or 12 h. This microarray analysis of AHR activation identified 108 distinct gene probes that were significantly modulated in the TN1–4 thymocyte progenitor stages. Although most of the genes identified have specific AHR recognition sequences, only seven genes were altered exclusively in the two T-cell committed stages of early thymocyte development (TN3 and TN4) in which the decline of S-phase cells is seen. Moreover, all seven of these genes were reduced in expression, and five of the seven are associated with cell cycle regulatory processes. These seven genes are novel targets for modulation by the TCDD-activated AHR and may be involved in the observed cell-cycle arrest and suppression of early thymocyte development. PMID:20159946

  14. Identification of stage-specific gene modulation during early thymocyte development by whole-genome profiling analysis after aryl hydrocarbon receptor activation.

    PubMed

    Laiosa, Michael D; Mills, Jeffrey H; Lai, Zhi-Wei; Singh, Kameshwar P; Middleton, Frank A; Gasiewicz, Thomas A; Silverstone, Allen E

    2010-05-01

    The aryl hydrocarbon receptor (AHR) is a basic helix-loop-helix transcription factor, implicated as an important modulator of the immune system and of early thymocyte development. We have shown previously that AHR activation by the environmental contaminant and potent AHR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) leads to a significant decline in the percentage of S-phase cells in the CD3(-)CD4(-)CD8(-) triple-negative stage (TN) 3 and TN4 T-cell committed thymocytes 9 to 12 h after exposure. In the more immature TN1- or TN2-stage cells, no effect on cell cycle was observed. To identify early molecular targets, which could provide insight into how the AHR acts as a modulator of thymocyte development and cell cycle regulation, we performed gene-profiling experiments using RNA isolated from four intrathymic progenitor populations in which the AHR was activated for 6 or 12 h. This microarray analysis of AHR activation identified 108 distinct gene probes that were significantly modulated in the TN1-4 thymocyte progenitor stages. Although most of the genes identified have specific AHR recognition sequences, only seven genes were altered exclusively in the two T-cell committed stages of early thymocyte development (TN3 and TN4) in which the decline of S-phase cells is seen. Moreover, all seven of these genes were reduced in expression, and five of the seven are associated with cell cycle regulatory processes. These seven genes are novel targets for modulation by the TCDD-activated AHR and may be involved in the observed cell-cycle arrest and suppression of early thymocyte development.

  15. Molecular basis for agonist selectivity and activation of the orphan BRS-3-receptor

    PubMed Central

    Gonzalez, Nieves; Hocart, Simon J.; Portal-Nuñez, Sergio; Mantey, Samuel A.; Nakagawa, Tomoo; Zudaire, Enrique; Coy, David H.; Jensen, Robert T.

    2008-01-01

    Bombesin receptor subtype-3(BRS-3), a G protein-coupled orphan receptor, shares 51% identity with the mammalian bombesin(Bn) receptor for gastrin-releasing peptide(GRPR). There is increasing interest in BRS-3 because it is important in energy metabolism, glucose control,motility and tumor-growth. BRS-3 has low affinity for all Bn-related peptides, however, recently synthetic high-affinity agonists[D-Tyr6/D-Phe6,βAla11,Phe13,Nle14]Bn-(6–14) were described, but they are nonselective for BRS-3 over other Bn-receptors. Based on these peptides, three BRS-3 selective-ligands were developed: peptide#2,[D-Tyr6(R)-Apa11,Phe13,Nle14]Bn(6–14); peptide#3,[D-Tyr6,(R)-Apa11,4Cl-Phe13,Nle14]Bn(6–14); peptide #4,Ac-Phe-Trp-Ala-His(tBzl)-Nip-Gly-Arg-NH2. Their molecular determinants of selectivity/high affinity for BRS-3 are unknown. To address this we used a chimeric/site-mutagenesis approach. Substitution of extracellular domain2(EC2) of BRS-3 by the comparable GRPR domain decreased 26-,4,0-fold affinity for peptides#4,3,2. Substitution of EC3 decreased affinity 4-,11-,0-fold affinity for peptides#2,3,4. Ten point mutations in the EC2 and adjacent transmembrane regions (TM2) 2 and 3 of BRS-3 were made. His107(EC2-BRS-3) for lysine(H107K)(EC2-GRPR), decreased affinity(25-,0-fold) for peptide#4,1; however it could not be activated by either peptide. Its combination with Val101(TM2),Gly112(EC2),Arg127(TM3) resulted in complete loss-of-affinity of peptide#4. Receptor-modeling showed that each of these residues face inward and are within 4Å of the binding-pocket. These results demonstrate [Val101,His107,Gly112,Arg127] in the EC2/adjacent upper TMs of BRS-3 are critical for the high BRS3-selectivity of peptide#4. His107 in EC2 is essential for BRS-3 activation, suggesting amino-aromatic ligand/receptor interactions with peptide#4 are critical for both binding/ activation. Furthermore, these result demonstrate that even though these three BRS-3 selective agonists were developed

  16. Spinal neuronal activation during locomotor-like activity enabled by epidural stimulation and 5-HT agonists in spinal rats

    PubMed Central

    Duru, Paul O.; Tillakaratne, Niranjala J.K.; Kim, Jung A.; Zhong, Hui; Stauber, Stacey M.; Pham, Trinh T.; Xiao, Mei S.; Edgerton, V. Reggie; Roy, Roland R.

    2015-01-01

    The neural networks that generate stepping in complete spinal adult rats remain poorly defined. To address this problem we used c-fos (an activity-dependent marker) to identify active interneurons and motoneurons in the lumbar spinal cord of adult spinal rats during a 30-minute bout of bipedal stepping. Spinal rats were either step trained (30 min/day, 3 days/week for 7.5 weeks) or not step-trained. Stepping was enabled by epidural stimulation and the administration of the serotonergic agonists quipazine and 8-OHDPAT. A third group of spinal rats served as untreated (no stimulation, drugs, or stepping) controls. The number of activated cholinergic central canal cluster cells and partition neurons was higher in both step-trained and non-trained than untreated rats, and higher in non-trained than step-trained rats. The latter finding suggests that daily treatment with epidural stimulation plus serotonergic agonist treatment without step training enhanced the excitability of a broader cholinergic interneuronal population than step training. The number of activated interneurons in laminae II-VI of lumbar cross sections was higher in both step-trained and non-trained than untreated rats, and highest in step-trained rats. This finding suggests that this population of interneurons was responsive to epidural stimulation plus serotonergic treatment and that load-bearing induced when stepping had an additive effect. The number of activated motoneurons of all size categories was higher in the step-trained than the other two groups, reflecting a strong effect of loading on motoneuron recruitment. In general, these results indicate that the spinal networks for locomotion are similar with and without brain input. PMID:25789848

  17. The Nuclear Receptor AhR Controls Bone Homeostasis by Regulating Osteoclast Differentiation via the RANK/c-Fos Signaling Axis

    PubMed Central

    Izawa, Takashi; Arakaki, Rieko; Mori, Hiroki; Tsunematsu, Takaaki; Kudo, Yasusei; Tanaka, Eiji

    2016-01-01

    The aryl hydrocarbon receptor (AhR) pathway plays a key role in receptor activator of NF-κB ligand (RANKL)–mediated osteoclastogenesis. However, the mechanism underlying the regulation of AhR expression in osteoclasts and the signaling pathway through which AhR controls osteoclastogenesis remain unclear. We found that the expression of AhR in bone marrow–derived osteoclasts was upregulated by RANKL at an earlier stage than was the expression of signature osteoclast genes such as those encoding cathepsin K and NFAT, cytoplasmic, calcineurin-dependent 1. In response to RANKL, bone marrow macrophages isolated from AhR−/− mice exhibited impaired phosphorylation of Akt and MAPK as well as NF-κB, whereas their response to M-CSF remained unchanged. Osteoclast differentiation mediated by the AhR signaling pathway was also regulated in an RANKL/c-Fos–dependent manner. Furthermore, ligand activation of AhR by the smoke toxin benzo[a]pyrene accelerated osteoclast differentiation in a receptor-dependent manner, and AhR-dependent regulation of mitochondrial biogenesis in osteoclasts was observed. Moreover, AhR−/− mice exhibited impaired bone healing with delayed endochondral ossification. Taken together, the present results suggest that the RANKL/AhR/c-Fos signaling axis plays a critical role in osteoclastogenesis, thereby identifying the potential of AhR in treating pathological, inflammatory, or metabolic disorders of the bone. PMID:27849171

  18. In vitro activation of rat cardiac glucocorticoid antagonist- versus agonist-receptor complexes.

    PubMed

    Schmidt, T J; Diehl, E E

    1988-06-30

    The synthetic antiglucocorticoid RU 38486 interacts with cardiac cytoplasmic glucocorticoid receptors and competes for in vitro binding with the potent agonist triamcinolone acetonide. In addition to binding to receptors with high affinity, RU 38486 also facilitates the in vitro conformational change in the receptor which is a consequence of the physiologically relevant activation step during which the receptor is converted from a non DNA- to a DNA-binding form. This ability of RU 38486 to promote receptor activation is reflected by both the appropriate shift in the elution profile of [3H]RU 38486-receptor complexes from DEAE-cellulose as well as by an increased binding of these complexes to DNA-cellulose. Although less effective than triamcinolone acetonide, RU 38486 promotes in vitro receptor activation under a variety of experimental conditions, including incubation of labeled cardiac cytosols at 25 degrees C for 30 min or at 15 degrees C for 30 min in the presence of 5 mM pyridoxal 5'-phosphate. Once thermally activated, the cardiac [3H]triamcinolone acetonide and [3H]RU 38486-receptor complexes bind to nonspecific DNA-cellulose with the same relative affinities, as evidenced by the fact that 50% of both activated complexes are eluted at approx. 215-250 mM NaCl. Thus, this pure antiglucocorticoid does promote, at least to some extent, many of the crucial in vitro events including high-affinity binding, activation, and DNA binding which have been shown to be required to elicit a physiological response in vivo.

  19. The glucagon-like peptide 1 receptor agonist enhances intrinsic peroxisome proliferator-activated receptor γ activity in endothelial cells

    SciTech Connect

    Onuma, Hirohisa; Inukai, Kouichi Kitahara, Atsuko; Moriya, Rie; Nishida, Susumu; Tanaka, Toshiaki; Katsuta, Hidenori; Takahashi, Kazuto; Sumitani, Yoshikazu; Hosaka, Toshio; Ishida, Hitoshi

    2014-08-22

    Highlights: • PPARγ activation was involved in the GLP-1-mediated anti-inflammatory action. • Exendin-4 enhanced endogenous PPARγ transcriptional activity in HUVECs. • H89, a PKA inhibitor, abolished GLP-1-induced PPARγ enhancement. • The anti-inflammatory effects of GLP-1 may be explained by PPARγ activation. - Abstract: Recent studies have suggested glucagon-like peptide-1 (GLP-1) signaling to exert anti-inflammatory effects on endothelial cells, although the precise underlying mechanism remains to be elucidated. In the present study, we investigated whether PPARγ activation is involved in the GLP-1-mediated anti-inflammatory action on endothelial cells. When we treated HUVEC cells with 0.2 ng/ml exendin-4, a GLP-1 receptor agonist, endogenous PPARγ transcriptional activity was significantly elevated, by approximately 20%, as compared with control cells. The maximum PPARγ activity enhancing effect of exendin-4 was observed 12 h after the initiation of incubation with exendin-4. As H89, a PKA inhibitor, abolished GLP-1-induced PPARγ enhancement, the signaling downstream from GLP-1 cross-talk must have been involved in PPARγ activation. In conclusion, our results suggest that GLP-1 has the potential to induce PPARγ activity, partially explaining the anti-inflammatory effects of GLP-1 on endothelial cells. Cross-talk between GLP-1 signaling and PPARγ activation would have major impacts on treatments for patients at high risk for cardiovascular disease.

  20. The central action of salbutamol, a beta-agonist with a potential antidepressant activity.

    PubMed

    Przegalinski, E; Baran, L; Kedrek, G

    1980-01-01

    The pharmacological profile of salbutamol, an agonist of beta-adrenergic receptors and a potential antidepressant drug, and its effect on the central serotonin system were studied. It was found that salbutamol either had no effect, or, at higher doses, inhibited the spontaneous activity of mice and rats; it did not influence significantly either the produced by amphetamine locomotor stimulation (in mice and rats) or amphetamine stereotype (in rats). Salbutamol while not affecting body temperature of normal mice reversed hypothermia but not ptosis induced by reserpine, and counteracted the hypothermic action of apomorphine in mice. It neither affected the spiperone-induced catalepsy nor was active in the behavioural despair test in rats. Salbutamol had no effect either, on the fenfluramine-induced hyperthermia in rabbits, on the 5-hydroxytryptophan-induced head twitch reaction in mice, on the tryptamine-induced clonic convulsions of forepaw in rats on the flexor reflex in spinal rats, or on the quipazine- or fenfluramine-induced stimulation of this reflex. The above findings indicate that the pharmacological profile of salbutamol resembles that of classical imipramine-like antidepressant drugs to a very small extent and it does not affect the central serotonergic transmission.

  1. Fenofibrate, a peroxisome proliferator-activated receptor-alpha agonist, exerts anticonvulsive properties.

    PubMed

    Porta, Natacha; Vallée, Louis; Lecointe, Cécile; Bouchaert, Emmanuel; Staels, Bart; Bordet, Régis; Auvin, Stéphane

    2009-04-01

    The underlying mechanisms of the ketogenic diet (KD) remain unknown. Involvement of peroxisome proliferator-activated receptor-alpha (PPARalpha) has been suggested. The aim of this study was to assess the anticonvulsant properties of fenofibrate, a PPARalpha agonist. Wistar rats were fed at libitum during 14 days by regular diet, KD, regular diet containing 0.2% fenofibrate (F), or KD containing 0.2% fenofibrate (KD + F). Pentylenetetrazol (PTZ) threshold and latencies to the onset of status epilepticus induced by lithium-pilocarpine were used to assess diet treatments with anticonvulsive effects. Myoclonic and generalized seizure PTZ thresholds were increased in F- and KD-treated animals in comparison to control. No difference was observed between KD + F group and the others groups (control, F, KD). Latencies to the onset of status epilepticus were increased in F and KD groups compared to control. Fenofibrate exerts anticonvulsive properties comparable to KD in adult rats using PTZ and lithium-pilocarpine models. The underlying mechanisms such as PPARalpha activation and others should be investigated. These findings may provide insights into future directions to simplify KD protocols.

  2. T-cell activation: A queuing theory analysis at low agonist density.

    PubMed

    Wedagedera, J R; Burroughs, N J

    2006-09-01

    We analyze a simple linear triggering model of the T-cell receptor (TCR) within the framework of queuing theory, in which TCRs enter the queue upon full activation and exit by downregulation. We fit our model to four experimentally characterized threshold activation criteria and analyze their specificity and sensitivity: the initial calcium spike, cytotoxicity, immunological synapse formation, and cytokine secretion. Specificity characteristics improve as the time window for detection increases, saturating for time periods on the timescale of downregulation; thus, the calcium spike (30 s) has low specificity but a sensitivity to single-peptide MHC ligands, while the cytokine threshold (1 h) can distinguish ligands with a 30% variation in the complex lifetime. However, a robustness analysis shows that these properties are degraded when the queue parameters are subject to variation-for example, under stochasticity in the ligand number in the cell-cell interface and population variation in the cellular threshold. A time integration of the queue over a period of hours is shown to be able to control parameter noise efficiently for realistic parameter values when integrated over sufficiently long time periods (hours), the discrimination characteristics being determined by the TCR signal cascade kinetics (a kinetic proofreading scheme). Therefore, through a combination of thresholds and signal integration, a T cell can be responsive to low ligand density and specific to agonist quality. We suggest that multiple threshold mechanisms are employed to establish the conditions for efficient signal integration, i.e., coordinate the formation of a stable contact interface.

  3. Type I IFN-mediated synergistic activation of mouse and human DC subsets by TLR agonists.

    PubMed

    Kreutz, Martin; Bakdash, Ghaith; Dolen, Yusuf; Sköld, Annette E; van Hout-Kuijer, Maaike A; de Vries, I Jolanda M; Figdor, Carl G

    2015-10-01

    Novel approaches of dendritic cell (DC) based cancer immunotherapy aim at harnessing the unique attributes of different DC subsets. Classical monocyte-derived DC vaccines are currently being replaced by either applying primary DCs or specifically targeting antigens and adjuvants to these subsets in vivo. Appropriate DC activation in both strategies is essential for optimal effect. For this purpose TLR agonists are favorable adjuvant choices, with TLR7 triggering being essential for inducing strong Th1 responses. However, mouse CD8α(+) DCs, considered to be the major cross-presenting subset, lack TLR7 expression. Interestingly, this DC subset can respond to TLR7 ligand upon concurrent TLR3 triggering. Nevertheless, the mechanism underlying this synergy remains obscure. We now show that TLR3 ligation results in the production of IFN-α, which rapidly induces the expression of TLR7, resulting in synergistic activation. Moreover, we demonstrate that this mechanism conversely holds for plasmacytoid DCs that respond to TLR3 ligation when TLR7 pathway is mobilized. We further demonstrate that this mechanism of sharpening DC senses is also conserved in human BDCA1(+) DCs and plasmacytoid DCs. These findings have important implications for future clinical trials as it suggests that combinations of TLR ligands should be applied irrespective of initial TLR expression profiles on natural DC subsets for optimal stimulation.

  4. Identification of an orally active small-molecule PTHR1 agonist for the treatment of hypoparathyroidism

    PubMed Central

    Tamura, Tatsuya; Noda, Hiroshi; Joyashiki, Eri; Hoshino, Maiko; Watanabe, Tomoyuki; Kinosaki, Masahiko; Nishimura, Yoshikazu; Esaki, Tohru; Ogawa, Kotaro; Miyake, Taiji; Arai, Shinichi; Shimizu, Masaru; Kitamura, Hidetomo; Sato, Haruhiko; Kawabe, Yoshiki

    2016-01-01

    Parathyroid hormone (PTH) is essential for calcium homeostasis and its action is mediated by the PTH type 1 receptor (PTHR1), a class B G-protein-coupled receptor. Hypoparathyroidism and osteoporosis can be treated with PTH injections; however, no orally effective PTH analogue is available. Here we show that PCO371 is a novel, orally active small molecule that acts as a full agonist of PTHR1. PCO371 does not affect the PTH type 2 receptor (PTHR2), and analysis using PTHR1–PTHR2 chimeric receptors indicated that Proline 415 of PTHR1 is critical for PCO371-mediated PTHR1 activation. Oral administration of PCO371 to osteopenic rats provokes a significant increase in bone turnover with limited increase in bone mass. In hypocalcemic rats, PCO371 restores serum calcium levels without increasing urinary calcium, and with stronger and longer-lasting effects than PTH injections. These results strongly suggest that PCO371 can provide a new treatment option for PTH-related disorders, including hypoparathyroidism. PMID:27857062

  5. Organization and activation of sexual and agonistic behavior in the leopard gecko, Eublepharis macularius.

    PubMed

    Rhen, T; Crews, D

    2000-04-01

    Gonadal sex is determined by the temperature experienced during incubation in the leopard gecko (Eublepharis macularius). Furthermore, both factors, incubation temperature and gonadal sex, influence adult sexual and agonistic behavior in this species. Yet it is unclear whether such differences in behavior are irreversibly organized during development or are mediated by differences in hormone levels in adulthood. To address this question, we gonadectomized adult females and males generated from a female-biased (30 degrees C) and a male-biased (32.5 degrees C) incubation temperature and treated them with equivalent levels of various sex steroids. We found that 17beta-estradiol (E(2)) activated sexual receptivity in females but not males, suggesting an organized sex difference in behavioral sensitivity to E(2). There were also organized and activated sex differences in attractivity to stimulus males. Although females were more attractive than males when treated with E(2), both sexes were equally unattractive when treated with dihydrotestosterone (DHT) or testosterone (T). Likewise, sex differences in aggressive and submissive behavior were organized and activated. Attacks on stimulus males were activated by T in males but not in females. In contrast, hormones did not influence flight behavior in males but did affect female submissiveness. Overall, males also evoked more attacks by stimulus males than did females. Nevertheless, females and males treated with androgens evoked more attacks than animals of the same sex that were treated with cholesterol or E(2). Incubation temperature had some weak effects on certain behaviors and no effect on others. This suggests that temperature effects in gonadally intact geckos may be due primarily to differences in circulating levels of hormones in adulthood. We conclude that gonadal sex has both organizational and activational effects on various behaviors in the leopard gecko.

  6. Phosphatidylinositol turnover (PI) during synaptic activation results from the release of a stimulatory and in inhibitory agonist

    SciTech Connect

    Bencherif, M.; Rubio, R.; Berne, R.M.

    1986-03-05

    PI has been implicated in the process of synaptic transmission and is increased by agonists. It has been suggested that PI is involved in cellular Ca/sup + +/ mobilization and the process represents a series of hydrolytic reactions with inositol as the final product. Hence, the rate of release of /sup 3/H-inositol (/sup 3/H-Ins) from prelabelled inositol phospholipids can be used as an index of PI. In the /sup 3/H-inositol prelabelled frog sympathetic ganglia, they studied the effect of synaptic activity on PI. PI did not change during orthodromic stimulation (20 Hz, 5 min). However, upon cessation of the stimulation, PI increased rapidly and remained elevated for at least 30 min. This increase in PI was reduced by suffusing the ganglia with either acetylcholine or adenosine. In the presence of atropine (5 ..mu..M), orthodromic stimulation increased PI. They hypothesized that synaptic activation releases a long-lasting stimulatory agonist and a short-lived inhibitory (Ach/adenosine) agonist(s) affecting PI. To test this idea, 2 sympathetic ganglia were used. One was prelabelled with /sup 3/H-inositol and the other was not. The two ganglia were placed together in a 5 ..mu..l drop of Ringers solution containing atropine. Orthodromic stimuli were applied to the non-labelled ganglion and elicited release of /sup 3/H-Ins from the non-stimulated ganglion.

  7. Simulator for SUPO, a Benchmark Aqueous Homogeneous Reactor (AHR)

    SciTech Connect

    Klein, Steven Karl; Determan, John C.

    2015-10-14

    A simulator has been developed for SUPO (Super Power) an aqueous homogeneous reactor (AHR) that operated at Los Alamos National Laboratory (LANL) from 1951 to 1974. During that period SUPO accumulated approximately 600,000 kWh of operation. It is considered the benchmark for steady-state operation of an AHR. The SUPO simulator was developed using the process that resulted in a simulator for an accelerator-driven subcritical system, which has been previously reported.

  8. Rational design of orally-active, pyrrolidine-based progesterone receptor partial agonists

    SciTech Connect

    Thompson, Scott K.; Washburn, David G.; Frazee, James S.; Madauss, Kevin P.; Hoang, Tram H.; Lapinski, Leahann; Grygielko, Eugene T.; Glace, Lindsay E.; Trizna, Walter; Williams, Shawn P.; Duraiswami, Chaya; Bray, Jeffrey D.; Laping, Nicholas J.

    2010-09-03

    Using the X-ray crystal structure of an amide-based progesterone receptor (PR) partial agonist bound to the PR ligand binding domain, a novel PR partial agonist class containing a pyrrolidine ring was designed. Members of this class of N-alkylpyrrolidines demonstrate potent and highly selective partial agonism of the progesterone receptor, and one of these analogs was shown to be efficacious upon oral dosing in the OVX rat model of estrogen opposition.

  9. Salvinorin A, an active component of the hallucinogenic sage salvia divinorum is a highly efficacious kappa-opioid receptor agonist: structural and functional considerations.

    PubMed

    Chavkin, Charles; Sud, Sumit; Jin, Wenzhen; Stewart, Jeremy; Zjawiony, Jordan K; Siebert, Daniel J; Toth, Beth Ann; Hufeisen, Sandra J; Roth, Bryan L

    2004-03-01

    The diterpene salvinorin A from Salvia divinorum has recently been reported to be a high-affinity and selective kappa-opioid receptor agonist (Roth et al., 2002). Salvinorin A and selected derivatives were found to be potent and efficacious agonists in several measures of agonist activity using cloned human kappa-opioid receptors expressed in human embryonic kidney-293 cells. Thus, salvinorin A, salvinorinyl-2-propionate, and salvinorinyl-2-heptanoate were found to be either full (salvinorin A) or partial (2-propionate, 2-heptanoate) agonists for inhibition of forskolin-stimulated cAMP production. Additional studies of agonist potency and efficacy of salvinorin A, performed by cotransfecting either the chimeric G proteins Gaq-i5 or the universal G protein Ga16 and quantification of agonist-evoked intracellular calcium mobilization, affirmed that salvinorin A was a potent and effective kappa-opioid agonist. Results from structure-function studies suggested that the nature of the substituent at the 2-position of salvinorin A was critical for kappa-opioid receptor binding and activation. Because issues of receptor reserve complicate estimates of agonist efficacy and potency, we also examined the agonist actions of salvinorin A by measuring potassium conductance through G protein-gated K(+) channels coexpressed in Xenopus oocytes, a system in which receptor reserve is minimal. Salvinorin A was found to be a full agonist, being significantly more efficacious than (trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl] benzeneacetamide methane-sulfonate hydrate (U50488) or (trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl] benzeneacetamide methane-sulfonate hydrate (U69593) (two standard kappa-opioid agonists) and similar in efficacy to dynorphin A (the naturally occurring peptide ligand for kappa-opioid receptors). Salvinorin A thus represents the first known naturally occurring non-nitrogenous full agonist at kappa-opioid receptors.

  10. The PPARalpha Agonist Fenofibrate Preserves Hippocampal Neurogenesis and Inhibits Microglial Activation After Whole-Brain Irradiation

    SciTech Connect

    Ramanan, Sriram; Kooshki, Mitra; Zhao Weiling; Hsu, F.-C.; Riddle, David R.; Robbins, Mike E.

    2009-11-01

    Purpose: Whole-brain irradiation (WBI) leads to cognitive impairment months to years after radiation. Numerous studies suggest that decreased hippocampal neurogenesis and microglial activation are involved in the pathogenesis of WBI-induced brain injury. The goal of this study was to investigate whether administration of the peroxisomal proliferator-activated receptor (PPAR) alpha agonist fenofibrate would prevent the detrimental effect of WBI on hippocampal neurogenesis. Methods and Materials: For this study, 129S1/SvImJ wild-type and PPARalpha knockout mice that were fed either regular or 0.2% wt/wt fenofibrate-containing chow received either sham irradiation or WBI (10-Gy single dose of {sup 137}Cs gamma-rays). Mice were injected intraperitoneally with bromodeoxyuridine to label the surviving cells at 1 month after WBI, and the newborn neurons were counted at 2 months after WBI by use of bromodeoxyuridine/neuronal nuclei double immunofluorescence. Proliferation in the subgranular zone and microglial activation were measured at 1 week and 2 months after WBI by use of Ki-67 and CD68 immunohistochemistry, respectively. Results: Whole-brain irradiation led to a significant decrease in the number of newborn hippocampal neurons 2 months after it was performed. Fenofibrate prevented this decrease by promoting the survival of newborn cells in the dentate gyrus. In addition, fenofibrate treatment was associated with decreased microglial activation in the dentate gyrus after WBI. The neuroprotective effects of fenofibrate were abolished in the knockout mice, indicating a PPARalpha-dependent mechanism or mechanisms. Conclusions: These data highlight a novel role for PPARalpha ligands in improving neurogenesis after WBI and offer the promise of improving the quality of life for brain cancer patients receiving radiotherapy.

  11. A novel natural Nrf2 activator with PPARγ-agonist (monascin) attenuates the toxicity of methylglyoxal and hyperglycemia

    SciTech Connect

    Hsu, Wei-Hsuan; Lee, Bao-Hong; Chang, Yu-Ying; Hsu, Ya-Wen; Pan, Tzu-Ming

    2013-11-01

    Methylglyoxal (MG) is a toxic-glucose metabolite and a major precursor of advanced glycation endproducts (AGEs). MG has been reported to result in inflammation by activating receptor for AGEs (RAGE). We recently found that Monascus-fermented metabolite monascin acts as a novel natural peroxisome proliferator-activated receptor-γ (PPARγ) agonist that improves insulin sensitivity. We investigated the metabolic, biochemical, and molecular abnormalities characteristic of type 2 diabetes in MG-treated Wistar rats treated with oral administration of monascin or rosiglitazone. Monascin (a novel PPARγ agonist) activated nuclear factor-erythroid 2-related factor 2 (Nrf2) and down-regulated hyperinsulinmia in oral glucose tolerance test (OGTT). Monascin was able to elevate glyoxalase-1 expression via activation of hepatic Nrf2, hence, resulting in MG metabolism to D-lactic acid and protected from AGEs production in MG-treated rats. Rosiglitazone did not activate Nrf2 nor glyoxalase expression to lower serum and hepatic AGEs levels. Monascin acts as a novel natural Nrf2 activator with PPARγ-agonist activity were confirmed by Nrf2 and PPARγ reporter assays in Hep G2 cells. These findings suggest that monascin acts as an anti-diabetic and anti-oxidative stress agent to a greater degree than rosiglitazone and thus may have therapeutic potential for the prevention of diabetes. - Highlights: • Monascin acts as a PPARgamma agonist. • Monascin activates Nrf2 and AMPK. • Monascin promotes MG metabolism into D-lactic acid. • Monascin attenuates inflammation and diabetes in vivo.

  12. TLR3 agonist and Sorafenib combinatorial therapy promotes immune activation and controls hepatocellular carcinoma progression.

    PubMed

    Ho, Victor; Lim, Tong Seng; Lee, Justin; Steinberg, Jeffrey; Szmyd, Radoslaw; Tham, Muly; Yaligar, Jadegoud; Kaldis, Philipp; Abastado, Jean-Pierre; Chew, Valerie

    2015-09-29

    Hepatocellular carcinoma (HCC) is associated with high mortality and the current therapy for advanced HCC, Sorafenib, offers limited survival benefits. Here we assessed whether combining the TLR3 agonist: lysine-stabilized polyinosinic-polycytidylic-acid (poly-ICLC) with Sorafenib could enhance tumor control in HCC. Combinatorial therapy with poly-ICLC and Sorafenib increased apoptosis and reduced proliferation of HCC cell lines in vitro, in association with impaired phosphorylation of AKT, MEK and ERK. In vivo, the combinatorial treatment enhanced control of tumor growth in two mouse models: one transplanted with Hepa 1-6 cells, and the other with liver tumors induced using the Sleeping beauty transposon. Tumor cell apoptosis and host immune responses in the tumor microenvironment were enhanced. Particularly, the activation of local NK cells, T cells, macrophages and dendritic cells was enhanced. Decreased expression of the inhibitory signaling molecules PD-1 and PD-L1 was observed in tumor-infiltrating CD8+ T cells and tumor cells, respectively. Tumor infiltration by monocytic-myeloid derived suppressor cells (Mo-MDSC) was also reduced indicating the reversion of the immunosuppressive tumor microenvironment. Our data demonstrated that the combinatorial therapy with poly-ICLC and Sorafenib enhances tumor control and local immune response hence providing a rationale for future clinical studies.

  13. Evidence for Noncanonical Neurotransmitter Activation: Norepinephrine as a Dopamine D2-Like Receptor Agonist.

    PubMed

    Sánchez-Soto, Marta; Bonifazi, Alessandro; Cai, Ning Sheng; Ellenberger, Michael P; Newman, Amy Hauck; Ferré, Sergi; Yano, Hideaki

    2016-04-01

    The Gαi/o-coupled dopamine D2-like receptor family comprises three subtypes: the D2 receptor (D2R), with short and long isoform variants (D2SR and D2LR), D3 receptor (D3R), and D4 receptor (D4R), with several polymorphic variants. The common overlap of norepinephrine innervation and D2-like receptor expression patterns prompts the question of a possible noncanonical action by norepinephrine. In fact, previous studies have suggested that norepinephrine can functionally interact with D4R. To our knowledge, significant interactions between norepinephrine and D2R or D3R receptors have not been demonstrated. By using radioligand binding and bioluminescent resonance energy transfer (BRET) assays in transfected cells, the present study attempted a careful comparison between dopamine and norepinephrine in their possible activation of all D2-like receptors, including the two D2R isoforms and the most common D4R polymorphic variants. Functional BRET assays included activation of G proteins with all Gαi/o subunits, adenylyl cyclase inhibition, and β arrestin recruitment. Norepinephrine acted as a potent agonist for all D2-like receptor subtypes, with the general rank order of potency of D3R > D4R ≥ D2SR ≥ D2L. However, for both dopamine and norepinephrine, differences depended on the Gαi/o protein subunit involved. The most striking differences were observed with Gαi2, where the rank order of potencies for both dopamine and norepinephrine were D4R > D2SR = D2LR > D3R. Furthermore the results do not support the existence of differences in the ability of dopamine and norepinephrine to activate different human D4R variants. The potency of norepinephrine for adrenergic α2A receptor was only about 20-fold higher compared with D3R and D4R across the three functional assays.

  14. Pharmacological properties of novel cyclic pentapeptides with µ-opioid receptor agonist activity.

    PubMed

    Perlikowska, Renata; Piekielna, Justyna; Fichna, Jakub; do-Rego, Jean Claude; Toth, Geza; Janecki, Tomasz; Janecka, Anna

    2014-03-01

    In our previous paper we have reported the synthesis and biological activity of a cyclic analog, Tyr-c(D-Lys- Phe-Phe-Asp)-NH2, based on endomorphin-2 (EM-2) structure. This analog displayed high affinity for the µ-opioid receptor, was much more stable than EM-2 in rat brain homogenate and showed remarkable antinociceptive activity after intracerebroventricular (i.c.v.) injection. Even more importantly, the cyclic analog elicited weak analgesia also after peripheral administration, giving evidence that it was able to cross, at least to some extent, the blood-brain barrier (BBB). Here we describe further modifications of this analog aimed at enhancing brain delivery by increasing lipophilicity. Two new cyclic pentapeptides, Tyr-c(D-Lys-D-1-Nal-Phe-Asp)-NH2 and Tyr-c(D-Lys-D-2-Nal-Phe-Asp)-NH2 (where 1-Nal=1- naphthyl-3-alanine, 2-Nal=2-naphthyl-3-alanine) were synthesized and evaluated in biological assays. Both analogs showed high µ-opioid receptor affinity and agonist activity and were stable in the rat brain homogenates. Unfortunately, the increase of lipophilicity was achieved at the expense of water solubility. The analog with D-2-Nal residue showed strong analgesic effect when given i.c.v. but could not be tested after intravenous (i.v.) administration where higher concentrations of the compound are required. However, this analog showed inhibitory effect on gastrointestinal (GI) motility in vivo, providing an interesting approach to the development of peripherally restricted agents that could be useful for studying gastrointestinal disorders in animal models.

  15. Evidence for Noncanonical Neurotransmitter Activation: Norepinephrine as a Dopamine D2-Like Receptor Agonist

    PubMed Central

    Sánchez-Soto, Marta; Bonifazi, Alessandro; Cai, Ning Sheng; Ellenberger, Michael P.; Newman, Amy Hauck

    2016-01-01

    The Gαi/o-coupled dopamine D2-like receptor family comprises three subtypes: the D2 receptor (D2R), with short and long isoform variants (D2SR and D2LR), D3 receptor (D3R), and D4 receptor (D4R), with several polymorphic variants. The common overlap of norepinephrine innervation and D2-like receptor expression patterns prompts the question of a possible noncanonical action by norepinephrine. In fact, previous studies have suggested that norepinephrine can functionally interact with D4R. To our knowledge, significant interactions between norepinephrine and D2R or D3R receptors have not been demonstrated. By using radioligand binding and bioluminescent resonance energy transfer (BRET) assays in transfected cells, the present study attempted a careful comparison between dopamine and norepinephrine in their possible activation of all D2-like receptors, including the two D2R isoforms and the most common D4R polymorphic variants. Functional BRET assays included activation of G proteins with all Gαi/o subunits, adenylyl cyclase inhibition, and β arrestin recruitment. Norepinephrine acted as a potent agonist for all D2-like receptor subtypes, with the general rank order of potency of D3R > D4R ≥ D2SR ≥ D2L. However, for both dopamine and norepinephrine, differences depended on the Gαi/o protein subunit involved. The most striking differences were observed with Gαi2, where the rank order of potencies for both dopamine and norepinephrine were D4R > D2SR = D2LR >> D3R. Furthermore the results do not support the existence of differences in the ability of dopamine and norepinephrine to activate different human D4R variants. The potency of norepinephrine for adrenergic α2A receptor was only about 20-fold higher compared with D3R and D4R across the three functional assays. PMID:26843180

  16. Selective anxiolytics: are the actions related to partial "agonist" activity or a preferential affinity for benzodiazepine receptor subtypes?

    PubMed

    Gee, K W; Yamamura, H I

    1983-01-01

    Both pharmacological and biochemical evidence support the existence of BZ receptor subtypes. Determination of the molecular basis of BZ receptor heterogeneity requires additional research. The physiological significance of BZ receptor subtypes is not currently understood. One hypothesis presented to explain the unique pharmacological effects of CL 218872 suggests that CL 218872 has preferential affinity for a BZ receptor subtype (i.e., type I sites) that mediates the anxiolytic effects of the clinically active BZs. An alternative hypothesis has been proposed to account for these observations and is based upon the possibility that CL 218872 may act as a partial agonist at the BZ receptor. The partial agonist theory is supported by behavioral evidence and the relatively small differences in affinity of the BZ receptor subtypes discriminated by CL 218872 at physiological temperatures. In addition, in vivo binding studies suggest that occupancy of type II BZ receptor subtypes (i.e., those with low affinity for CL 218872) is necessary for CL 218872 to produce minimal anticonflict activity (4). Unlike certain other neurotransmitter systems, it is difficult to correlate the heterogeneous binding properties of BZ receptor ligands with their agonist/antagonist potential at BZ receptor. For example, CL 218872 discriminates BZ receptor subtypes and acts as an agonist at the BZ receptor. Beta-carbolines such as PCC also discriminate receptor subtypes, yet they act as antagonists at the BZ receptor. Compounding the complexity, neither the nature nor the existence of an endogenous ligand is known. So, the designation of agonist or antagonist effects is made on a purely functional basis.(ABSTRACT TRUNCATED AT 250 WORDS)

  17. Cytochrome P450s in human immune cells regulate IL-22 and c-Kit via an AHR feedback loop

    PubMed Central

    Effner, Renate; Hiller, Julia; Eyerich, Stefanie; Traidl-Hoffmann, Claudia; Brockow, Knut; Triggiani, Massimo; Behrendt, Heidrun; Schmidt-Weber, Carsten B.; Buters, Jeroen T. M.

    2017-01-01

    The mechanisms how environmental compounds influence the human immune system are unknown. The environmentally sensitive transcription factor aryl hydrocarbon receptor (AHR) has immune-modulating functions and responds to small molecules. Cytochrome P4501 enzymes (CYP1) act downstream of the AHR and metabolize small molecules. However, it is currently unknown whether CYP1 activity is relevant for immune modulation. We studied the interdependence of CYP1 and AHR in human primary immune cells using pharmacological methods. CYP1 inhibition increased the expression levels of the stem cell factor receptor (c-Kit) and interleukin (IL)-22 but decreased IL-17. Single cell analyses showed that CYP1 inhibition especially promoted CD4+ helper T (Th) cells that co-express c-Kit and IL-22 simultaneously. The addition of an AHR antagonist reversed all these effects. In addition to T cells, we screened other human immune cells for CYP and found cell-specific fingerprints, suggesting that similar mechanisms are present in multiple immune cells. We describe a feedback loop yet unknown in human immune cells where CYP1 inhibition resulted in an altered AHR-dependent immune response. This mechanism relates CYP1-dependent metabolism of environmental small molecules to human immunity. PMID:28276465

  18. Deletion of Aryl Hydrocarbon Receptor AHR in Mice Leads to Subretinal Accumulation of Microglia and RPE Atrophy

    PubMed Central

    Kim, Soo-Young; Yang, Hyun-Jin; Chang, Yi-Sheng; Kim, Jung-Woong; Brooks, Matthew; Chew, Emily Y.; Wong, Wai T.; Fariss, Robert N.; Rachel, Rivka A.; Cogliati, Tiziana; Qian, Haohua; Swaroop, Anand

    2014-01-01

    Purpose. The aryl hydrocarbon receptor (AHR) is a ligand-activated nuclear receptor that regulates cellular response to environmental signals, including UV and blue wavelength light. This study was undertaken to elucidate AHR function in retinal homeostasis. Methods. RNA-seq data sets were examined for Ahr expression in the mouse retina and rod photoreceptors. The Ahr−/− mice were evaluated by fundus imaging, optical coherence tomography, histology, immunohistochemistry, and ERG. For light damage experiments, adult mice were exposed to 14,000 to 15,000 lux of diffuse white light for 2 hours. Results. In mouse retina, Ahr transcripts were upregulated during development, with continued increase in aging rod photoreceptors. Fundus examination of 3-month-old Ahr−/− mice revealed subretinal autofluorescent spots, which increased in number with age and following acute light exposure. Ahr−/− retina also showed subretinal microglia accumulation that correlated with autofluorescence changes, RPE abnormalities, and reactivity against immunoglobulin, complement factor H, and glial fibrillary acidic protein. Functionally, Ahr−/− mice displayed reduced ERG c-wave amplitudes. Conclusions. The Ahr−/− mice exhibited subretinal accumulation of microglia and focal RPE atrophy, phenotypes observed in AMD. Together with a recently published report on another Ahr−/− mouse model, our study suggests that AHR has a protective role in the retina as an environmental stress sensor. As such, its altered function may contribute to human AMD progression and provide a target for pharmacological intervention. PMID:25159211

  19. Cerebral radioprotection by pentobarbital: Dose-response characteristics and association with GABA agonist activity

    SciTech Connect

    Olson, J.J.; Friedman, R.; Orr, K.; Delaney, T.; Oldfield, E.H. )

    1990-05-01

    Pentobarbital reduces cerebral radiation toxicity; however, the mechanism of this phenomenon remains unknown. As an anesthetic and depressant of cerebral metabolism, pentobarbital induces its effects on the central nervous system by stimulating the binding of gamma-aminobutyric acid (GABA) to its receptor and by inhibiting postsynaptic excitatory amino acid activity. The purpose of this study is to investigate the role of these actions as well as other aspects of the radioprotective activity of pentobarbital. Fischer 344 rats were separated into multiple groups and underwent two dose-response evaluations. In one set of experiments to examine the relationship of radioprotection to pentobarbital dose, a range of pentobarbital doses (0 to 75 mg/kg) were given intraperitoneally prior to a constant-level radiation dose (70 Gy). In a second series of experiments to determine the dose-response relationship of radiation protection to radiation dose, a range of radiation doses (10 to 90 Gy) were given with a single pentobarbital dose. Further groups of animals were used to evaluate the importance of the timing of pentobarbital administration, the function of the (+) and (-) isomers of pentobarbital, and the role of an alternative GABA agonist (diazepam). In addition, the potential protective effects of alternative methods of anesthesia (ketamine) and induction of cerebral hypometabolism (hypothermia) were examined. Enhancement of survival time from acute radiation injury due to high-dose single-fraction whole-brain irradiation was maximal with 60 mg/kg of pentobarbital, and occurred over the range of all doses examined between 30 to 90 Gy. Protection was seen only in animals that received the pentobarbital before irradiation. Administration of other compounds that enhance GABA binding (Saffan and diazepam) also significantly enhanced survival time.

  20. Polyacetylenes from Notopterygium incisum–New Selective Partial Agonists of Peroxisome Proliferator-Activated Receptor-Gamma

    PubMed Central

    Liu, Xin; Noha, Stefan M.; Malainer, Clemens; Kramer, Matthias P.; Cocic, Amina; Kunert, Olaf; Schinkovitz, Andreas; Heiss, Elke H.; Schuster, Daniela

    2013-01-01

    Peroxisome proliferator-activated receptor gamma (PPARγ) is a key regulator of glucose and lipid metabolism and therefore an important pharmacological target to combat metabolic diseases. Since the currently used full PPARγ agonists display serious side effects, identification of novel ligands, particularly partial agonists, is highly relevant. Searching for new active compounds, we investigated extracts of the underground parts of Notopterygium incisum, a medicinal plant used in traditional Chinese medicine, and observed significant PPARγ activation using a PPARγ-driven luciferase reporter model. Activity-guided fractionation of the dichloromethane extract led to the isolation of six polyacetylenes, which displayed properties of selective partial PPARγ agonists in the luciferase reporter model. Since PPARγ activation by this class of compounds has so far not been reported, we have chosen the prototypical polyacetylene falcarindiol for further investigation. The effect of falcarindiol (10 µM) in the luciferase reporter model was blocked upon co-treatment with the PPARγ antagonist T0070907 (1 µM). Falcarindiol bound to the purified human PPARγ receptor with a Ki of 3.07 µM. In silico docking studies suggested a binding mode within the ligand binding site, where hydrogen bonds to Cys285 and Glu295 are predicted to be formed in addition to extensive hydrophobic interactions. Furthermore, falcarindiol further induced 3T3-L1 preadipocyte differentiation and enhanced the insulin-induced glucose uptake in differentiated 3T3-L1 adipocytes confirming effectiveness in cell models with endogenous PPARγ expression. In conclusion, we identified falcarindiol-type polyacetylenes as a novel class of natural partial PPARγ agonists, having potential to be further explored as pharmaceutical leads or dietary supplements. PMID:23630612

  1. Design, synthesis, and biological evaluation of novel investigational nonapeptide KISS1R agonists with testosterone-suppressive activity.

    PubMed

    Asami, Taiji; Nishizawa, Naoki; Matsui, Hisanori; Nishibori, Kimiko; Ishibashi, Yoshihiro; Horikoshi, Yasuko; Nakayama, Masaharu; Matsumoto, Shin-ichi; Tarui, Naoki; Yamaguchi, Masashi; Matsumoto, Hirokazu; Ohtaki, Tetsuya; Kitada, Chieko

    2013-11-14

    Metastin/kisspeptin is a 54 amino acid peptide ligand of the KISS1R receptor and is a critical regulator of GnRH secretion. The N-terminally truncated peptide, metastin(45-54), possesses a 10-fold higher receptor-binding affinity than full-length metastin and agonistic KISS1R activity but is rapidly inactivated in rodent plasma. We have developed a decapeptide analog [D-Tyr(45),D-Trp(47),azaGly(51),Arg(Me)(53)]metastin(45-54) with improved serum stability compared with metastin(45-54) but with decreased KISS1R agonistic activity. Amino acid replacements at positions 45-47 led to an enhancement of KISS1R agonistic activity and metabolic stability. N-terminal truncation resulted in a stable nonapeptide, [D-Tyr(46),D-Pya(4)(47),azaGly(51),Arg(Me)(53)]metastin(46-54), compound 26, which displayed KISS1R binding affinities comparable to metastin(45-54) and had improved serum stability. Compound 26 reduced plasma testosterone in male rats and is the first short-length metastin analog to possess testosterone suppressive activities. Compound 26 has led to the elucidation of investigational analogs TAK-683 and TAK-448, both of which have undergone clinical evaluation for hormone-dependent diseases such as prostate cancer.

  2. Peroxisome proliferator-activated receptorα agonists differentially regulate inhibitor of DNA binding expression in rodents and human cells.

    PubMed

    González, María Del Carmen; Corton, J Christopher; Acero, Nuria; Muñoz-Mingarro, Dolores; Quirós, Yolanda; Alvarez-Millán, Juan José; Herrera, Emilio; Bocos, Carlos

    2012-01-01

    Inhibitor of DNA binding (Id2) is a helix-loop-helix (HLH) transcription factor that participates in cell differentiation and proliferation. Id2 has been linked to the development of cardiovascular diseases since thiazolidinediones, antidiabetic agents and peroxisome proliferator-activated receptor (PPAR) gamma agonists, have been reported to diminish Id2 expression in human cells. We hypothesized that PPARα activators may also alter Id2 expression. Fenofibrate diminished hepatic Id2 expression in both late pregnant and unmated rats. In 24 hour fasted rats, Id2 expression was decreased under conditions known to activate PPARα. In order to determine whether the fibrate effects were mediated by PPARα, wild-type mice and PPARα-null mice were treated with Wy-14,643 (WY). WY reduced Id2 expression in wild-type mice without an effect in PPARα-null mice. In contrast, fenofibrate induced Id2 expression after 24 hours of treatment in human hepatocarcinoma cells (HepG2). MK-886, a PPARα antagonist, did not block fenofibrate-induced activation of Id2 expression, suggesting a PPARα-independent effect was involved. These findings confirm that Id2 is a gene responsive to PPARα agonists. Like other genes (apolipoprotein A-I, apolipoprotein A-V), the opposite directional transcriptional effect in rodents and a human cell line further emphasizes that PPARα agonists have different effects in rodents and humans.

  3. Peroxisome Proliferator-Activated Receptorα Agonists Differentially Regulate Inhibitor of DNA Binding Expression in Rodents and Human Cells

    PubMed Central

    González, María del Carmen; Corton, J. Christopher; Acero, Nuria; Muñoz-Mingarro, Dolores; Quirós, Yolanda; Álvarez-Millán, Juan José; Herrera, Emilio; Bocos, Carlos

    2012-01-01

    Inhibitor of DNA binding (Id2) is a helix-loop-helix (HLH) transcription factor that participates in cell differentiation and proliferation. Id2 has been linked to the development of cardiovascular diseases since thiazolidinediones, antidiabetic agents and peroxisome proliferator-activated receptor (PPAR) gamma agonists, have been reported to diminish Id2 expression in human cells. We hypothesized that PPARα activators may also alter Id2 expression. Fenofibrate diminished hepatic Id2 expression in both late pregnant and unmated rats. In 24 hour fasted rats, Id2 expression was decreased under conditions known to activate PPARα. In order to determine whether the fibrate effects were mediated by PPARα, wild-type mice and PPARα-null mice were treated with Wy-14,643 (WY). WY reduced Id2 expression in wild-type mice without an effect in PPARα-null mice. In contrast, fenofibrate induced Id2 expression after 24 hours of treatment in human hepatocarcinoma cells (HepG2). MK-886, a PPARα antagonist, did not block fenofibrate-induced activation of Id2 expression, suggesting a PPARα-independent effect was involved. These findings confirm that Id2 is a gene responsive to PPARα agonists. Like other genes (apolipoprotein A-I, apolipoprotein A-V), the opposite directional transcriptional effect in rodents and a human cell line further emphasizes that PPARα agonists have different effects in rodents and humans. PMID:22701468

  4. A novel peroxisome proliferator-activated receptor alpha/gamma dual agonist demonstrates favorable effects on lipid homeostasis.

    PubMed

    Guo, Qiu; Sahoo, Soumya P; Wang, Pei-Ran; Milot, Denise P; Ippolito, Marc C; Wu, Margaret S; Baffic, Joanne; Biswas, Chhabi; Hernandez, Melba; Lam, My-Hanh; Sharma, Neelam; Han, Wei; Kelly, Linda J; MacNaul, Karen L; Zhou, Gaochao; Desai, Ranjit; Heck, James V; Doebber, Thomas W; Berger, Joel P; Moller, David E; Sparrow, Carl P; Chao, Yu-Sheng; Wright, Samuel D

    2004-04-01

    Patients with type 2 diabetes mellitus exhibit hyperglycemia and dyslipidemia as well as a markedly increased incidence of atherosclerotic cardiovascular disease. Here we report the characterization of a novel arylthiazolidinedione capable of lowering both glucose and lipid levels in animal models. This compound, designated TZD18, is a potent agonist with dual human peroxisome proliferator-activated receptor (PPAR)-alpha/gamma activities. In keeping with its PPARgamma activity, TZD18 caused complete normalization of the elevated glucose in db/db mice and Zucker diabetic fatty rats. TZD18 lowered both cholesterol and triglycerides in hamsters and dogs. TZD18 inhibited cholesterol biosynthesis at steps before mevalonate and reduced hepatic levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity. Moreover, TZD18 significantly suppressed gene expression of fatty acid synthesis and induced expression of genes for fatty acid degradation and triglyceride clearance. Studies on 17 additional PPARalpha or PPARalpha/gamma agonists showed that lipid lowering in hamsters correlated with the magnitude of hepatic gene expression changes. Importantly, the presence of PPARgamma agonism did not affect the relationship between hepatic gene expression and lipid lowering. Taken together, these data suggest that PPARalpha/gamma agonists, such as TZD18, affect lipid homeostasis, leading to an antiatherogenic plasma lipid profile. Agents with these properties may provide favorable means for treatment of type 2 diabetes and dyslipidemia and the prevention of atherosclerotic cardiovascular disease.

  5. Selective CB2 receptor agonists. Part 2: Structure-activity relationship studies and optimization of proline-based compounds.

    PubMed

    Riether, Doris; Zindell, Renee; Wu, Lifen; Betageri, Raj; Jenkins, James E; Khor, Someina; Berry, Angela K; Hickey, Eugene R; Ermann, Monika; Albrecht, Claudia; Ceci, Angelo; Gemkow, Mark J; Nagaraja, Nelamangala V; Romig, Helmut; Sauer, Achim; Thomson, David S

    2015-02-01

    Through a ligand-based pharmacophore model (S)-proline based compounds were identified as potent cannabinoid receptor 2 (CB2) agonists with high selectivity over the cannabinoid receptor 1 (CB1). Structure-activity relationship investigations for this compound class lead to oxo-proline compounds 21 and 22 which combine an impressive CB1 selectivity profile with good pharmacokinetic properties. In a streptozotocin induced diabetic neuropathy model, 22 demonstrated a dose-dependent reversal of mechanical hyperalgesia.

  6. Design of Potent and Orally Active GPR119 Agonists for the Treatment of Type II Diabetes

    PubMed Central

    2015-01-01

    We report herein the design and synthesis of a series of potent and selective GPR119 agonists. Our objective was to develop a GPR119 agonist with properties that were suitable for fixed-dose combination with a DPP4 inhibitor. Starting from a phenoxy analogue (1), medicinal chemistry efforts directed toward reducing half-life and increasing solubility led to the synthesis of a series of benzyloxy analogues. Compound 28 was chosen for further profiling because of its favorable physicochemical properties and excellent GPR119 potency across species. This compound exhibited a clean off-target profile in counterscreens and good in vivo efficacy in mouse oGTT. PMID:26288697

  7. Peroxisome proliferator-activated receptor-gamma agonist rosiglitazone attenuates postincisional pain by regulating macrophage polarization

    SciTech Connect

    Hasegawa-Moriyama, Maiko; Ohnou, Tetsuya; Godai, Kohei; Kurimoto, Tae; Nakama, Mayo; Kanmura, Yuichi

    2012-09-14

    Highlights: Black-Right-Pointing-Pointer Rosiglitazone attenuated postincisional pain. Black-Right-Pointing-Pointer Rosiglitazone alters macrophage polarization to F4/80{sup +}CD206{sup +} M2 macrophages at the incisional sites. Black-Right-Pointing-Pointer Transplantation of rosiglitazone-treated macrophages produced analgesic effects. -- Abstract: Acute inflammation triggered by macrophage infiltration to injured tissue promotes wound repair and may induce pain hypersensitivity. Peroxisome proliferator-activated receptor {gamma} (PPAR){gamma} signaling is known to regulate heterogeneity of macrophages, which are often referred to as classically activated (M1) and alternatively activated (M2) macrophages. M1 macrophages have considerable antimicrobial activity and produce a wide variety of proinflammatory cytokines. In contrast, M2 macrophages are involved in anti-inflammatory and homeostatic functions linked to wound healing and tissue repair. Although it has been suggested that PPAR{gamma} agonists attenuate pain hypersensitivity, the molecular mechanism of macrophage-mediated effects of PPAR{gamma} signaling on pain development has not been explored. In this study, we investigated the link between the phenotype switching of macrophage polarization induced by PPAR{gamma} signaling and the development of acute pain hypersensitivity. Local administration of rosiglitazone significantly ameliorated hypersensitivity to heat and mechanical stimuli, and paw swelling. Consistent with the down-regulation of nuclear factor {kappa}B (NF{kappa}B) phosphorylation by rosiglitazone at the incisional sites, the number of F4/80{sup +}iNOS{sup +} M1 macrophages was decreased whereas numbers of F4/80{sup +}CD206{sup +} M2 macrophages were increased in rosiglitazone-treated incisional sites 24 h after the procedure. In addition, gene induction of anti-inflammatory M2-macrophage-associated markers such as arginase1, FIZZ1 and interleukin (IL)-10 were significantly increased, whereas

  8. Diabetes or peroxisome proliferator-activated receptor alpha agonist increases mitochondrial thioesterase I activity in heart

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Peroxisome proliferator-activated receptor alpha (PPAR alpha) is a transcriptional regulator of the expression of mitochondrial thioesterase I (MTE-I) and uncoupling protein 3 (UCP3), which are induced in the heart at the mRNA level in response to diabetes. Little is known about the regulation of pr...

  9. Efficient help for autoreactive B-cell activation requires CD4+ T-cell recognition of an agonist peptide at the effector stage.

    PubMed

    Hondowicz, Brian D; Batheja, Amrita O; Metzgar, Michele H; Pagán, Antonio J; Perng, Olivia A; Willms, Simone; Caton, Andrew J; Erikson, Jan

    2009-09-01

    T-cell recognition of peptide/MHC complexes is flexible and can lead to differential activation, but how interactions with agonist (full activation) or partial agonist (suboptimal activation) peptides can shape immune responses in vivo is not well characterized. We investigated the effect of stimulation by agonist or partial agonist ligands during initial CD4(+) T-cell priming, and subsequent T-B-cell cognate interactions, on antibody production by anti-chromatin B cells. We found that autoantibody production required TCR recognition of an agonist peptide at the effector stage of B-cell activation. However, interaction with a weak agonist ligand at this effector stage failed to promote efficient autoantibody production, even if the CD4(+) T cells were fully primed by an agonist peptide. These studies suggest that the reactivity of the TCR for a target self-peptide during CD4(+) T-B-cell interaction can be a critical determinant in restraining anti-chromatin autoantibody production.

  10. Thrombospondin-1 Mimetic Agonist Peptides Induce Selective Death in Tumor Cells: Design, Synthesis, and Structure-Activity Relationship Studies.

    PubMed

    Denèfle, Thomas; Boullet, Héloise; Herbi, Linda; Newton, Clara; Martinez-Torres, Ana-Carolina; Guez, Alexandre; Pramil, Elodie; Quiney, Claire; Pourcelot, Marilyne; Levasseur, Mikail D; Lardé, Eva; Moumné, Roba; Ogi, François-Xavier; Grondin, Pascal; Merle-Beral, Hélène; Lequin, Olivier; Susin, Santos A; Karoyan, Philippe

    2016-09-22

    Thrombospondin-1 (TSP-1) is a glycoprotein considered as a key actor within the tumor microenvironment. Its binding to CD47, a cell surface receptor, triggers programmed cell death. Previous studies allowed the identification of 4N1K decapeptide derived from the TSP-1/CD47 binding epitope. Here, we demonstrate that this peptide is able to induce selective apoptosis of various cancer cell lines while sparing normal cells. A structure-activity relationship study led to the design of the first serum stable TSP-1 mimetic agonist peptide able to trigger selective programmed cell death (PCD) of at least lung, breast, and colorectal cancer cells. Altogether, these results will be of valuable interest for further investigation in the design of potent CD47 agonist peptides, opening new perspectives for the development of original anticancer therapies.

  11. TCDD dysregulation of 13 AHR-target genes in rat liver

    SciTech Connect

    Watson, John D.; Prokopec, Stephenie D.; Smith, Ashley B.; Okey, Allan B.; Pohjanvirta, Raimo; Boutros, Paul C.

    2014-02-01

    Despite several decades of research, the complete mechanism by which 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and other xenobiotic agonists of the aryl hydrocarbon receptor (AHR) cause toxicity remains unclear. While it has been shown that the AHR is required for all major manifestations of toxicity, the specific downstream changes involved in the development of toxic phenotypes remain unknown. Here we examine a panel of 13 genes that are AHR-regulated in many species and tissues. We profiled their hepatic mRNA abundances in two rat strains with very different sensitivities to TCDD: the TCDD-sensitive Long–Evans (Turku/AB; L–E) and the TCDD-resistant Han/Wistar (Kuopio; H/W). We evaluated doses ranging from 0 to 3000 μg/kg at 19 h after TCDD exposure and time points ranging from 1.5 to 384 h after exposure to 100 μg/kg TCDD. Twelve of 13 genes responded to TCDD in at least one strain, and seven of these showed statistically significant inter-strain differences in the time course analysis (Aldh3a1, Cyp1a2, Cyp1b1, Cyp2a1, Fmo1, Nfe2l2 and Nqo1). Cyp2s1 did not respond to TCDD in either rat strain. Five genes exhibited biphasic responses to TCDD insult (Ahrr, Aldh3a1, Cyp1b1, Nfe2l2 and Nqo1), suggesting a secondary event, such as association with additional transcriptional modulators. Of the 12 genes that responded to TCDD during the dose–response analysis, none had an ED{sub 50} equivalent to that of Cyp1a1, the most sensitive gene in this study, while nine genes responded to doses at least 10–100 fold higher, in at least one strain (Ahrr (L–E), Aldh3a1 (both), Cyp1a2 (both), Cyp1b1 (both), Cyp2a1 (L–E), Inmt (both), Nfe2l2 (L–E), Nqo1 (L–E) and Tiparp (both)). These data shed new light on the association of the AHR target genes with TCDD toxicity, and in particular the seven genes exhibiting strain-specific differences represent strong candidate mediators of Type-II toxicities. - Highlights: • NanoString measured hepatic mRNA molecules

  12. Complex pharmacology of natural cannabinoids: evidence for partial agonist activity of delta9-tetrahydrocannabinol and antagonist activity of cannabidiol on rat brain cannabinoid receptors.

    PubMed

    Petitet, F; Jeantaud, B; Reibaud, M; Imperato, A; Dubroeucq, M C

    1998-01-01

    Delta9-tetrahydrocannabinol (delta9-THC), cannabinol and cannabidiol are three important natural cannabinoids from the Marijuana plant (Cannabis sativa). Using [35S]GTP-gamma-S binding on rat cerebellar homogenate as an index of cannabinoid receptor activation we show that: delta9-THC does not induce the maximal effect obtained by classical cannabinoid receptor agonists such as CP55940. Moreover at high concentration delta9-THC exhibits antagonist properties. Cannabinol is a weak agonist on rat cerebellar cannabinoid receptors and cannabidiol behaves as an antagonist acting in the micromolar range.

  13. Correction for Inhibition Leads to an Allosteric Co-Agonist Model for Pentobarbital Modulation and Activation of α1β3γ2L GABAA Receptors

    PubMed Central

    Ziemba, Alexis M.; Forman, Stuart A.

    2016-01-01

    Background Pentobarbital, like propofol and etomidate, produces important general anesthetic effects through GABAA receptors. Photolabeling also indicates that pentobarbital binds to some of the same sites where propofol and etomidate act. Quantitative allosteric co-agonist models for propofol and etomidate account for modulatory and agonist effects in GABAA receptors and have proven valuable in establishing drug site characteristics and for functional analysis of mutants. We therefore sought to establish an allosteric co-agonist model for pentobarbital activation and modulation of α1β3γ2L receptors, using a novel approach to first correct pentobarbital activation data for inhibitory effects in the same concentration range. Methods Using oocyte-expressed α1β3γ2L GABAA receptors and two-microelectrode voltage-clamp, we quantified modulation of GABA responses by a low pentobarbital concentration and direct effects of high pentobarbital concentrations, the latter displaying mixed agonist and inhibitory effects. We then isolated and quantified pentobarbital inhibition in activated receptors using a novel single-sweep “notch” approach, and used these results to correct steady-state direct activation for inhibition. Results Combining results for GABA modulation and corrected direct activation, we estimated receptor open probability and optimized parameters for a Monod-Wyman-Changeux allosteric co-agonist model. Inhibition by pentobarbital was consistent with two sites with IC50s near 1 mM, while co-agonist model parameters suggest two allosteric pentobarbital agonist sites characterized by KPB ≈ 5 mM and high efficacy. The results also indicate that pentobarbital may be a more efficacious agonist than GABA. Conclusions Our novel approach to quantifying both inhibitory and co-agonist effects of pentobarbital provides a basis for future structure-function analyses of GABAA receptor mutations in putative pentobarbital binding sites. PMID:27110714

  14. Human Receptor Activation by Aroclor 1260, a Polychlorinated Biphenyl Mixture

    PubMed Central

    Wahlang, Banrida; Falkner, K. Cameron; Clair, Heather B.; Al-Eryani, Laila; Prough, Russell A.; States, J. Christopher; Coslo, Denise M.; Omiecinski, Curtis J.; Cave, Matthew C.

    2014-01-01

    Polychlorinated biphenyls (PCBs) are persistent environmental toxicants, present in 100% of U.S. adults and dose-dependently associated with obesity and non-alcoholic fatty liver disease (NAFLD). PCBs are predicted to interact with receptors previously implicated in xenobiotic/energy metabolism and NAFLD. These receptors include the aryl hydrocarbon receptor (AhR), pregnane xenobiotic receptor (PXR), constitutive androstane receptor (CAR), peroxisome proliferator-activated receptors (PPARs), liver-X-receptor (LXRα), and farnesoid-X-receptor (FXR). This study evaluates Aroclor 1260, a PCB mixture with congener composition mimicking that of human adipose tissue, and selected congeners, as potential ligands for these receptors utilizing human hepatoma-derived (HepG2) and primate-derived (COS-1) cell lines, and primary human hepatocytes. Aroclor 1260 (20 μg/ml) activated AhR, and PCB 126, a minor component, was a potent inducer. Aroclor 1260 activated PXR in a simple concentration-dependent manner at concentrations ≥10 μg/ml. Among the congeners tested, PCBs 138, 149, 151, 174, 183, 187, and 196 activated PXR. Aroclor 1260 activated CAR2 and CAR3 variants at lower concentrations and antagonize CAR2 activation by the CAR agonist, CITCO, at higher concentrations (≥20 μg/ml). Additionally, Aroclor 1260 induced CYP2B6 in primary hepatocytes. At subtoxic doses, Aroclor 1260 did not activate LXR or FXR and had no effect on LXR- or FXR-dependent induction by the agonists T0901317 or GW4064, respectively. Aroclor 1260 (20 μg/ml) suppressed PPARα activation by the agonist nafenopin, although none of the congeners tested demonstrated significant inhibition. The results suggest that Aroclor 1260 is a human AhR, PXR and CAR3 agonist, a mixed agonist/antagonist for CAR2, and an antagonist for human PPARα. PMID:24812009

  15. Activation of α7 nicotinic receptors by orthosteric and allosteric agonists: influence on single-channel kinetics and conductance.

    PubMed

    Pałczyńska, Magda M; Jindrichova, Marie; Gibb, Alasdair J; Millar, Neil S

    2012-11-01

    Nicotinic acetylcholine receptors (nAChRs) are oligomeric transmembrane proteins in which five subunits coassemble to form a central ion channel pore. Conventional agonists, such as acetylcholine (ACh), bind to an orthosteric site, located at subunit interfaces in the extracellular domain. More recently, it has been demonstrated that nAChRs can also be activated by ligands binding to an allosteric transmembrane site. In the case of α7 nAChRs, ACh causes rapid activation and almost complete desensitization. In contrast, allosteric agonists such as 4-(4-bromophenyl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c] quin oline-8-sulfonamide (4BP-TQS) activate α7 nAChRs more slowly and cause only low levels of apparent desensitization. In the present study, single-channel patch-clamp recording has been used to investigate differences in the mechanism of activation of α7 nAChRs by ACh and 4BP-TQS. The most striking difference between activation by ACh and 4BP-TQS is in single-channel kinetics. In comparison with activation by ACh, single-channel open times and burst lengths are substantially longer (~160-800-fold, respectively), and shut times are shorter (~8-fold) when activated by 4BP-TQS. In addition, coapplication of ACh and 4BP-TQS results in a further increase in single-channel burst lengths. Mean burst lengths seen when the two agonists are coapplied (3099 ± 754 ms) are ~2.5-fold longer than with 4BP-TQS alone and ∼370-fold longer than with ACh alone. Intriguingly, the main single-channel conductance of α7 nAChRs, was significantly larger when activated by 4BP-TQS (100.3 ± 2.4 pS) than when activated by ACh (90.0 ± 2.7 pS), providing evidence that activation by allosteric and orthosteric agonists results in different α7 nAChRs open-channel conformations.

  16. Activation of α7 Nicotinic Receptors by Orthosteric and Allosteric Agonists: Influence on Single-Channel Kinetics and Conductance

    PubMed Central

    Pałczyńska, Magda M.; Jindrichova, Marie; Gibb, Alasdair J.

    2012-01-01

    Nicotinic acetylcholine receptors (nAChRs) are oligomeric transmembrane proteins in which five subunits coassemble to form a central ion channel pore. Conventional agonists, such as acetylcholine (ACh), bind to an orthosteric site, located at subunit interfaces in the extracellular domain. More recently, it has been demonstrated that nAChRs can also be activated by ligands binding to an allosteric transmembrane site. In the case of α7 nAChRs, ACh causes rapid activation and almost complete desensitization. In contrast, allosteric agonists such as 4-(4-bromophenyl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c] quin oline-8-sulfonamide (4BP-TQS) activate α7 nAChRs more slowly and cause only low levels of apparent desensitization. In the present study, single-channel patch-clamp recording has been used to investigate differences in the mechanism of activation of α7 nAChRs by ACh and 4BP-TQS. The most striking difference between activation by ACh and 4BP-TQS is in single-channel kinetics. In comparison with activation by ACh, single-channel open times and burst lengths are substantially longer (∼160–800-fold, respectively), and shut times are shorter (∼8-fold) when activated by 4BP-TQS. In addition, coapplication of ACh and 4BP-TQS results in a further increase in single-channel burst lengths. Mean burst lengths seen when the two agonists are coapplied (3099 ± 754 ms) are ∼2.5-fold longer than with 4BP-TQS alone and ∼370-fold longer than with ACh alone. Intriguingly, the main single-channel conductance of α7 nAChRs, was significantly larger when activated by 4BP-TQS (100.3 ± 2.4 pS) than when activated by ACh (90.0 ± 2.7 pS), providing evidence that activation by allosteric and orthosteric agonists results in different α7 nAChRs open-channel conformations. PMID:22874415

  17. Characterizing the role of endothelin-1 in the progression of cardiac hypertrophy in aryl hydrocarbon receptor (AhR) null mice

    SciTech Connect

    Lund, Amie K.; Goens, M. Beth; Nunez, Bethany A.; Walker, Mary K. . E-mail: mkwalker@unm.edu

    2006-04-15

    The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor characterized to play a role in detection and adaptation to environmental stimuli. Genetic deletion of AhR results in hypertension, and cardiac hypertrophy and fibrosis, associated with elevated plasma angiotensin II (Ang II) and endothelin-1 (ET-1), thus AhR appears to contribute to cardiovascular homeostasis. In these studies, we tested the hypothesis that ET-1 mediates cardiovascular pathology in AhR null mice via ET{sub A} receptor activation. First, we determine the time courses of cardiac hypertrophy, and of plasma and tissue ET-1 expression in AhR wildtype and null mice. AhR null mice exhibited increases in heart-to-body weight ratio and age-related expression of cardiac hypertrophy markers, {beta}-myosin heavy chain ({beta}-MHC), and atrial natriuretic factor (ANF), which were significant at 2 months. Similarly, plasma and tissue ET-1 expression was significantly elevated at 2 months and increased further with age. Second, AhR null mice were treated with ET{sub A} receptor antagonist, BQ-123 (100 nmol/kg/day), for 7, 28, or 58 days and blood pressure, cardiac fibrosis, and cardiac hypertrophy assessed, respectively. BQ-123 for 7 days significantly reduced mean arterial pressure in conscious, catheterized mice. BQ-123 for 28 days significantly reduced the histological appearance of cardiac fibrosis. Treatment for 58 days significantly reduced cardiac mass, assessed by heart weight, echocardiography, and {beta}-MHC and ANF expression; and reduced cardiac fibrosis as determined by osteopontin and collagen I mRNA expression. These findings establish ET-1 and the ET{sub A} receptor as primary determinants of hypertension and cardiac pathology in AhR null mice.

  18. A novel AhR ligand, 2AI, protects the retina from environmental stress

    PubMed Central

    Gutierrez, Mark A.; Davis, Sonnet S.; Rosko, Andrew; Nguyen, Steven M.; Mitchell, Kylie P.; Mateen, Samiha; Neves, Joana; Garcia, Thelma Y.; Mooney, Shaun; Perdew, Gary H.; Hubbard, Troy D.; Lamba, Deepak A.; Ramanathan, Arvind

    2016-01-01

    Various retinal degenerative diseases including dry and neovascular age-related macular degeneration (AMD), retinitis pigmentosa, and diabetic retinopathy are associated with the degeneration of the retinal pigmented epithelial (RPE) layer of the retina. This consequently results in the death of rod and cone photoreceptors that they support, structurally and functionally leading to legal or complete blindness. Therefore, developing therapeutic strategies to preserve cellular homeostasis in the RPE would be a favorable asset in the clinic. The aryl hydrocarbon receptor (AhR) is a conserved, environmental ligand-dependent, per ARNT-sim (PAS) domain containing bHLH transcription factor that mediates adaptive response to stress via its downstream transcriptional targets. Using in silico, in vitro and in vivo assays, we identified 2,2′-aminophenyl indole (2AI) as a potent synthetic ligand of AhR that protects RPE cells in vitro from lipid peroxidation cytotoxicity mediated by 4-hydroxynonenal (4HNE) as well as the retina in vivo from light-damage. Additionally, metabolic characterization of this molecule by LC-MS suggests that 2AI alters the lipid metabolism of RPE cells, enhancing the intracellular levels of palmitoleic acid. Finally, we show that, as a downstream effector of 2AI-mediated AhR activation, palmitoleic acid protects RPE cells from 4HNE-mediated stress, and light mediated retinal degeneration in mice. PMID:27364765

  19. Molecular mechanisms of p21 and p27 induction by 3-methylcholanthrene, an aryl-hydrocarbon receptor agonist, involved in antiproliferation of human umbilical vascular endothelial cells.

    PubMed

    Pang, Pai-Huei; Lin, Ying-Hsi; Lee, Yi-Hsuan; Hou, Hsing-Han; Hsu, Sung-Po; Juan, Shu-Hui

    2008-04-01

    We previously reported that 3-methylcholanthrene (3MC), an aryl-hydrocarbon receptor (AhR) agonist, inhibits the proliferation of human umbilical vascular endothelial cells (HUVECs; Juan et al., 2006, Eur J Pharmacol 530: 1-8). Herein, pretreatment of HUVECs with p21 or p27 small interfering (si)RNA reduced 3MC-induced elimination of [(3)H]thymidine incorporation, demonstrating their essential roles in the antiproliferation of HUVECs. The molecular mechanisms of p21 and p27 involved in the antiproliferative effects of 3MC were elucidated in this study. 3MC time- and concentration-dependently increased p21 and p27 levels, and decreased the protein level of CDK2 with no apparent alteration of p53. Interestingly, 3MC-mediated p21 and p27 inductions were eliminated by resveratrol, an AhR antagonist, suggesting their AhR dependency, further confirmed by AhR siRNA. Among the relevant pathways, p38MAPK activation sustained the levels of p21 and p27 induced by 3MC, which was eliminated by AhR antagonists and N-acetylcysteine (NAC), an antioxidant. 3MC concentration-dependently enhanced not only the consensus dioxin-responsive element (DRE)-driven luciferase activity, but also the binding activity of the AhR to the putative DRE derived from the p21 and p27 promoters. A deletion of the DRE (-285/-270) in p21 (-2,300/+8) only partially alleviated the 3MC-induced luciferase activity unless NAC was added, suggesting that there may be a DRE-independent mechanism associated with oxidative stress. However, a deletion of the DRE (-660/-645) in p27 (-1,358/-100) almost completely abrogated the activation. Our study demonstrated that both the functional DRE and the phosphorylation of p38MAPK are essential for the induction of p21 and p27, resulting in the antiproliferative action of 3MC in HUVECs.

  20. Promotion of Wound Healing by an Agonist of Adenosine A2A Receptor Is Dependent on Tissue Plasminogen Activator.

    PubMed

    Montesinos, M Carmen; Desai-Merchant, Avani; Cronstein, Bruce N

    2015-12-01

    Impaired wound healing, as it occurs in diabetes mellitus or long-term corticoid treatment, is commonly associated with disability, diminished quality of life, and high economic costs. Selective agonists of the A2A receptor subtype of adenosine, an endogenous regulator of inflammation, promote tissue repair in animal models, both healthy and with impaired healing. Plasmin-mediated proteolysis of fibrin and other matrix proteins is essential for cell migration at sites of injury. Since adenosine A2A receptor activation increases plasminogen activator release from macrophages and mast cells, we studied the effect of a selective agonist, CGS-21680, on full-thickness excisional wound closure in wild-type, urokinase plasminogen activator (uPA)-deficient, and tissue plasminogen activator (tPA)-deficient mice. Wound closure was impaired in tPA- and uPA-deficient mice as compared with wild-type mice, and topical application of CGS-21680 significantly increased the rate at which wounds closed in wild-type mice and uPA-deficient mice, but not in tPA-deficient mice. Immunostaining of tissue sections showed that tPA was present in endothelial cells and histiocytes by day 3 post-wound and also by day 6. In contrast, uPA was more prominent in these cell types only by day 6 post-wound. Our results confirm that plasminogen activation contributes to wound repair and are consistent with the hypothesis that adenosine A2A receptor activation promotes wound closure by a mechanism that depends upon tPA, but not uPA. Moreover, our results suggest that topical adenosine A2A receptor agonists may be useful in promotion of wound closure in patients with impaired wound healing.

  1. Sequence-Specific Modifications Enhance the Broad-Spectrum Antiviral Response Activated by RIG-I Agonists

    PubMed Central

    Chiang, Cindy; Beljanski, Vladimir; Yin, Kevin; Olagnier, David; Ben Yebdri, Fethia; Steel, Courtney; Goulet, Marie-Line; DeFilippis, Victor R.; Streblow, Daniel N.; Haddad, Elias K.; Trautmann, Lydie; Ross, Ted; Lin, Rongtuan

    2015-01-01

    ABSTRACT The cytosolic RIG-I (retinoic acid-inducible gene I) receptor plays a pivotal role in the initiation of the immune response against RNA virus infection by recognizing short 5′-triphosphate (5′ppp)-containing viral RNA and activating the host antiviral innate response. In the present study, we generated novel 5′ppp RIG-I agonists of varieous lengths, structures, and sequences and evaluated the generation of the antiviral and inflammatory responses in human epithelial A549 cells, human innate immune primary cells, and murine models of influenza and chikungunya viral pathogenesis. A 99-nucleotide, uridine-rich hairpin 5′pppRNA termed M8 stimulated an extensive and robust interferon response compared to other modified 5′pppRNA structures, RIG-I aptamers, or poly(I·C). Interestingly, manipulation of the primary RNA sequence alone was sufficient to modulate antiviral activity and inflammatory response, in a manner dependent exclusively on RIG-I and independent of MDA5 and TLR3. Both prophylactic and therapeutic administration of M8 effectively inhibited influenza virus and dengue virus replication in vitro. Furthermore, multiple strains of influenza virus that were resistant to oseltamivir, an FDA-approved therapeutic treatment for influenza, were highly sensitive to inhibition by M8. Finally, prophylactic M8 treatment in vivo prolonged survival and reduced lung viral titers of mice challenged with influenza virus, as well as reducing chikungunya virus-associated foot swelling and viral load. Altogether, these results demonstrate that 5′pppRNA can be rationally designed to achieve a maximal RIG-I-mediated protective antiviral response against human-pathogenic RNA viruses. IMPORTANCE The development of novel therapeutics to treat human-pathogenic RNA viral infections is an important goal to reduce spread of infection and to improve human health and safety. This study investigated the design of an RNA agonist with enhanced antiviral and inflammatory

  2. High agonist-independent activity is a distinguishing feature of the dopamine D1B receptor subtype.

    PubMed

    Tiberi, M; Caron, M G

    1994-11-11

    Dopamine D1A and D1B receptor subtypes belong to the superfamily of G protein-coupled receptors. Both receptors are coupled to the activation of adenylyl cyclase and exhibit distinct brain distribution. To identify functional differences, binding and stimulation of adenylyl cyclase were assessed in 293 cells expressing transiently either dopamine D1A or D1B receptors. Membranes expressing D1B receptors displayed higher affinities for agonists than those expressing D1A receptors, whereas antagonist affinities were lower at the D1B than at the D1A receptor. Basal activity of adenylyl cyclase in whole 293 cells expressing various levels of D1B receptors was significantly higher than the basal activity measured in cells expressing D1A receptors. Maximal activation of adenylyl cyclase resulting from stimulation of the D1B receptor was less than that obtained following agonist activation of the D1A receptor. In cells expressing D1B receptors, agonists displayed an increased potency for stimulating adenylyl cyclase in comparison with the potencies determined for the D1A receptor. On the other hand, certain antagonists displayed "negative efficacy" at both receptor subtypes but had a more profound inhibition on the agonist-independent signaling activity of the D1B receptor. The properties described here are reminiscent of those of constitutively active G protein-coupled receptors obtained by site-directed mutations. Thus, the D1B receptor may represent a naturally occurring receptor subtype with properties akin to those of constitutively active G protein-coupled receptors. The different anatomical distribution and biochemical properties of these D1 receptors strengthen the functional distinctions between the two subtypes and could account for the basis of heterogeneity within a given class of neurotransmitter or hormone receptors. In addition, if these properties are recapitulated in cells expressing the D1B receptors, they may underlie important role in the regulation of

  3. Parallel Functional Activity Profiling Reveals Valvulopathogens Are Potent 5-Hydroxytryptamine2B Receptor Agonists: Implications for Drug Safety Assessment

    PubMed Central

    Huang, Xi-Ping; Setola, Vincent; Yadav, Prem N.; Allen, John A.; Rogan, Sarah C.; Hanson, Bonnie J.; Revankar, Chetana; Robers, Matt; Doucette, Chris

    2009-01-01

    Drug-induced valvular heart disease (VHD) is a serious side effect of a few medications, including some that are on the market. Pharmacological studies of VHD-associated medications (e.g., fenfluramine, pergolide, methysergide, and cabergoline) have revealed that they and/or their metabolites are potent 5-hydroxytryptamine2B (5-HT2B) receptor agonists. We have shown that activation of 5-HT2B receptors on human heart valve interstitial cells in vitro induces a proliferative response reminiscent of the fibrosis that typifies VHD. To identify current or future drugs that might induce VHD, we screened approximately 2200 U.S. Food and Drug Administration (FDA)-approved or investigational medications to identify 5-HT2B receptor agonists, using calcium-based high-throughput screening. Of these 2200 compounds, 27 were 5-HT2B receptor agonists (hits); 14 of these had previously been identified as 5-HT2B receptor agonists, including seven bona fide valvulopathogens. Six of the hits (guanfacine, quinidine, xylometazoline, oxymetazoline, fenoldopam, and ropinirole) are approved medications. Twenty-three of the hits were then “functionally profiled” (i.e., assayed in parallel for 5-HT2B receptor agonism using multiple readouts to test for functional selectivity). In these assays, the known valvulopathogens were efficacious at concentrations as low as 30 nM, whereas the other compounds were less so. Hierarchical clustering analysis of the pEC50 data revealed that ropinirole (which is not associated with valvulopathy) was clearly segregated from known valvulopathogens. Taken together, our data demonstrate that patterns of 5-HT2B receptor functional selectivity might be useful for identifying compounds likely to induce valvular heart disease. PMID:19570945

  4. Dual agonist Surrobody™ simultaneously activates death receptors DR4 and DR5 to induce cancer cell death

    PubMed Central

    Milutinovic, Snezana; Kashyap, Arun K.; Yanagi, Teruki; Wimer, Carina; Zhou, Sihong; O' Neil, Ryann; Kurtzman, Aaron L.; Faynboym, Alexsandr; Xu, Li; Hannum, Charles H.; Diaz, Paul W.; Matsuzawa, Shu-ichi; Horowitz, Michael; Horowitz, Lawrence; Bhatt, Ramesh R.; Reed, John C.

    2015-01-01

    Death receptors of the Tumor Necrosis Factor (TNF) family are found on surface of most cancer cells and their activation typically kills cancer cells through the stimulation of the extrinsic apoptotic pathway. The endogenous ligand for death receptors-4 and -5 (DR4 and DR5) is Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand, TRAIL (Apo2L). Since most untransformed cells are not susceptible to TRAIL-induced apoptosis, death receptor activators have emerged as promising cancer therapeutic agents. One strategy to stimulate death receptors in cancer patients is to use soluble human recombinant TRAIL protein, but this agent has limitations of a short half-life and decoy receptor sequestration. Another strategy that attempted to evade decoy receptor sequestration and to provide improved pharmacokinetic properties was to generate DR4 or DR5 agonist antibodies. The resulting monoclonal agonist antibodies overcame the limitations of short half-life and avoided decoy receptor sequestration, but are limited by activating only one of the two death receptors. Here, we describe a DR4 and DR5 dual agonist produced using Surrobody™ technology that activates both DR4 and DR5 to induce apoptotic death of cancer cells in vitro and in vivo and also avoids decoy receptor sequestration. This fully human anti-DR4/DR5 Surrobody displays superior potency to DR4- and DR5-specific antibodies, even when combined with TRAIL-sensitizing pro-apoptotic agents. Moreover, cancer cells were less likely to acquire resistance to Surrobody than either anti-DR4 or anti-DR5 mono-specific antibodies. Taken together, Surrobody shows promising preclinical pro-apoptotic activity against cancer cells, meriting further exploration of its potential as a novel cancer therapeutic agent. PMID:26516157

  5. Neurosteroids shift partial agonist activation of GABA(A) receptor channels from low- to high-efficacy gating patterns.

    PubMed

    Bianchi, Matt T; Macdonald, Robert L

    2003-11-26

    Although GABA activates synaptic (alphabetagamma) GABA(A) receptors with high efficacy, partial agonist activation of alphabetagamma isoforms and GABA activation of the primary extrasynaptic (alphabetadelta) GABA(A) receptors are limited to low-efficacy activity, characterized by minimal desensitization and brief openings. The unusual sensitivity of alphabetadelta receptor channels to neurosteroid modulation prompted investigation of whether this high sensitivity was dependent on the delta subunit or the low-efficacy channel function that it confers. We show that the isoform specificity (alphabetadelta > alphabetagamma) of neurosteroid modulation could be reversed by conditions that reversed isoform-specific activity modes, including the use of beta-alanine to achieve increased efficacy with alphabetadelta receptors and taurine to render alphabetagamma receptors low efficacy. We suggest that neurosteroids preferentially enhance low-efficacy GABA(A) receptor activity independent of subunit composition. Allosteric conversion of partial to full agonism may be a general mechanism for reversibly scaling the efficacy of GABA(A) receptors to endogenous partial agonists.

  6. Paired Ig-Like Type 2 Receptor-Derived Agonist Ligands Ameliorate Inflammatory Reactions by Downregulating β1 Integrin Activity

    PubMed Central

    Lee, Kyoung-Jin; Lim, Dongyoung; Yoo, Yeon Ho; Park, Eun-Ji; Lee, Sun-Hee; Yadav, Birendra Kumar; Lee, Yong-Ki; Park, Jeong Hyun; Kim, Daejoong; Park, Kyeong Han; Hahn, Jang-Hee

    2016-01-01

    The paired immunoglobulin-like type 2 receptor (PILR) family consists of two functionally opposite members, inhibitory PILRα and activating PILRβ receptors. PILRs are widely expressed in various immune cells and interact with their ligands, especially CD99 expressed on activated T cells, to participate in immune responses. Here we investigated whether PILR-derived agonists inhibit β1 integrin activity as ligands for CD99. PILR-derived peptides as well as PILR-Fc fusion proteins prevented cell adhesion to fibronectin through the regulation of β1 integrin activity. Especially, PILRpep3, a representative 3-mer peptide covering the conserved motifs of the PILR extracellular domain, prevented the clustering and activation of β1 integrin by dephosphorylating FAK and vinculin, which are major components of focal adhesion. In addition, PILRpep3 inhibited transendothelial migration of monocytes as well as endothelial cell tube formation. Furthermore, upon intraperitoneal injection of PILRpep3 into mice with collagen-induced arthritis, the inflammatory response of rheumatoid arthritis was strongly suppressed. Taken together, these results suggest that PILR-derived agonist ligands may prevent the inflammatory reactions of rheumatoid arthritis by activating CD99. PMID:27306643

  7. Integration of Genome-Wide Computation DRE Search, AhR ChIP-chip and Gene Expression Analyses of TCDD-Elicited Responses in the Mouse Liver

    PubMed Central

    2011-01-01

    Background The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor (TF) that mediates responses to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Integration of TCDD-induced genome-wide AhR enrichment, differential gene expression and computational dioxin response element (DRE) analyses further elucidate the hepatic AhR regulatory network. Results Global ChIP-chip and gene expression analyses were performed on hepatic tissue from immature ovariectomized mice orally gavaged with 30 μg/kg TCDD. ChIP-chip analysis identified 14,446 and 974 AhR enriched regions (1% false discovery rate) at 2 and 24 hrs, respectively. Enrichment density was greatest in the proximal promoter, and more specifically, within ± 1.5 kb of a transcriptional start site (TSS). AhR enrichment also occurred distal to a TSS (e.g. intergenic DNA and 3' UTR), extending the potential gene expression regulatory roles of the AhR. Although TF binding site analyses identified over-represented DRE sequences within enriched regions, approximately 50% of all AhR enriched regions lacked a DRE core (5'-GCGTG-3'). Microarray analysis identified 1,896 number of TCDD-responsive genes (|fold change| ≥ 1.5, P1(t) > 0.999). Integrating this gene expression data with our ChIP-chip and DRE analyses only identified 625 differentially expressed genes that involved an AhR interaction at a DRE. Functional annotation analysis of differentially regulated genes associated with AhR enrichment identified overrepresented processes related to fatty acid and lipid metabolism and transport, and xenobiotic metabolism, which are consistent with TCDD-elicited steatosis in the mouse liver. Conclusions Details of the AhR regulatory network have been expanded to include AhR-DNA interactions within intragenic and intergenic genomic regions. Moreover, the AhR can interact with DNA independent of a DRE core suggesting there are alternative mechanisms of AhR-mediated gene regulation. PMID:21762485

  8. Nicotinic acetylcholine receptor agonist attenuates ILC2-dependent airway hyperreactivity

    PubMed Central

    Galle-Treger, Lauriane; Suzuki, Yuzo; Patel, Nisheel; Sankaranarayanan, Ishwarya; Aron, Jennifer L.; Maazi, Hadi; Chen, Lin; Akbari, Omid

    2016-01-01

    Allergic asthma is a complex and chronic inflammatory disorder that is associated with airway hyperreactivity (AHR) and driven by Th2 cytokine secretion. Type 2 innate lymphoid cells (ILC2s) produce large amounts of Th2 cytokines and contribute to the development of AHR. Here, we show that ILC2s express the α7-nicotinic acetylcholine receptor (α7nAChR), which is thought to have an anti-inflammatory role in several inflammatory diseases. We show that engagement of a specific agonist with α7nAChR on ILC2s reduces ILC2 effector function and represses ILC2-dependent AHR, while decreasing expression of ILC2 key transcription factor GATA-3 and critical inflammatory modulator NF-κB, and reducing phosphorylation of upstream kinase IKKα/β. Additionally, the specific α7nAChR agonist reduces cytokine production and AHR in a humanized ILC2 mouse model. Collectively, our data suggest that α7nAChR expressed by ILC2s is a potential therapeutic target for the treatment of ILC2-mediated asthma. PMID:27752043

  9. Characterization of a new synthetic isoflavonoid with inverse agonist activity at the central benzodiazepine receptor.

    PubMed

    Lopes, Daniele V S; Caruso, Rodrigo R B; Castro, Newton G; Costa, Paulo R R; da Silva, Alcides J M; Noël, François

    2004-07-14

    Research aimed at developing selective drugs acting on gamma-aminobutyric acid (GABA)A receptors introduced compounds from diverse chemical classes unrelated to the 1,4-benzodiazepines, including flavonoids. These studies also revealed the potential use of inverse agonists as cognition-enhancing agents. Here we report pharmacological properties of the novel synthetic isoflavonoid 2-methoxy-3,8,9-trihydroxy coumestan (PCALC36). PCALC36 displaced [3H]flunitrazepam binding to rat brain synaptosomes with an IC50 of 13.8 microM. Scatchard analysis of the effect of PCALC36 showed a concentration-dependent reduction of the Bmax of [3H]flunitrazepam, without a marked change in Kd. This effect could be reversed by diluting and washing the preparation. Addition of 20-microM GABA shifted to the right the inhibition curve of PCALC36 on [3H]flunitrazepam binding (IC50 ratio of 0.68), which is characteristic for inverse agonists. PCALC36 produced little change in the GABAergic tonic currents recorded by whole-cell patch clamp in cultured rat hippocampal neurones, but it caused a 20% reduction in miniature inhibitory postsynaptic current amplitude and completely antagonised the full (direct) agonist midazolam in a quickly reversible manner. The data suggest that the coumestan backbone can be useful for developing novel ligands at the GABAA receptor.

  10. NF-κB activation primes cells to a pro-inflammatory polarized response to a TLR7 agonist

    PubMed Central

    Lee, Jongdae; Hayashi, Masaaki; Lo, Jeng-Fan; Fearns, Colleen; Chu, Wen-Ming; Luo, Yunping; Xiang, Rong; Chuang, Tsung-Hsien

    2009-01-01

    Toll-like receptor 7 (TLR7) mediates anti-viral immunity by recognizing ssRNA viruses. Small molecular weight TLR7 agonists have been approved, or are being evaluated, for treatment of cancers or infectious diseases. Although TLR7 is predominantly expressed in a restricted set of immune cell types including plasmacytoid dendritic cells (pDCs), it is also expressed in non-native expressing cells (e.g., hepatocytes) under certain circumstances. To elucidate the molecular basis of TLR7 induction by pro-inflammatory stimulation and the subsequent cellular responses in these non-native TLR7-expressing cell types, we firstly cloned and characterized the 5′-promoter region of TLR7. The proximal region of this promoter drives the transcription of the TLR7 gene. Pro-inflammatory stimuli activated TLR7 transcription via a NF-κB binding motif in this region, and this activation could be blocked by mutation of the NF-κB binding site or addition of NF-κB inhibitors. Further studies showed that pretreatment of the Hep3B hepatocytes with TNF-α or IL-1 rendered them responsive to TLR7 activation by a TLR7 agonist. However, distinct from TLR7 activation in pDCs, which respond to stimulation with Th1 polarized cytokine production, TLR7 induction by pro-inflammatory signals in hepatocytes reconstitutes the NF-κB-dependent cascade but not the IRF7-dependent cascade, resulting in a pro-inflammatory polarized response rather than a Th1 polarized response. These results indicate that inflammatory stimulation is capable of priming cells to respond to TLR7 agonist with an immune response that differs from that in native TLR7-expressing cells. PMID:19426145

  11. Acute fatigue impairs neuromuscular activity of anterior cruciate ligament-agonist muscles in female team handball players.

    PubMed

    Zebis, M K; Bencke, J; Andersen, L L; Alkjaer, T; Suetta, C; Mortensen, P; Kjaer, M; Aagaard, P

    2011-12-01

    In sports, like team handball, fatigue has been associated with an increased risk of anterior cruciate ligament (ACL) injury. While effects of fatigue on muscle function are commonly assessed during maximal isometric voluntary contraction (MVC), such measurements may not relate to the muscle function during match play. The purpose of this study was to investigate the effect of muscle fatigue induced by a simulated handball match on neuromuscular strategy during a functional sidecutting movement, associated with the incidence of ACL injury. Fourteen female team handball players were tested for neuromuscular activity [electromyography (EMG)] during a sidecutting maneuver on a force plate, pre and post a simulated handball match. MVC was obtained during maximal isometric quadriceps and hamstring contraction. The simulated handball match consisted of exercises mimicking handball match activity. Whereas the simulated handball match induced a decrease in MVC strength for both the quadriceps and hamstring muscles (P<0.05), a selective decrease in hamstring neuromuscular activity was seen during sidecutting (P<0.05). This study shows impaired ACL-agonist muscle (i.e. hamstring) activity during sidecutting in response to acute fatigue induced by handball match play. Thus, screening procedures should involve functional movements to reveal specific fatigue-induced deficits in ACL-agonist muscle activation during high-risk phases of match play.

  12. Prevention of RhoA activation and cofilin-mediated actin polymerization mediates the antihypertrophic effect of adenosine receptor agonists in angiotensin II- and endothelin-1-treated cardiomyocytes.

    PubMed

    Zeidan, Asad; Gan, Xiaohong Tracey; Thomas, Ashley; Karmazyn, Morris

    2014-01-01

    Adenosine receptor activation has been shown to be associated with diminution of cardiac hypertrophy and it has been suggested that endogenously produced adenosine may serve to blunt pro-hypertrophic processes. In the present study, we determined the effects of two pro-hypertrophic stimuli, angiotensin II (Ang II, 100 nM) and endothelin-1 (ET-1, 10 nM) on Ras homolog gene family, member A (RhoA)/Rho-associated, coiled-coil containing protein kinase (ROCK) activation in cultured neonatal rat ventricular myocytes and whether the latter serves as a target for the anti-hypertrophic effect of adenosine receptor activation. Both hypertrophic stimuli potently increased RhoA activity with peak activation occurring 15-30 min following agonist addition. These effects were associated with significantly increased phosphorylation (inactivation) of cofilin, a downstream mediator of RhoA, an increase in actin polymerization, and increased activation and nuclear import of p38 mitogen activated protein kinase. The ability of both Ang II and ET-1 to activate the RhoA pathway was completely prevented by the adenosine A1 receptor agonist N (6)-cyclopentyladenosine, the A2a receptor agonist 2-p-(2-carboxyethyl)-phenethylamino-5'-N-ethylcarboxamidoadenosine, the A3 receptor agonist N (6)-(3-iodobenzyl)adenosine-5'-methyluronamide as well as the nonspecific adenosine analog 2-chloro adenosine. All effects of specific receptor agonists were prevented by their respective receptor antagonists. Moreover, all adenosine agonists prevented either Ang II- or ET-1-induced hypertrophy, a property shared by the RhoA inhibitor Clostridium botulinum C3 exoenzyme, the ROCK inhibitor Y-27632 or the actin depolymerizing agent latrunculin B. Our study therefore demonstrates that both Ang II and ET-1 can activate the RhoA pathway and that prevention of the hypertrophic response to both agonists by adenosine receptor activation is mediated by prevention of RhoA stimulation and actin polymerization.

  13. Green fluorescent protein (GFP) as a marker of aryl hydrocarbon receptor (AhR) function in developing zebrafish (Danio rerio).

    PubMed

    Mattingly, C J; McLachlan, J A; Toscano, W A

    2001-08-01

    We developed an inducible in vivo reporter system to examine expression of the aryl hydrocarbon receptor (AhR) during development in zebrafish (Danio rerio). AhR is a ligand-activated transcription factor that mediates the toxic actions of environmental contaminants such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Induction of cytochrome P4501A1 (CYP1A1) is an early biomarker of AhR activation. A 1905 base pair region of the human CYP1A1 promoter/enhancer region was regulated by AhR in zebrafish liver cells after exposure to TCDD (10 nM) in a transient transfection assay. This regulatory region was fused to the cDNA sequence encoding green fluorescent protein (GFP) of jellyfish (Aequorea victoria). Transgenic zebrafish were generated to express this AhR-regulated GFP construct. Injected fish exposed to TCDD exhibited induction of GFP in the eye, nose, and vertebrae of zebrafish embryos (48 and 72 hr after fertilization) compared to vehicle controls (DMSO), which did not express GFP. To investigate whether AhR-regulated GFP expression correlated with sites of TCDD toxicity, we exposed wild-type zebrafish to DMSO or TCDD and examined them for morphologic abnormalities. By 5 days after fertilization, TCDD-exposed fish exhibited gross dysmorphogenesis in cranio-facial and vertebral development.

  14. Differential regulation of the mitogen-activated protein and stress-activated protein kinase cascades by adrenergic agonists in quiescent and regenerating adult rat hepatocytes.

    PubMed Central

    Spector, M S; Auer, K L; Jarvis, W D; Ishac, E J; Gao, B; Kunos, G; Dent, P

    1997-01-01

    To study the mechanisms by which catecholamines regulate hepatocyte proliferation after partial hepatectomy (PHX), hepatocytes were isolated from adult male rats 24 h after sham operation or two-thirds PHX and treated with catecholamines and other agonists. In freshly isolated sham cells, p42 mitogen-activated protein (MAP) kinase activity was stimulated by the alpha1-adrenergic agonist phenylephrine (PHE). Activation of p42 MAP kinase by growth factors was blunted by pretreatment of sham hepatocytes with glucagon but not by that with the beta2-adrenergic agonist isoproterenol (ISO). In PHX cells, the ability of PHE to activate p42 MAP kinase was dramatically reduced, whereas ISO became competent to inhibit p42 MAP kinase activation. PHE treatment of sham but not PHX and ISO treatment of PHX but not sham hepatocytes also activated the stress-activated protein (SAP) kinases p46/54 SAP kinase and p38 SAP kinase. These data demonstrate that an alpha1- to beta2-adrenergic receptor switch occurs upon PHX and results in an increase in SAP kinase versus MAP kinase signaling by catecholamines. In primary cultures of hepatocytes, ISO treatment of PHX but not sham cells inhibited [3H]thymidine incorporation. In contrast, PHE treatment of sham but not PHX cells stimulated [3H]thymidine incorporation, which was reduced by approximately 25 and approximately 95% with specific inhibitors of p42 MAP kinase and p38 SAP kinase function, respectively. Inhibition of the p38 SAP kinase also dramatically reduced basal [3H]thymidine incorporation. These data suggest that p38 SAP kinase plays a permissive role in liver regeneration. Alterations in the abilities of catecholamines to modulate the activities of protein kinase A and the MAP and SAP kinase pathways may represent one physiological mechanism by which these agonists can regulate hepatocyte proliferation after PHX. PMID:9199291

  15. [Development of agonists/antagonists for protease-activated receptors (PARs) and the possible therapeutic application to gastrointestinal diseases].

    PubMed

    Sekiguchi, Fumiko

    2005-06-01

    Protease-activated receptors (PARs), a family of G-protein-coupled seven-transmembrane-domain receptors, are activated by proteolytic unmasking of the N-terminal cryptic tethered ligand by certain serine proteases. Among four PAR family members cloned to date, PAR-1, PAR-2, and PAR-4 can also be activated through a non-enzymatic mechanism, which is achieved by direct binding of exogenously applied synthetic peptides based on the tethered ligand sequence, known as PARs-activating peptides, to the body of the receptor. Various peptide mimetics have been synthesized as agonists for PARs with improved potency, selectivity, and stability. Some peptide mimetics and/or nonpeptide compounds have also been developed as antagonists for PAR-1 and PAR-4. PARs are widely distributed in the mammalian body, especially throughout the alimentary systems, and play various roles in physiological/pathophysiological conditions, i.e., modulation of salivary, gastric, or pancreatic glandular exocrine secretion, gastrointestinal smooth muscle motility, gastric mucosal cytoprotection, suppression/facilitation of visceral pain and inflammation, etc. Thus PARs are now considered novel therapeutic targets, and development of selective agonists and/or antagonists for PARs might provide a novel strategy for the treatment of various diseases that are resistant to current therapeutics.

  16. PPAR-γ agonist stabilizes KLF4 protein via activating Akt signaling and reducing KLF4 ubiquitination

    SciTech Connect

    Sun, Yan; Zheng, Bin; Zhang, Xin-hua; He, Ming; Guo, Zong-wei; Wen, Jin-kun

    2014-01-10

    Highlights: •PPAR-γ increases KLF4 protein level but does not influence KLF4 gene transcription. •The increase of KLF4 protein levels induced by pioglitazone is PPAR-γ-dependent. •Pioglitazone stabilizes KLF4 protein via activating Akt signaling and reducing KLF4 ubiquitination. -- Abstract: Peroxisome proliferator activated receptor γ (PPAR-γ) plays important roles in cell cycle regulation, differentiation and apoptosis. Krüppel-like factor 4 (KLF4) modulates vascular smooth muscle cell (VSMC) phenotype. Both KLF4 and PPAR-γ are involved in VSMC proliferation and differentiation. However, the actual relationship between KLF4 and PPAR-γ in VSMCs is not clear. In this study, we found that PPAR-γ agonist pioglitazone increases KLF4 protein levels but does not influence KLF4 gene transcription. PPAR-γ overexpression increases, while PPAR-γ knockdown reduces KLF4 expression, suggesting that the increase in KLF4 protein levels induced by pioglitazone is PPAR-γ-dependent. Further study showed that pioglitazone enhances KLF4 protein stability through reducing KLF4 ubiquitination. Furthermore, we demonstrated that stabilization of KLF4 by pioglitazone was related to the activation of Akt signaling pathway. Taken together, we revealed that PPAR-γ agonist pioglitazone stabilizes KLF4 protein via activating Akt signaling and reducing KLF4 ubiquitination, providing further insights into PPAR-γ and KLF4 in regulating each other’s expression in VSMCs.

  17. Discovery of new dual PPARγ-GPR40 agonists with robust antidiabetic activity: Design, synthesis and in combo drug evaluation.

    PubMed

    Hidalgo-Figueroa, Sergio; Navarrete-Vázquez, Gabriel; Estrada-Soto, Samuel; Giles-Rivas, Diana; Alarcón-Aguilar, Francisco J; León-Rivera, Ismael; Giacoman-Martínez, Abraham; Miranda Pérez, Elizabeth; Almanza-Pérez, Julio C

    2017-03-22

    The design of compounds 1 and 2 was based on the similar scaffold of pharmacophoric groups for PPARγ and GPR40 agonists. In order to find new compounds with improved biological activity, the current manuscript describes a new dual PPARγ-GPR40 agonist. We synthesized two compounds, which were prepared following a multistep synthetic route, and the relative mRNA expression levels of PPARγ, GLUT4, and GPR40 were quantified in cell culture, as well as insulin secretion and [Ca(2+)] intracellular levels. Compound 1 showed a 7-times increase in the mRNA expression of PPARγ, which in turn enhanced the expression levels of GLUT4 respect to control and pioglitazone. It also showed an increase of 2-fold in the [Ca(2+)]i level allowing an increment on insulin release, being as active as the positive control (glibenclamide), causing also an increase of 2-fold in mRNA expression of GPR40. Furthermore, the compound 2 showed lower activity than the compound 1. The ester of 1 showed antidiabetic activity at a 50mg/kg single dose in streptozotocin-nicotinamide-induced diabetic mice model. In addition, we achieved a molecular docking study of compound 1 on PPARγ and GPR40 receptors, showing a great affinity for both targets. We observed important polar interactions between the carboxylic group and main residues into the binding pocket. Therefore, the compound 1 has a potential for the development of antidiabetic agents with newfangled dual action.

  18. Design and implementation of SMO for a nonlinear MIMO AHRS

    NASA Astrophysics Data System (ADS)

    Doostdar, Parisa; Keighobadi, Jafar

    2012-10-01

    In a low-cost attitude heading reference system (AHRS), the measurements made by MEMS inertial and magnetic sensors are affected by large parameter uncertainties, stochastic noises and unknown disturbances. In this paper, considering the robustness of the sliding mode observers (SMO) against both structured and unstructured uncertainties as well as exogenous inputs, the process of design and implementation of a nonlinear SMO is proposed for a low-cost AHRS. For simultaneous estimation of orientation variables and calibration biases of gyroscopes, a nonlinear and non-affine model of the AHRS is considered. Therefore, based on the Lie-algebraic method, the estimation algorithm is designed for a general class of non-affine nonlinear MIMO systems. In the proposed observer, owing to decreasing the required assumptions for coordinate transformation in recent literatures, the design process of the SMO is simplified. The gain matrices of the proposed SMO are obtained through ensuring the stability and the convergence of estimation errors based on Lyapunov's direct method. The expected tracking performance of the robust state and parameter estimation algorithm compared to that of the extended Kalman filter (EKF) is evaluated through simulations and real experiments of a strapped AHRS on a ground vehicle.

  19. An intrinsic agonist mechanism for activation of glucagon-like peptide-1 receptor by its extracellular domain

    PubMed Central

    Yin, Yanting; Zhou, X Edward; Hou, Li; Zhao, Li-Hua; Liu, Bo; Wang, Gaihong; Jiang, Yi; Melcher, Karsten; Xu, H Eric

    2016-01-01

    The glucagon-like peptide-1 receptor is a class B G protein coupled receptor (GPCR) that plays key roles in glucose metabolism and is a major therapeutic target for diabetes. The classic two-domain model for class B GPCR activation proposes that the apo-state receptor is auto-inhibited by its extracellular domain, which physically interacts with the transmembrane domain. The binding of the C-terminus of the peptide hormone to the extracellular domain allows the N-terminus of the hormone to insert into the transmembrane domain to induce receptor activation. In contrast to this model, here we demonstrate that glucagon-like peptide-1 receptor can be activated by N-terminally truncated glucagon-like peptide-1 or exendin-4 when fused to the receptor, raising the question regarding the role of N-terminal residues of peptide hormone in glucagon-like peptide-1 receptor activation. Mutations of cysteine 347 to lysine or arginine in intracellular loop 3 transform the receptor into a G protein-biased receptor and allow it to be activated by a nonspecific five-residue linker that is completely devoid of exendin-4 or glucagon-like peptide-1 sequence but still requires the presence of an intact extracellular domain. Moreover, the extracellular domain can activate the receptor in trans in the presence of an intact peptide hormone, and specific mutations in three extracellular loops abolished this extracellular domain trans-activation. Together, our data reveal a dominant role of the extracellular domain in glucagon-like peptide-1 receptor activation and support an intrinsic agonist model of the extracellular domain, in which peptide binding switches the receptor from the auto-inhibited state to the auto-activated state by releasing the intrinsic agonist activity of the extracellular domain. PMID:27917297

  20. An intrinsic agonist mechanism for activation of glucagon-like peptide-1 receptor by its extracellular domain.

    PubMed

    Yin, Yanting; Zhou, X Edward; Hou, Li; Zhao, Li-Hua; Liu, Bo; Wang, Gaihong; Jiang, Yi; Melcher, Karsten; Xu, H Eric

    2016-01-01

    The glucagon-like peptide-1 receptor is a class B G protein coupled receptor (GPCR) that plays key roles in glucose metabolism and is a major therapeutic target for diabetes. The classic two-domain model for class B GPCR activation proposes that the apo-state receptor is auto-inhibited by its extracellular domain, which physically interacts with the transmembrane domain. The binding of the C-terminus of the peptide hormone to the extracellular domain allows the N-terminus of the hormone to insert into the transmembrane domain to induce receptor activation. In contrast to this model, here we demonstrate that glucagon-like peptide-1 receptor can be activated by N-terminally truncated glucagon-like peptide-1 or exendin-4 when fused to the receptor, raising the question regarding the role of N-terminal residues of peptide hormone in glucagon-like peptide-1 receptor activation. Mutations of cysteine 347 to lysine or arginine in intracellular loop 3 transform the receptor into a G protein-biased receptor and allow it to be activated by a nonspecific five-residue linker that is completely devoid of exendin-4 or glucagon-like peptide-1 sequence but still requires the presence of an intact extracellular domain. Moreover, the extracellular domain can activate the receptor in trans in the presence of an intact peptide hormone, and specific mutations in three extracellular loops abolished this extracellular domain trans-activation. Together, our data reveal a dominant role of the extracellular domain in glucagon-like peptide-1 receptor activation and support an intrinsic agonist model of the extracellular domain, in which peptide binding switches the receptor from the auto-inhibited state to the auto-activated state by releasing the intrinsic agonist activity of the extracellular domain.

  1. Constitutive IDO expression in human cancer is sustained by an autocrine signaling loop involving IL-6, STAT3 and the AHR

    PubMed Central

    Sahm, Felix; Rauschenbach, Katharina J.; Trump, Saskia; Winter, Marcus; Ott, Martina; Ochs, Katharina; Lutz, Christian; Liu, Xiangdong; Anastasov, Natasa; Lehmann, Irina; Höfer, Thomas; von Deimling, Andreas; Wick, Wolfgang; Platten, Michael

    2014-01-01

    Indoleamine-2,3-dioxygenase (IDO) inhibitors have entered clinical trials based on their ability to restore anti-tumor immunity in preclinical studies. However, the mechanisms leading to constitutive expression of IDO in human tumors are largely unknown. Here we analyzed the pathways mediating constitutive IDO expression in human cancer. IDO-positive tumor cells and tissues showed basal phosphorylation and acetylation of STAT3 as evidenced by western blotting and immunoprecipitation. Inhibition of IL-6 or STAT3 using siRNA and/or pharmacological inhibitors reduced IDO mRNA and protein expression as well as kynurenine formation. In turn, IDO enzymatic activity activated the AHR as shown by the induction of AHR target genes. IDO-mediated AHR activation induced IL-6 expression, while inhibition or knockdown of the AHR reduced IL-6 expression. IDO activity thus sustains its own expression via an autocrine AHR–IL-6–STAT3 signaling loop. Inhibition of the AHR–IL-6–STAT3 signaling loop restored T-cell proliferation in mixed leukocyte reactions performed in the presence of IDO-expressing human cancer cells. Identification of the IDO-AHR-IL-6-STAT3 signaling loop maintaining IDO expression in human cancers reveals novel therapeutic targets for the inhibition of this core pathway promoting immunosuppression of human cancers. The relevance of the IDO-AHR-IL-6-STAT3 transcriptional circuit is underscored by the finding that high expression of its members IDO, STAT3 and the AHR target gene CYP1B1 is associated with reduced relapse-free survival in lung cancer patients. PMID:24657910

  2. Inhibition of indoleamine 2,3-dioxygenase activity enhances the anti-tumour effects of a Toll-like receptor 7 agonist in an established cancer model.

    PubMed

    Ito, Hiroyasu; Ando, Tatsuya; Arioka, Yuko; Saito, Kuniaki; Seishima, Mitsuru

    2015-04-01

    Toll-like receptor (TLR) agonists have been shown to have anti-tumour activity in basic research and clinical studies. However, TLR agonist monotherapy does not sufficiently eliminate tumours. Activation of the innate immune response by TLR agonists is effective at driving adaptive immunity via interleukin-12 (IL-12) or IL-1, but is counteracted by the simultaneous induction of immunosuppressive cytokines and other molecules, including IL-10, transforming growth factor-β, and indoleamine 2,3-dioxygenase (IDO). In the present study, we evaluated the anti-cancer effect of the TLR7 agonist, imiquimod (IMQ), in the absence of IDO activity. The administration of IMQ in IDO knockout (KO) mice inoculated with tumour cells significantly suppressed tumour progression compared with that in wild-type (WT) mice, and improved the survival rate. Moreover, injection with IMQ enhanced the tumour antigen-specific T helper type 1 response in IDO-KO mice with tumours. Combination therapy with IMQ and an IDO inhibitor also significantly inhibited tumour growth. Our results indicated that the enhancement of IDO expression with TLR agonists in cancer treatment might impair host anti-tumour immunity while the inhibition of IDO could enhance the therapeutic efficacy of TLR agonists via the increase of T helper type 1 immune response.

  3. CBLB502, an agonist of Toll-like receptor 5, has antioxidant and scavenging free radicals activities in vitro.

    PubMed

    Li, Weiguang; Ge, Changhui; Yang, Liu; Wang, Ruixue; Lu, Yiming; Gao, Yan; Li, Zhihui; Wu, Yonghong; Zheng, Xiaofei; Wang, Zhaoyan; Zhang, Chenggang

    2016-01-01

    The bacterial protein flagellin is the known agonist of Toll-like receptor 5 (TLR5). It has been reported that CBLB502, a novel agonist of TLR5 derived from Salmonella flagellin, could reduce radiation toxicity in mouse and primate models, protect mice from dermatitis and oral mucositis caused by radiation, inhibit acute renal ischemic failure, and inhibit the growth of A549 lung cancer cell. The property of CBLB502 is able to bind to TLR5 and activates NF-κB signaling. In this study, we investigated the antioxidant potential and free radicals scavenging properties of CBLB502 in vitro. Interestingly, we found that CBLB502 has a direct and distinct antioxidant capacity and can efficiently scavenge a variety of free radicals, including superoxide anion, hydroxyl radical, and ABTS cation (ABTS(+)). Through wave scanning and kinetic evaluation of scavenging ABTS(+), we found that the ABTS(+) scavenging process of CBLB502 is relatively slow, and the ABTS(+) scavenging activity of CBLB502 has a consistently kinetics characteristics. In conclusion, our results suggested that CBLB502 has antioxidant and scavenging free radicals activities in vitro. It is implied that CBLB502 might partially promote the beneficial protective effect through its scavenging free radicals.

  4. An Orally Active Allosteric GLP-1 Receptor Agonist Is Neuroprotective in Cellular and Rodent Models of Stroke

    PubMed Central

    Qu, Di; Wang, Ling; Wang, Xinshang; Li, Xubo; Zhou, Shimeng; Zhou, Ying; Wang, Ning; Meng, Jingru; Ma, Xue

    2016-01-01

    Diabetes is a major risk factor for the development of stroke. Glucagon-like peptide-1 receptor (GLP-1R) agonists have been in clinical use for the treatment of diabetes and also been reported to be neuroprotective in ischemic stroke. The quinoxaline 6,7-dichloro-2-methylsulfonyl-3-N-tert- butylaminoquinoxaline (DMB) is an agonist and allosteric modulator of the GLP-1R with the potential to increase the affinity of GLP-1 for its receptor. The aim of this study was to evaluate the neuroprotective effects of DMB on transient focal cerebral ischemia. In cultured cortical neurons, DMB activated the GLP-1R, leading to increased intracellular cAMP levels with an EC50 value about 100 fold that of exendin-4. Pretreatment of neurons with DMB protected against necrotic and apoptotic cell death was induced by oxygen-glucose deprivation (OGD). The neuroprotective effects of DMB were blocked by GLP-1R knockdown with shRNA but not by GLP-1R antagonism. In C57BL/6 mice, DMB was orally administered 30 min prior to middle cerebral artery occlusion (MCAO) surgery. DMB markedly reduced the cerebral infarct size and neurological deficits caused by MCAO and reperfusion. The neuroprotective effects were mediated by activation of the GLP-1R through the cAMP-PKA-CREB signaling pathway. DMB exhibited anti-apoptotic effects by modulating Bcl-2 family members. These results provide evidence that DMB, a small molecular GLP-1R agonist, attenuates transient focal cerebral ischemia injury and inhibits neuronal apoptosis induced by MCAO. Taken together, these data suggest that DMB is a potential neuroprotective agent against cerebral ischemia. PMID:26863436

  5. Characterization testing of a 40 Ahr bipolar nickel hydrogen battery

    NASA Technical Reports Server (NTRS)

    Brewer, Jeffrey C.; Manzo, Michelle A.; Gahn, Randall F.

    1989-01-01

    In a continuing effort to develop NiH2 bipolar technology to a point where it can be used efficiently in space flight, testing of a second 40 Ahr, 10-cell bipolar battery has begun. This battery has undergone extensive characterization testing to determine the effects of such operating parameters as charge and discharge rates, temperature, and pressure. The fundamental design of this actively cooled bipolar battery is the same as the first battery. Most of the individual components, however, are from different manufacturers. Different testing procedures as well as certain unique battery characteristics make it difficult to directly compare the two sets of results. In general, the performance of this battery throughout characterization produced expected results. The main differences seen between the first and second batteries occurred during the high-rate discharge portion of the test matrix. The first battery also had poor high-rate discharge results, although better than those of the second battery. Minor changes were made to the battery frame design used for the first battery in an attempt to allow better gas access to the reaction sites for the second build and hopefully improve performance. The changes, however, did not improve the performance of the second battery and could have possibly contributed to the poorer performance that was observed. There are other component differences that could have contributed to the poorer performance of the second battery. The H2 electrode in the second battery was constructed with a Goretex backing which could have limited the high-rate current flow. The gas screen in the second battery had a larger mesh which again could have limited the high-rate current flow. Small scale 2 x 2 batteries are being tested to evaluate the effects of the component variations.

  6. Characterization of CM572, a Selective Irreversible Partial Agonist of the Sigma-2 Receptor with Antitumor Activity.

    PubMed

    Nicholson, Hilary; Comeau, Anthony; Mesangeau, Christophe; McCurdy, Christopher R; Bowen, Wayne D

    2015-08-01

    The sigma-2 receptors are promising therapeutic targets because of their significant upregulation in tumor cells compared with normal tissue. Here, we characterize CM572 [3-(4-(4-(4-fluorophenyl)piperazin-1-yl)butyl)-6-isothiocyanatobenzo[d]oxazol-2(3H)-one] (sigma-1 Ki ≥ 10 µM, sigma-2 Ki = 14.6 ± 6.9 nM), a novel isothiocyanate derivative of the putative sigma-2 antagonist, SN79 [6-acetyl-3-(4-(4-(4-fluorophenyl)piperazin-1-yl)butyl)benzo[d]oxazol-2(3H)-one]. CM572 bound irreversibly to sigma-2 receptors by virtue of the isothiocyanate moiety but not to sigma-1. Studies in human SK-N-SH neuroblastoma cells revealed that CM572 induced an immediate dose-dependent increase in cytosolic calcium concentration. A 24-hour treatment of SK-N-SH cells with CM572 induced dose-dependent cell death, with an EC50 = 7.6 ± 1.7 µM. This effect was sustained over 24 hours even after a 60-minute pretreatment with CM572, followed by extensive washing to remove ligand, indicating an irreversible effect consistent with the irreversible binding data. Western blot analysis revealed that CM572 also induced cleavage activation of proapoptotic BH3-interacting domain death agonist. These data suggest irreversible agonist-like activity. Low concentrations of CM572 that were minimally effective were able to attenuate significantly the calcium signal and cell death induced by the sigma-2 agonist CB-64D [(+)-1R,5R-(E)-8-benzylidene-5-(3-hydroxyphenyl)-2-methylmorphan-7-one]. CM572 was also cytotoxic against PANC-1 pancreatic and MCF-7 breast cancer cell lines. The cytotoxic activity of CM572 was selective for cancer cells over normal cells, being much less potent against primary human melanocytes and human mammary epithelial cells. Taken together, these data show that CM572 is a selective, irreversible sigma-2 receptor partial agonist. This novel irreversible ligand may further our understanding of the endogenous role of this receptor, in addition to having potential use in targeted

  7. Characterization of CM572, a Selective Irreversible Partial Agonist of the Sigma-2 Receptor with Antitumor Activity

    PubMed Central

    Nicholson, Hilary; Comeau, Anthony; Mesangeau, Christophe; McCurdy, Christopher R.

    2015-01-01

    The sigma-2 receptors are promising therapeutic targets because of their significant upregulation in tumor cells compared with normal tissue. Here, we characterize CM572 [3-(4-(4-(4-fluorophenyl)piperazin-1-yl)butyl)-6-isothiocyanatobenzo[d]oxazol-2(3H)-one] (sigma-1 Ki ≥ 10 µM, sigma-2 Ki = 14.6 ± 6.9 nM), a novel isothiocyanate derivative of the putative sigma-2 antagonist, SN79 [6-acetyl-3-(4-(4-(4-fluorophenyl)piperazin-1-yl)butyl)benzo[d]oxazol-2(3H)-one]. CM572 bound irreversibly to sigma-2 receptors by virtue of the isothiocyanate moiety but not to sigma-1. Studies in human SK-N-SH neuroblastoma cells revealed that CM572 induced an immediate dose-dependent increase in cytosolic calcium concentration. A 24-hour treatment of SK-N-SH cells with CM572 induced dose-dependent cell death, with an EC50 = 7.6 ± 1.7 µM. This effect was sustained over 24 hours even after a 60-minute pretreatment with CM572, followed by extensive washing to remove ligand, indicating an irreversible effect consistent with the irreversible binding data. Western blot analysis revealed that CM572 also induced cleavage activation of proapoptotic BH3-interacting domain death agonist. These data suggest irreversible agonist-like activity. Low concentrations of CM572 that were minimally effective were able to attenuate significantly the calcium signal and cell death induced by the sigma-2 agonist CB-64D [(+)-1R,5R-(E)-8-benzylidene-5-(3-hydroxyphenyl)-2-methylmorphan-7-one]. CM572 was also cytotoxic against PANC-1 pancreatic and MCF-7 breast cancer cell lines. The cytotoxic activity of CM572 was selective for cancer cells over normal cells, being much less potent against primary human melanocytes and human mammary epithelial cells. Taken together, these data show that CM572 is a selective, irreversible sigma-2 receptor partial agonist. This novel irreversible ligand may further our understanding of the endogenous role of this receptor, in addition to having potential use in targeted

  8. 4-Nitrophenol exposure alters the AhR signaling pathway and related gene expression in the rat liver.

    PubMed

    Li, Ruonan; Song, Meiyan; Li, Zhi; Li, Yansen; Watanabe, Gen; Nagaoka, Kentaro; Taya, Kazuyoshi; Li, Chunmei

    2017-02-01

    4-Nitrophenol (PNP) is well known as an environmental endocrine disruptor. The aim of this study was to clarify the mechanism of PNP-induced liver damage and determine the regulatory involvement of the aryl hydrocarbon receptor (AhR) signaling pathway and associated gene expression. Immature male Wistar-Imamichi rats (28 days old) were randomly divided into control and PNP groups, which consisted of 1- and 3-day exposure (1 DE and 3 DE, respectively) and 3-day exposure followed by 3-day recovery (3 DE + 3 DR), groups. Each group was administered the vehicle or PNP (200 mg kg(-1) body weight). The body and liver weight were significantly decreased in the 3 DE group. The mRNA expression levels of estrogen receptor-α (ERα), glutathione S-transferase (GST) and AhR exhibited a significant increase in the 1 DE group whereas, in contrast, that of cytochrome P450 (CYP) 1A1 decreased significantly in the 3 DE +3 DR group. AhR and CYP1A1 proteins were detected in the cytoplasm of hepatocytes of the 1 DE and 3 DE +3 DR groups whereas the ERα protein was found in the hepatocyte nuclei of the 1 DE and 3 DE groups. The present study demonstrates that PNP activated the AhR signaling pathway and regulated related CYP1A1 and GST gene expression in the liver. Copyright © 2016 John Wiley & Sons, Ltd.

  9. Analysis of the AHR gene proximal promoter GGGGC-repeat polymorphism in lung, breast, and colon cancer

    SciTech Connect

    Spink, Barbara C.; Bloom, Michael S.; Wu, Susan; Sell, Stewart; Schneider, Erasmus; Ding, Xinxin; Spink, David C.

    2015-01-01

    The aryl hydrocarbon receptor (AhR) regulates expression of numerous genes, including those of the CYP1 gene family. With the goal of determining factors that control AHR gene expression, our studies are focused on the role of the short tandem repeat polymorphism, (GGGGC){sub n}, located in the proximal promoter of the human AHR gene. When luciferase constructs containing varying GGGGC repeats were transfected into cancer cell lines derived from the lung, colon, and breast, the number of GGGGC repeats affected AHR promoter activity. The number of GGGGC repeats was determined in DNA from 327 humans and from 38 samples representing 5 species of non-human primates. In chimpanzees and 3 species of macaques, only (GGGGC){sub 2} alleles were observed; however, in western gorilla, (GGGGC){sub n} alleles with n = 2, 4, 5, 6, 7, and 8 were identified. In all human populations examined, the frequency of (GGGGC){sub n} was n = 4 > 5 ≫ 2, 6. When frequencies of the (GGGGC){sub n} alleles in DNA from patients with lung, colon, or breast cancer were evaluated, the occurrence of (GGGGC){sub 2} was found to be 8-fold more frequent among lung cancer patients in comparison with its incidence in the general population, as represented by New York State neonates. Analysis of matched tumor and non-tumor DNA samples from the same individuals provided no evidence of microsatellite instability. These studies indicate that the (GGGGC){sub n} short tandem repeats are inherited, and that the (GGGGC){sub 2} allele in the AHR proximal promoter region should be further investigated with regard to its potential association with lung cancer susceptibility. - Highlights: • The AHR proximal promoter contains a polymorphism, (GGGGC){sub n}, where n = 4 > 5 ≫ 2, 6 • Matched tumor and non-tumor DNA did not show (GGGGC){sub n} microsatellite instability • AHR promoter activity of a construct with (GGGGC){sub 2} was lower than that of (GGGGC){sub 4} • The frequency of (GGGGC){sub 2} in lung

  10. Definition of two agonist types at the mammalian cold-activated channel TRPM8

    PubMed Central

    Janssens, Annelies; Gees, Maarten; Toth, Balazs Istvan; Ghosh, Debapriya; Mulier, Marie; Vennekens, Rudi; Vriens, Joris; Talavera, Karel; Voets, Thomas

    2016-01-01

    Various TRP channels act as polymodal sensors of thermal and chemical stimuli, but the mechanisms whereby chemical ligands impact on TRP channel gating are poorly understood. Here we show that AITC (allyl isothiocyanate; mustard oil) and menthol represent two distinct types of ligands at the mammalian cold sensor TRPM8. Kinetic analysis of channel gating revealed that AITC acts by destabilizing the closed channel, whereas menthol stabilizes the open channel, relative to the transition state. Based on these differences, we classify agonists as either type I (menthol-like) or type II (AITC-like), and provide a kinetic model that faithfully reproduces their differential effects. We further demonstrate that type I and type II agonists have a distinct impact on TRPM8 currents and TRPM8-mediated calcium signals in excitable cells. These findings provide a theoretical framework for understanding the differential actions of TRP channel ligands, with important ramifications for TRP channel structure-function analysis and pharmacology. DOI: http://dx.doi.org/10.7554/eLife.17240.001 PMID:27449282

  11. PPARγ agonists promote differentiation of cancer stem cells by restraining YAP transcriptional activity

    PubMed Central

    Rattanakorn, Kirk; Gadi, Abhilash; Verma, Narendra; Maurizi, Giulia; Gunaratne, Preethi H.; Coarfa, Cristian; Kennedy, Oran D.; Garabedian, Michael J.; Basilico, Claudio; Mansukhani, Alka

    2016-01-01

    Osteosarcoma (OS) is a highly aggressive pediatric bone cancer in which most tumor cells remain immature and fail to differentiate into bone-forming osteoblasts. However, OS cells readily respond to adipogenic stimuli suggesting they retain mesenchymal stem cell-like properties. Here we demonstrate that nuclear receptor PPARγ agonists such as the anti-diabetic, thiazolidinedione (TZD) drugs induce growth arrest and cause adipogenic differentiation in human, mouse and canine OS cells as well as in tumors in mice. Gene expression analysis reveals that TZDs induce lipid metabolism pathways while suppressing targets of the Hippo-YAP pathway, Wnt signaling and cancer-related proliferation pathways. Significantly, TZD action appears to be restricted to the high Sox2 expressing cancer stem cell population and is dependent on PPARγ expression. TZDs also affect growth and cell fate by causing the cytoplasmic sequestration of the transcription factors SOX2 and YAP that are required for tumorigenicity. Finally, we identify a TZD-regulated gene signature based on Wnt/Hippo target genes and PPARγ that predicts patient outcomes. Together, this work highlights a novel connection between PPARγ agonist in inducing adipogenesis and mimicking the tumor suppressive hippo pathway. It also illustrates the potential of drug repurposing for TZD-based differentiation therapy for osteosarcoma. PMID:27528232

  12. Use of a novel one-nostril mask-spacer device to evaluate airway hyperresponsiveness (AHR) in horses after chronic administration of albuterol.

    PubMed

    Mazan, Melissa R; Lascola, Kara; Bruns, Susan J; Hoffman, Andrew M

    2014-07-01

    Inflammatory airway disease (IAD) is very common in stabled horses. Short-acting beta agonist (SABA) drugs are often used to relieve clinical signs, although long-term exposure to these drugs may result in rebound bronchoconstriction. The purpose of this study was twofold: i) to describe the deposition of radiolabeled drugs using a novel one-nostril design mask-spacer combination with a breath-activated inhaler (BAI), and ii) to determine whether treatment for 10 d with inhaled albuterol using this device would impair the ability of albuterol to prevent bronchospasm during a histamine challenge test. The percentage of radio-aerosol deposited in the total lung was 12.39% ± 5.05%. All study horses demonstrated airway hyperresponsiveness (AHR) before enrollment in the study [mean provocative concentration eliciting 35% increase in delta flow (PC35) < 6 mg/mL histamine]. There was no significant difference in airway hyperresponsiveness to post-albuterol histamine challenge before or after treatment with albuterol. A 10-d treatment with placebo, however, caused a significant increase in airway hyperresponsiveness in all horses (P < 0.001). The results of this study show that the novel mask-spacer device was effective in delivering radiolabeled aerosolized drug to the lung and that delivery of a SABA for 10 d using this device did not result in increased airway hyperresponsiveness.

  13. A novel peroxisome proliferator-activated receptor (PPAR)gamma agonist, NIP-222, reduces urinary albumin excretion in streptozotocin-diabetic mice independent of PPARgamma activation.

    PubMed

    Yotsumoto, Takashi; Naitoh, Takeshi; Kanaki, Tatsuro; Matsuda, Maho; Tsuruzoe, Nobutomo

    2003-12-01

    NIP-222 is a novel peroxisome proliferator-activated receptor (PPAR)gamma agonist. This study provides evidence that NIP-222 decreases urinary albumin excretion (UAE) in diabetic mice independent of its PPARgamma activation. We compared the effect of NIP-222 and another PPARgamma agonist, troglitazone, on UAE, plasma glucose level, blood pressure, and creatinine clearance (C(cr)) in streptozotocin (STZ)-induced diabetic mice. Treatment for 3 weeks with NIP-222 (30 mg/kg) was associated with a significant decrease in UAE without any change in blood pressure, creatinine clearance, or plasma glucose level. In contrast, UAE did not decrease in mice treated with troglitazone (300 mg/kg). These results indicate that NIP-222 has PPARgamma independent effects on UAE in diabetic mice and suggest that this agent may have potential to minimize the development and progression of diabetic nephropathy.

  14. [Dmt(1)]DALDA analogues with enhanced μ opioid agonist potency and with a mixed μ/κ opioid activity profile.

    PubMed

    Bai, Longxiang; Li, Ziyuan; Chen, Jiajia; Chung, Nga N; Wilkes, Brian C; Li, Tingyou; Schiller, Peter W

    2014-04-01

    Analogues of [Dmt(1)]DALDA (H-Dmt-d-Arg-Phe-Lys-NH2; Dmt=2',6'-dimethyltyrosine), a potent μ opioid agonist peptide with mitochondria-targeted antioxidant activity, were prepared by replacing Phe(3) with various 2',6'-dialkylated Phe analogues, including 2',6'-dimethylphenylalanine (Dmp), 2',4',6'-trimethylphenylalanine (Tmp), 2'-isopropyl-6'-methylphenylalanine (Imp) and 2'-ethyl-6'-methylphenylalanine (Emp), or with the bulky amino acids 3'-(1-naphthyl)alanine (1-Nal), 3'-(2-naphthyl)alanine (2-Nal) or Trp. Several compounds showed significantly increased μ agonist potency, retained μ receptor selectivity and are of interest as drug candidates for neuropathic pain treatment. Surprisingly, the Dmp(3)-, Imp(3)-, Emp(3)- and 1-Nal(3)-containing analogues showed much increased κ receptor binding affinity and had mixed μ/κ properties. In these cases, molecular dynamics studies indicated conformational preorganization of the unbound peptide ligands due to rotational restriction around the C(β)C(γ) bond of the Xxx(3) residue, in correlation with the observed κ receptor binding enhancement. Compounds with a mixed μ/κ opioid activity profile are known to have therapeutic potential for treatment of cocaine abuse.

  15. Lobeglitazone, a Novel Peroxisome Proliferator-Activated Receptor γ Agonist, Attenuates Renal Fibrosis Caused by Unilateral Ureteral Obstruction in Mice

    PubMed Central

    Seo, Jung Beom; Jung, Yun-A; Seo, Hye-Young; Kang, Sun Hee; Jeon, Hui-Jeon; Lee, Jae Man; Lee, Sungwoo; Kim, Jung-Guk; Lee, In-Kyu

    2017-01-01

    Background Renal tubulointerstitial fibrosis is a common feature of the final stage of nearly all cause types of chronic kidney disease. Although classic peroxisome proliferator-activated receptor γ (PPARγ) agonists have a protective effect on diabetic nephropathy, much less is known about their direct effects in renal fibrosis. This study aimed to investigate possible beneficial effects of lobeglitazone, a novel PPARγ agonist, on renal fibrosis in mice. Methods We examined the effects of lobeglitazone on renal tubulointerstitial fibrosis in unilateral ureteral obstruction (UUO) induced renal fibrosis mice. We further defined the role of lobeglitazone on transforming growth factor (TGF)-signaling pathways in renal tubulointerstitial fibrosis through in vivo and in vitro study. Results Through hematoxylin/eosin and sirius red staining, we observed that lobeglitazone effectively attenuates UUO-induced renal atrophy and fibrosis. Immunohistochemical analysis in conjunction with quantitative reverse transcription polymerase chain reaction and Western blot analysis revealed that lobeglitazone treatment inhibited UUO-induced upregulation of renal Smad-3 phosphorylation, α-smooth muscle actin, plasminogen activator inhibitor 1, and type 1 collagen. In vitro experiments with rat mesangial cells and NRK-49F renal fibroblast cells suggested that the effects of lobeglitazone on UUO-induced renal fibrosis are mediated by inhibition of the TGF-β/Smad signaling pathway. Conclusion The present study demonstrates that lobeglitazone has a protective effect on UUO-induced renal fibrosis, suggesting that its clinical applications could extend to the treatment of non-diabetic origin renal disease. PMID:28256116

  16. Activation of the gut calcium-sensing receptor by peptide agonists reduces rapid elevation of plasma glucose in response to oral glucose load in rats.

    PubMed

    Muramatsu, Maya; Hira, Tohru; Mitsunaga, Arimi; Sato, Eri; Nakajima, Shingo; Kitahara, Yoshiro; Eto, Yuzuru; Hara, Hiroshi

    2014-06-15

    The calcium-sensing receptor (CaSR) is expressed in various tissues, including the gastrointestinal tract. To investigate the role of gut CaSR on glycemic control, we examined whether single oral administration of CaSR agonist peptides affected the glycemic response in rats. Glucose tolerance tests were performed under oral or duodenal administration of various CaSR agonist peptides (γGlu-Cys, protamine, and poly-d-lysine hydrobromide) in conscious rats. Involvement of CaSR was determined by using a CaSR antagonist. Signaling pathways underlying CaSR agonist-modified glycemia were investigated using gut hormone receptor antagonists. The gastric emptying rate after the administration of CaSR agonist peptides was measured by the phenol red recovery method. Oral and duodenal administration of CaSR agonist peptides attenuated glycemic responses under the oral glucose tolerance test, but the administration of casein did not. The promotive effect on glucose tolerance was weakened by luminal pretreatment with a CaSR antagonist. Treatment with a 5-HT3 receptor antagonist partially diminished the glucose-lowering effect of peptides. Furthermore, the gastric emptying rate was decreased by duodenal administration of CaSR agonist peptides. These results demonstrate that activation of the gut CaSR by peptide agonists promotes glucose tolerance in conscious rats. 5-HT3 receptor and the delayed gastric emptying rate appear to be involved in the glucose-lowering effect of CaSR agonist peptides. Thus, activation of gut CaSR by dietary peptides reduces glycemic responses so that gut CaSR may be a potential target for the improvement of postprandial glycemia.

  17. The peroxisome proliferator–activated receptor β/δ agonist GW0742 has direct protective effects on right heart hypertrophy

    PubMed Central

    2013-01-01

    Abstract Pulmonary hypertension is a debilitating disease with no cure. We have previously shown that peroxisome proliferator–activated receptor (PPAR) β/δ agonists protect the right heart in hypoxia-driven pulmonary hypertension without affecting vascular remodeling. PPARβ/δ is an important receptor in lipid metabolism, athletic performance, and the sensing of prostacyclin. Treatment of right heart hypertrophy and failure in pulmonary hypertension is an emerging target for future therapy. Here we have investigated the potential of GW0742, a PPARβ agonist, to act directly on the right heart in vivo and what transcriptomic signatures are associated with its actions. Right heart hypertrophy and failure was induced in mice using a pulmonary artery banding (PAB) model. GW0742 was administered throughout the study. Cardiovascular parameters were measured using echocardiography and pressure monitoring. Fibrosis and cellular changes were measured using immunohistochemistry. Transcriptomics were measured using the Illumina MouseRef-8v3 BeadChip array and analyzed using GeneSpring GX (ver. 11.0). PAB resulted in right heart hypertrophy and failure and in increased fibrosis. GW0742 reduced or prevented the effects of PAB on all parameters measured. GW0742 altered a number of genes in the transcriptome, with Angptl4 emerging as the top gene altered (increased) in animals with PAB. In conclusion, the PPARβ/δ agonist GW0742 has direct protective effects on the right heart in vivo. These observations identify PPARβ/δ as a viable therapeutic target to treat pulmonary hypertension that may complement current and future vasodilator drugs. PMID:25006409

  18. [Blocking action of Nephila clavata spider toxin on ionic currents activated by glutamate and its agonists in isolated hippocampal neurons].

    PubMed

    Kiskin, N I; Kliuchko, E M; Kryshtal', O A; Tsyndrenko, A Ia; Akaike, N

    1989-01-01

    The blocking action of the Nephila clavata spider neurotoxin was studied using the concentration clamp method in isolated neurons of the rat hippocampus. Crude venom JSTX blocked L-glutamate-, quisqualate- and kainate-activated ionic currents mediated by activation of the non-N-methyl-D-aspartate (non-NMDA) membrane receptors. Ionic currents elicited by all agonists were depressed by crude JSTX venom to 34-35% of its initial amplitude with no recovery during prolonged washing. An active fraction of JSTX venom blocked ionic currents almost completely, but its action was partially reversible. The concentration dependences of blocking kinetics allowed determining the rate constants of JSTX interaction with glutamate receptors. It is supposed that JSTX blocks the non-NMDA ionic channels in some of their open states and may be one of useful tools in further biochemical and electrophysiological characterization of the glutamate-mediated synaptic transmission.

  19. Deficiency in Aryl Hydrocarbon Receptor (AHR) Expression throughout Aging Alters Gene Expression Profiles in Murine Long-Term Hematopoietic Stem Cells

    PubMed Central

    Bennett, John A.; Singh, Kameshwar P.; Unnisa, Zeenath; Welle, Stephen L.; Gasiewicz, Thomas A.

    2015-01-01

    Dysregulation of hematopoietic stem cell (HSC) signaling can contribute to the development of diseases of the blood system. Lack of aryl hydrocarbon receptor (AhR) has been associated with alterations in gene expression related to HSC function and the subsequent development of a myeloproliferative disorder in aging female mice. We sorted the most primitive population of HSCs with the highest stem cell potential (Long-term, or LT-HSCs) from 18-month-old AhR-null-allele (AhR-KO) and WT mice and analyzed gene expression using microarray to determine alterations in gene expression and cell signaling networks in HSCs that could potentially contribute to the aging phenotype of AhR-KO mice. Comparisons with previous array data from 8-week old mice indicated that aging alone is sufficient to alter gene expression. In addition, a significant number of gene expression differences were observed in aged LT-HSCs that are dependent on both aging and lack of AhR. Pathway analysis of these genes revealed networks related to hematopoietic stem cell activity or function. qPCR was used to confirm the differential expression of a subset of these genes, focusing on genes that may represent novel AhR targets due to the presence of a putative AhR binding site in their upstream regulatory region. We verified differential expression of PDGF-D, Smo, Wdfy1, Zbtb37 and Zfp382. Pathway analysis of this subset of genes revealed overlap between cellular functions of the novel AhR targets and AhR itself. Lentiviral-mediated knockdown of AhR in lineage-negative hematopoietic cells was sufficient to induce changes in all five of the candidate AhR targets identified. Taken together, these data suggest a role for AhR in HSC functional regulation, and identify novel HSC AhR target genes that may contribute to the phenotypes observed in AhR-KO mice. PMID:26208102

  20. Activation of AhR-mediated toxicity pathway by emerging ...

    EPA Pesticide Factsheets

    Polychlorinated diphenyl sulfides (PCDPSs) are a group of environmental pollutants for which limited toxicological information is available. This study tested the hypothesis that PCDPSs could activate the mammalian aryl hydrocarbon receptor (AhR) mediated toxicity pathways. Eighteen PCDPSs were tested in the H4IIE-luc transactivation assay, with 13/18 causing concentration-dependent AhR activation. Potencies of several congeners were similar to those of mono-ortho substituted polychlorinated biphenyls. A RNA sequencing (RNA-seq)-based transcriptomic analysis was performed on H4IIE cells treated with two PCDPS congeners, 2,2',3,3',4,5,6-hepta-CDPS, and 2,4,4',5-tetra-CDPS. Results of RNA-seq revealed a remarkable modulation on a relatively short gene list by exposure to the tested concentrations of PCDPSs, among which, Cyp1 responded with the greatest fold up-regulation. Both the identities of the modulated transcripts and the associated pathways were consistent with targets and pathways known to be modulated by other types of AhR agonists and there was little evidence for significant off-target effects within the cellular context of the H4IIE bioassay. The results suggest AhR activation as a toxicologically relevant mode of action for PCDPSs suggests the utility of AhR-related toxicity pathways for predicting potential hazards associated with PCDPS exposure in mammals and potentially other vertebrates. Polychlorinated diphenyl sulfides (PCDPSs) are a group of en

  1. Benzimidazoisoquinolines: A New Class of Rapidly Metabolized Aryl Hydrocarbon Receptor (AhR) Ligands that Induce AhR-Dependent Tregs and Prevent Murine Graft-Versus-Host Disease

    PubMed Central

    Punj, Sumit; Kopparapu, Prasad; Jang, Hyo Sang; Phillips, Jessica L.; Pennington, Jamie; Rohlman, Diana; O’Donnell, Edmond; Iversen, Patrick L.; Kolluri, Siva Kumar; Kerkvliet, Nancy I.

    2014-01-01

    The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that plays multiple roles in regulation of immune and inflammatory responses. The ability of certain AhR ligands to induce regulatory T cells (Tregs) has generated interest in developing AhR ligands for therapeutic treatment of immune-mediated diseases. To this end, we designed a screen for novel Treg-inducing compounds based on our understanding of the mechanisms of Treg induction by the well-characterized immunosuppressive AhR ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). We screened a ChemBridge small molecule library and identified 10-chloro-7H-benzimidazo[2,1-a]benzo[de]Iso-quinolin-7-one (10-Cl-BBQ) as a potent AhR ligand that was rapidly metabolized and not cytotoxic to proliferating T cells. Like TCDD,10-Cl-BBQ altered donor CD4+ T cell differentiation during the early stages of a graft versus host (GVH) response resulting in expression of high levels of CD25, CTLA-4 and ICOS, as well as several genes associated with Treg function. The Treg phenotype required AhR expression in the donor CD4+ T cells. Foxp3 was not expressed in the AhR-induced Tregs implicating AhR as an independent transcription factor for Treg induction. Structure-activity studies showed that unsubstituted BBQ as well as 4, 11-dichloro-BBQ were capable of inducing AhR-Tregs. Other substitutions reduced activation of AhR. Daily treatment with 10-Cl-BBQ during the GVH response prevented development of GVH disease in an AhR-dependent manner with no overt toxicity. Together, our data provide strong support for development of select BBQs that activate the AhR to induce Tregs for treatment of immune-mediated diseases. PMID:24586378

  2. Functional impact of allosteric agonist activity of selective positive allosteric modulators of metabotropic glutamate receptor subtype 5 in regulating central nervous system function.

    PubMed

    Noetzel, Meredith J; Rook, Jerri M; Vinson, Paige N; Cho, Hyekyung P; Days, Emily; Zhou, Y; Rodriguez, Alice L; Lavreysen, Hilde; Stauffer, Shaun R; Niswender, Colleen M; Xiang, Zixiu; Daniels, J Scott; Jones, Carrie K; Lindsley, Craig W; Weaver, C David; Conn, P Jeffrey

    2012-02-01

    Positive allosteric modulators (PAMs) of metabotropic glutamate receptor subtype 5 (mGlu(5)) have emerged as an exciting new approach for the treatment of schizophrenia and other central nervous system (CNS) disorders. Of interest, some mGlu(5) PAMs act as pure PAMs, only potentiating mGlu(5) responses to glutamate whereas others [allosteric agonists coupled with PAM activity (ago-PAMs)] potentiate responses to glutamate and have intrinsic allosteric agonist activity in mGlu(5)-expressing cell lines. All mGlu(5) PAMs previously shown to have efficacy in animal models act as ago-PAMs in cell lines, raising the possibility that allosteric agonist activity is critical for in vivo efficacy. We have now optimized novel mGlu(5) pure PAMs that are devoid of detectable agonist activity and structurally related mGlu(5) ago-PAMs that activate mGlu(5) alone in cell lines. Studies of mGlu(5) PAMs in cell lines revealed that ago-PAM activity is dependent on levels of mGlu(5) receptor expression in human embryonic kidney 293 cells, whereas PAM potency is relatively unaffected by levels of receptor expression. Furthermore, ago-PAMs have no agonist activity in the native systems tested, including cortical astrocytes and subthalamic nucleus neurons and in measures of long-term depression at the hippocampal Schaffer collateral-CA1 synapse. Finally, studies with pure PAMs and ago-PAMs chemically optimized to provide comparable CNS exposure revealed that both classes of mGlu(5) PAMs have similar efficacy in a rodent model predictive of antipsychotic activity. These data suggest that the level of receptor expression influences the ability of mGlu(5) PAMs to act as allosteric agonists in vitro and that ago-PAM activity observed in cell-based assays may not be important for in vivo efficacy.

  3. Analysis of the AHR gene proximal promoter GGGGC-repeat polymorphism in lung, breast, and colon cancer.

    PubMed

    Spink, Barbara C; Bloom, Michael S; Wu, Susan; Sell, Stewart; Schneider, Erasmus; Ding, Xinxin; Spink, David C

    2015-01-01

    The aryl hydrocarbon receptor (AhR) regulates expression of numerous genes, including those of the CYP1 gene family. With the goal of determining factors that control AHR gene expression, our studies are focused on the role of the short tandem repeat polymorphism, (GGGGC)n, located in the proximal promoter of the human AHR gene. When luciferase constructs containing varying GGGGC repeats were transfected into cancer cell lines derived from the lung, colon, and breast, the number of GGGGC repeats affected AHR promoter activity. The number of GGGGC repeats was determined in DNA from 327 humans and from 38 samples representing 5 species of non-human primates. In chimpanzees and 3 species of macaques, only (GGGGC)2 alleles were observed; however, in western gorilla, (GGGGC)n alleles with n=2, 4, 5, 6, 7, and 8 were identified. In all human populations examined, the frequency of (GGGGC)n was n=4>5≫2, 6. When frequencies of the (GGGGC)n alleles in DNA from patients with lung, colon, or breast cancer were evaluated, the occurrence of (GGGGC)2 was found to be 8-fold more frequent among lung cancer patients in comparison with its incidence in the general population, as represented by New York State neonates. Analysis of matched tumor and non-tumor DNA samples from the same individuals provided no evidence of microsatellite instability. These studies indicate that the (GGGGC)n short tandem repeats are inherited, and that the (GGGGC)2 allele in the AHR proximal promoter region should be further investigated with regard to its potential association with lung cancer susceptibility.

  4. Analysis of the AHR gene proximal promoter GGGGC-repeat polymorphism in lung, breast, and colon cancer

    PubMed Central

    Spink, Barbara C.; Bloom, Michael S.; Wu, Susan; Sell, Stewart; Schneider, Erasmus; Ding, Xinxin; Spink, David C.

    2015-01-01

    The aryl hydrocarbon receptor (AhR) regulates expression of numerous genes, including those of the CYP1 gene family. With the goal of determining factors that control AHR gene expression, our studies are focused on the role of the short tandem repeat polymorphism, (GGGGC)n, located in the proximal promoter of the human AHR gene. When luciferase constructs containing varying GGGGC repeats were transfected into cancer cell lines derived from lung, colon, and breast, the number of GGGGC repeats affected AHR promoter activity. The number of GGGGC repeats was determined in DNA from 327 humans and from 38 samples representing 5 species of non-human primates. In chimpanzees and 3 species of macaques, only (GGGGC)2 alleles were observed; however, in western gorilla, (GGGGC)n alleles with n = 2, 4, 5, 6, 7, and 8 were identified. In all human populations examined, the frequency of (GGGGC)n was n = 4>5≫2, 6. When frequencies of the (GGGGC)n alleles in DNA from patients with lung, colon, or breast cancer were evaluated, the occurrence of (GGGGC)2 was found to be 8-fold more frequent among lung cancer patients in comparison with its incidence in the general population, as represented by New York State neonates. Analysis of matched tumor and non-tumor DNA samples from the same individuals provided no evidence of microsatellite instability. These studies indicate that the (GGGGC)n short tandem repeats are inherited, and that the (GGGGC)2 allele in the AHR proximal promoter region should be further investigated with regard to its potential association with lung cancer susceptibility. PMID:25447411

  5. Design, synthesis and in vitro evaluation of a series of α-substituted phenylpropanoic acid PPARγ agonists to further investigate the stereochemistry-activity relationship.

    PubMed

    Ohashi, Masao; Nakagome, Izumi; Kasuga, Jun-Ichi; Nobusada, Hiromi; Matsuno, Kenji; Makishima, Makoto; Hirono, Shuichi; Hashimoto, Yuichi; Miyachi, Hiroyuki

    2012-11-01

    We previously demonstrated that the α-benzylphenylpropanoic acid-type PPARγ-selective agonist 6 exhibited a reversed stereochemistry-activity relationship, that is, the (R)-enantiomer is a more potent PPARγ agonist than the (S)-enantiomer, compared with structurally similar α-ethylphenylpropanoic acid-type PPAR agonists. Here, we designed, synthesized and evaluated the optically active α-cyclohexylmethylphenylpropanoic acid derivatives 7 and α-phenethylphenylpropanoic acid derivatives 8, respectively. Interestingly, α-cyclohexylmethyl derivatives showed reversal of the stereochemistry-activity relationship [i.e., (R) more potent than (S)], like α-benzyl derivatives, whereas α-phenethyl derivatives showed the 'normal' relationship [(S) more potent than (R)]. These results suggested that the presence of a branched carbon atom at the β-position with respect to the carboxyl group is a critical determinant of the reversed stereochemistry-activity relationship.

  6. Combined inhalation of beta2 -agonists improves swim ergometer sprint performance but not high-intensity swim performance.

    PubMed

    Kalsen, A; Hostrup, M; Bangsbo, J; Backer, V

    2014-10-01

    There is a high prevalence of asthma and airway hyperresponsiveness (AHR) in elite athletes, which leads to a major use of beta2 -agonists. In a randomized double-blinded crossover study, we investigated the effects of combined inhalation of beta2 -agonists (salbutamol, formoterol, and salmeterol), in permitted doses within the World Anti-Doping Agency 2013 prohibited list, in elite swimmers with (AHR, n = 13) or without (non-AHR, n = 17) AHR. Maximal voluntary isometric contraction of m. quadriceps (MVC), sprint performance on a swim ergometer and performance in an exhaustive swim test at 110% of VO2max were determined. Venous plasma interleukin-6 (IL-6) and interleukin-8 (IL-8) were measured post-exercise. No improvement was observed in the exhaustive swim test, but swim ergometer sprint time was improved (P < 0.05) in both groups from 57 ± 1.7 to 56 ± 1.8 s in AHR and 58.3 ± 1 to 57.4 ± 1 s in non-AHR. MVC and post-exercise plasma IL-6 increased (P < 0.05) with beta2 -agonists in both groups, whereas IL-8 only increased in AHR. In summary, inhalation of beta2 -agonists, in permitted doses, did not improve swim performance in elite swimmers. However, swim ergometer sprint performance and MVC were increased, which should be considered when making future anti-doping regulations.

  7. Peroxisome proliferator-activated receptor-γ (PPAR-γ) agonist inhibits collagen synthesis in human hypertrophic scar fibroblasts by targeting Smad3 via miR-145

    SciTech Connect

    Zhu, Hua-Yu; Li, Chao; Zheng, Zhao; Zhou, Qin; Guan, Hao; Su, Lin-Lin; Han, Jun-Tao; Zhu, Xiong-Xiang; Wang, Shu-yue; Li, Jun Hu, Da-Hai

    2015-03-27

    The transcription factor peroxisome proliferator-activated receptor-γ (PPAR-γ) functions to regulate cell differentiation and lipid metabolism. Recently, its agonist has been documented to regulate extracellular matrix production in human dermal fibroblasts. This study explored the underlying molecular mechanisms and gene interactions in hypertrophic scar fibroblasts (HSFBs) in vitro. HSFBs were cultured and treated with or without PPAR-γ agonist or antagonist for gene expression. Bioinformatical analysis predicted that miR-145 could target Smad3 expression. Luciferase assay was used to confirm such an interaction. The data showed that PPAR-γ agonist troglitazone suppressed expression of Smad3 and Col1 in HSFBs. PPAR-γ agonist induced miR-145 at the gene transcriptional level, which in turn inhibited Smad3 expression and Col1 level in HSFBs. Furthermore, ELISA data showed that Col1 level in HSFBs was controlled by a feedback regulation mechanism involved in PPAR-γ agonist and antagonist-regulated expression of miR-145 and Smad3 in HSFBs. These findings indicate that PPAR-γ-miR-145-Smad3 axis plays a role in regulation of collagen synthesis in HSFBs. - Highlights: • PPAR-γ agonist inhibits collagen synthesis in HSFBs. • Smad3 and type I collagen expression are decreased by PPAR-γ agonist. • miR-145 expression is increased by PPAR-γ agonist in HSFBs. • Increased miR-145 inhibits collagen synthesis by targeting Smad3. • miR-145 regulates collagen synthesis.

  8. Macrophages exposed continuously to lipopolysaccharide and other agonists that act via toll-like receptors exhibit a sustained and additive activation state

    PubMed Central

    Hume, David A; Underhill, David M; Sweet, Matthew J; Ozinsky, Adrian O; Liew, Foo Y; Aderem, Alan

    2001-01-01

    Background Macrophages sense microorganisms through activation of members of the Toll-like receptor family, which initiate signals linked to transcription of many inflammation associated genes. In this paper we examine whether the signal from Toll-like receptors [TLRs] is sustained for as long as the ligand is present, and whether responses to different TLR agonists are additive. Results RAW264 macrophage cells were doubly-transfected with reporter genes in which the IL-12p40, ELAM or IL-6 promoter controls firefly luciferase, and the human IL-1β promoter drives renilla luciferase. The resultant stable lines provide robust assays of macrophage activation by TLR stimuli including LPS [TLR4], lipopeptide [TLR2], and bacterial DNA [TLR9], with each promoter demonstrating its own intrinsic characteristics. With each of the promoters, luciferase activity was induced over an 8 hr period, and thereafter reached a new steady state. Elevated expression required the continued presence of agonist. Sustained responses to different classes of agonist were perfectly additive. This pattern was confirmed by measuring inducible cytokine production in the same cells. While homodimerization of TLR4 mediates responses to LPS, TLR2 appears to require heterodimerization with another receptor such as TLR6. Transient expression of constitutively active forms of TLR4 or TLR2 plus TLR6 stimulated IL-12 promoter activity. The effect of LPS, a TLR4 agonist, was additive with that of TLR2/6 but not TLR4, whilst that of lipopeptide, a TLR2 agonist, was additive with TLR4 but not TLR2/6. Actions of bacterial DNA were additive with either TLR4 or TLR2/6. Conclusions These findings indicate that maximal activation by any one TLR pathway does not preclude further activation by another, suggesting that common downstream regulatory components are not limiting. Upon exposure to a TLR agonist, macrophages enter a state of sustained activation in which they continuously sense the presence of a

  9. Activation of cerebral function by CS-932, a functionally selective M1 partial agonist: neurochemical characterization and pharmacological studies.

    PubMed

    Iwata, N; Kozuka, M; Hara, T; Kanek, T; Tonohiro, T; Sugimoto, M; Niitsu, Y; Kondo, Y; Yamamoto, T; Sakai, J; Nagano, M

    2000-11-01

    A newly synthesized agonist for muscarinic acetylcholine (ACh) receptors CS-932, (R)-3-(3-iso-xazoloxy)-1-azabicyclo-[2.2.2]octane hydrochloride, showed a relatively higher affinity for M1 than M2 receptors expressed in Chinese hamster ovary (CHO)-cells in comparison with ACh. CS-932 elevated the intracellular Ca2+ level only in M1-CHO cells, although ACh increased the level in both M1- and M3-CHO cells. CS-932 and ACh reduced forskolin-stimulated accumulation of cAMP in M2-CHO cells by 20% and 80%, respectively. This neurochemical profile of CS-932 indicates that the compound can activate M1-receptor-mediated functions selectively. CS-932 increased firing of cholinoceptive neurons in rat hippocampal slices, and this excitation was antagonized by pirenzepine, but not by AF-DX 116. CS-932 increased awake and decreased slow wave sleep episodes of daytime EEG in free-moving rats. It counteracted scopolamine-induced slow waves in rat cortical EEG. CS-932 also increased the power of alpha- and beta-waves, but decreased delta-wave of the cortical EEG in anesthetized monkeys. It ameliorated scopolamine-induced impairment of working memory in rats. Orally administered CS-932 had the best penetration into the brain among the muscarinic agonists tested and caused the least salivary secretion among the cholinomimetics examined. These results indicate that CS-932 has potential as a cognitive enhancer with fewer side effects in therapy for Alzheimer disease.

  10. Design, synthesis and structure-activity relationship studies of novel free fatty acid receptor 1 agonists bearing amide linker.

    PubMed

    Yang, Jianyong; Li, Zheng; Li, Huilan; Liu, Chunxia; Wang, Nasi; Shi, Wei; Liao, Chen; Cai, Xingguang; Huang, Wenlong; Qian, Hai

    2017-04-15

    The free fatty acid receptor 1 (FFA1/GPR40) has attracted extensive attention as a novel antidiabetic target. Aiming to explore the chemical space of FFA1 agonists, a new series of lead compounds with amide linker were designed and synthesized by combining the scaffolds of NIH screened lead compound 1 and GW9508. Among them, the optimal lead compound 17 exhibited a considerable agonistic activity (45.78%) compared to the NIH screened compound 1 (15.32%). During OGTT in normal mice, the compound 17 revealed a significant glucose-lowering effect (-23.7%) at the dose of 50mg/kg, proximity to the hypoglycemic effect (-27.8%) of Metformin (200mg/kg). In addition, the compound 17 (100mg/kg) also exhibited a significant improvement in glucose tolerance with a 29.1% reduction of glucose AUC0-2h in type 2 diabetic mice. All of these results indicated that compound 17 was considered to be a promising lead structure suitable for further optimization.

  11. Pharmacological profile of the abeorphine 201-678, a potent orally active and long lasting dopamine agonist

    SciTech Connect

    Jaton, A.L.; Giger, R.K.A.; Vigouret, J.M.; Enz, A.; Frick, W.; Closse, A.; Markstein, R.

    1986-01-13

    The central dopaminergic effects of an abeorphine derivative 201-678 were compared to those of apomorphine and bromocriptine in different model systems. After oral administration, this compound induced contralateral turning in rats with 6-hydroxydopamine induced nigral lesions and exhibited strong anti-akinetic properties in rats with 6-hydroxydopamine induced hypothalamic lesions. It decreased dopamine metabolism in striatum and cortex, but did not modify noradrenaline and serotonin metabolism in the rat brain. 201-678 counteracted the in vivo increase of tyrosine hydroxylase activity induced by ..gamma..-butyrolactone. In vitro it stimulated DA-sensitive adenylate cyclase and inhibited acetylcholine release from rat striatal slices. This compound had high affinity for /sup 3/H-dopamine and /sup 3/H-clonidine binding sites. These results indicate that 201-678 is a potent, orally active dopamine agonist with a long duration of action. Furthermore it appears more selective than other dopaminergic drugs. 29 references, 5 figures, 3 tables.

  12. The endogenous mu-opioid receptor agonists endomorphins 1 and 2 have novel hypotensive activity in the rabbit.

    PubMed

    Champion, H C; Zadina, J E; Kastin, A J; Hackler, L; Ge, L J; Kadowitz, P J

    1997-06-27

    The endogenous peptides endomorphins 1 and 2 are newly isolated, potent, and selective mu-opioid receptor agonists. In the present study, responses to the endomorphin peptides were investigated in the systemic vascular bed of the rabbit. Endomorphins 1 and 2 induced dose-related decreases in systemic arterial pressure when injected in doses of 1-30 nmol/kg i.v. In terms of relative vasodepressor activity, endomorphins 1 and 2 were similar to the ORL1 receptor ligand, nociceptin (Orphanin FQ), and met-enkephalin in decreasing systemic arterial pressure. Vasodepressor responses to endomorphins 1 and 2 were inhibited by the opioid receptor antagonist, naloxone, in a dose of 2 mg/kg i.v. These results demonstrate that endomorphins 1 and 2 have significant naloxone-sensitive, vasodepressor activity in the rabbit.

  13. Sequence and in vitro function of chicken, ring-necked pheasant, and Japanese quail AHR1 predict in vivo sensitivity to dioxins.

    PubMed

    Farmahin, Reza; Wu, Dongmei; Crump, Doug; Hervé, Jessica C; Jones, Stephanie P; Hahn, Mark E; Karchner, Sibel I; Giesy, John P; Bursian, Steven J; Zwiernik, Matthew J; Kennedy, Sean W

    2012-03-06

    There are large differences in sensitivity to the toxic and biochemical effects of dioxins and dioxin-like compounds (DLCs) among vertebrates. Previously, we demonstrated that the difference in sensitivity between domestic chicken (Gallus gallus domesticus) and common tern (Sterna hirundo) to aryl hydrocarbon receptor 1 (AHR1)-dependent changes in gene expression following exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is based upon the identities of the amino acids at two sites within the ligand binding domain of AHR1 (chicken--highly sensitive; Ile324_Ser380 vs common tern--250-fold less sensitive than chicken; Val325_Ala381). Here, we tested the hypotheses that (i) the sensitivity of other avian species to TCDD, 2,3,4,7,8-pentachlorodibenzofuran (PeCDF), and 2,3,7,8-tetrachlorodibenzofuran (TCDF) is also determined by the amino acids at sites that are equivalent to sites 324 and 380 in chicken, and (ii) Ile324_Ala380 and Val324_Ser380 genotypes confer intermediate sensitivity to DLCs in birds. We compared ligand-induced transactivation function of full-length AHR1s from chicken, common tern, ring-necked pheasant (Phasianus colchicus; Ile324_Ala380) and Japanese quail (Coturnix japonica; Val324_Ala380), and three Japanese quail AHR1 mutants. The results support our hypothesis that avian species can be grouped into three general classes of sensitivity to DLCs. Both AHR1 genotype and in vitro transactivation assays predict in vivo sensitivity. Contrary to the assumption that TCDD is the most potent DLC, PeCDF was more potent than TCDD at activating Japanese quail (13- to 26-fold) and common tern (23- to 30-fold) AHR1. Our results support and expand previous in vitro and in vivo work that demonstrated ligand-dependent species differences in AHR1 affinity. The findings and methods will be of use for DLC risk assessments.

  14. The Role of AHR in Breast Cancer Development

    DTIC Science & Technology

    2005-07-01

    cancer, AhR, galangin 16. SECURITY CLASSIFICATION OF: 17. LIMITATION 18. NUMBER 19a. NAME OF RESPONSIBLE PERSON OF ABSTRACT OF PAGES USAMRMC a. REPORT...Z39.18 ABSTRACT The study described herein was designed to determine if and how a non-toxic, naturally occurring bioflavonoid, galangin , affects growth of...human mammary tumor cells. Our previous studies demonstrated that, in other cell types, galangin is a potent inhibitor of the aryl hydrocarbon

  15. Discovery of SMP-304, a novel benzylpiperidine derivative with serotonin transporter inhibitory activity and 5-HT1A weak partial agonistic activity showing the antidepressant-like effect.

    PubMed

    Yoshinaga, Hidefumi; Masumoto, Shuji; Koyama, Koji; Kinomura, Naoya; Matsumoto, Yuji; Kato, Taro; Baba, Satoko; Matsumoto, Kenji; Horisawa, Tomoko; Oki, Hitomi; Yabuuchi, Kazuki; Kodo, Toru

    2017-01-01

    We report the discovery of a novel benzylpiperidine derivative with serotonin transporter (SERT) inhibitory activity and 5-HT1A receptor weak partial agonistic activity showing the antidepressant-like effect. The 3-methoxyphenyl group and the phenethyl group of compound 1, which has weak SERT binding activity, but potent 5-HT1A binding activity, were optimized, leading to compound 35 with potent and balanced dual SERT and 5-HT1A binding activity, but also potent CYP2D6 inhibitory activity. Replacement of the methoxy group in the left part of compound 35 with a larger alkoxy group, such as ethoxy, isopropoxy or methoxy-ethoxy group ameliorated CYP2D6 inhibition, giving SMP-304 as a candidate. SMP-304 with serotonin uptake inhibitory activity and 5-HT1A weak partial agonistic activity, which could work as a 5-HT1A antagonist, displayed faster onset of antidepressant-like effect than a representative SSRI paroxetine in an animal model.

  16. Ahr2-dependence of PCB126 effects on the swim bladder in relation to expression of CYP1 and cox-2 genes in developing zebrafish

    SciTech Connect

    Jönsson, Maria E.; Kubota, Akira; Timme-Laragy, Alicia R.; Woodin, Bruce; Stegeman, John J.

    2012-12-01

    The teleost swim bladder is assumed a homolog of the tetrapod lung. Both swim bladder and lung are developmental targets of persistent aryl hydrocarbon receptor (AHR) agonists; in zebrafish (Danio rerio) the swim bladder fails to inflate with exposure to 3,3′,4,4′,5-pentachlorobiphenyl (PCB126). The mechanism for this effect is unknown, but studies have suggested roles of cytochrome P450 1 (CYP1) and cyclooxygenase 2 (Cox-2) in some Ahr-mediated developmental effects in zebrafish. We determined relationships between swim bladder inflation and CYP1 and Cox-2 mRNA expression in PCB126-exposed zebrafish embryos. We also examined effects on β-catenin dependent transcription, histological effects, and Ahr2 dependence of the effect of PCB126 on swim bladder using morpholinos targeting ahr2. One-day-old embryos were exposed to waterborne PCB126 or carrier (DMSO) for 24 h and then held in clean water until day 4, a normal time for swim bladder inflation. The effects of PCB126 were concentration-dependent with EC{sub 50} values of 1.4 to 2.0 nM for induction of the CYP1s, 3.7 and 5.1 nM (or higher) for cox-2a and cox-2b induction, and 2.5 nM for inhibition of swim bladder inflation. Histological defects included a compaction of the developing bladder. Ahr2-morpholino treatment rescued the effect of PCB126 (5 nM) on swim bladder inflation and blocked induction of CYP1A, cox-2a, and cox-2b. With 2 nM PCB126 approximately 30% of eleutheroembryos failed to inflate the swim bladder, but there was no difference in CYP1 or cox-2 mRNA expression between those embryos and embryos showing inflated swim bladder. Our results indicate that PCB126 blocks swim bladder inflation via an Ahr2-mediated mechanism. This mechanism seems independent of CYP1 or cox-2 mRNA induction but may involve abnormal development of swim bladder cells. -- Highlights: ► PCB126 caused cellular changes in the developing swim bladder. ► Swim bladder inflation was not related to expression of CYP1 or cox

  17. The acute anorexic effect of liraglutide, a GLP-1 receptor agonist, does not require functional leptin receptor, serotonin, and hypothalamic POMC and CART activities in mice.

    PubMed

    Nonogaki, Katsunori; Kaji, Takao

    2016-10-01

    The acute anorexic effect of liraglutide, a GLP-1 receptor agonist, did not require functional leptin receptor, serotonin, and hypothalamic proopiomelanocortin and cocaine amphetamine regulated transcript activities in mice, although decrease in functional hypothalamic orexin activity might be involved in the acute anorexic effect of liraglutide.

  18. Significant Correlation between TLR2 Agonist Activity and TNF-α Induction in J774.A1 Macrophage Cells by Different Medicinal Mushroom Products.

    PubMed

    Coy, Catherine; Standish, Leanna J; Bender, Geoff; Lu, Hailing

    2015-01-01

    In the US market, there is a variety of mushroom preparations available, even within the same species of mushroom. Nonetheless, little is known about whether species or the various extraction methods affect biological activity and potency of the immune modulatory activity of mushroom extracts. After discovering that protein-bound polysaccharide-K, a hot water extract from Trametes versicolor, was a potent Toll-like receptor (TLR)-2 agonist that stimulates both innate and adaptive immunity, this study was initiated to evaluate whether other medicinal mushroom products also have TLR2 agonist activity and immune-enhancing potential as measured by the induction of tumor necrosis factor (TNF)-α in J774.A1 murine macrophage cells. Furthermore, the products were divided by extraction method and species to determine whether these factors affect their immunomodulatory activity. The results showed that the majority (75%) of mushroom products tested had TLR2 agonist activity and that there was a significant correlation between TLR2 agonist activity and TNF-α induction potential in the mushroom products analyzed. In addition, the data demonstrated that hot water mushroom extracts are more potent than ground mushroom products in activating TLR2 and inducing TNF-α. These data provide evidence that extraction methods may affect the biological activity of mushroom products; thus, further studies are warranted to investigate the structural differences between various mushroom products.

  19. Metabolic Profiling of Chicken Embryos Exposed to Perfluorooctanoic Acid (PFOA) and Agonists to Peroxisome Proliferator-Activated Receptors

    PubMed Central

    Mattsson, Anna; Kärrman, Anna; Pinto, Rui; Brunström, Björn

    2015-01-01

    Untargeted metabolic profiling of body fluids in experimental animals and humans exposed to chemicals may reveal early signs of toxicity and indicate toxicity pathways. Avian embryos develop separately from their mothers, which gives unique possibilities to study effects of chemicals during embryo development with minimal confounding factors from the mother. In this study we explored blood plasma and allantoic fluid from chicken embryos as matrices for revealing metabolic changes caused by exposure to chemicals during embryonic development. Embryos were exposed via egg injection on day 7 to the environmental pollutant perfluorooctanoic acid (PFOA), and effects on the metabolic profile on day 12 were compared with those caused by GW7647 and rosiglitazone, which are selective agonists to peroxisome-proliferator activated receptor α (PPARα) and PPARγ, respectively. Analysis of the metabolite concentrations from allantoic fluid by Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA) showed clear separation between the embryos exposed to GW7647, rosiglitazone, and vehicle control, respectively. In blood plasma only GW7647 caused a significant effect on the metabolic profile. PFOA induced embryo mortality and increased relative liver weight at the highest dose. Sublethal doses of PFOA did not significantly affect the metabolic profile in either matrix, although single metabolites appeared to be altered. Neonatal mortality by PFOA in the mouse has been suggested to be mediated via activation of PPARα. However, we found no similarity in the metabolite profile of chicken embryos exposed to PFOA with those of embryos exposed to PPAR agonists. This indicates that PFOA does not activate PPAR pathways in our model at concentrations in eggs and embryos well above those found in wild birds. The present study suggests that allantoic fluid and plasma from chicken embryos are useful and complementary matrices for exploring effects on the metabolic profile resulting

  20. Central activation of the sympathetic nervous system including the adrenals in anaesthetized guinea pigs by the muscarinic agonist talsaclidine.

    PubMed

    Walland, A; Pieper, M P

    1998-04-01

    Talsaclidine, a novel M1-receptor selective muscarinic agonist for cholinergic substitution therapy of Alzheimer's disease, activates the sympathetic nervous system in guinea pigs and dogs at the orthosympathic ganglia and the paraganglionic adrenals. Results from guinea pigs provide indirect evidence for an additional central site of action. The present investigation in anaesthetized and vagotomized guinea pigs intended to demonstrate central activation of the sympathetic nervous system directly by comparing the blood pressure effects of intracerebroventricular and intravenous injections of small doses of talsaclidine. Increasing doses of 0.2 and 0.6 mg/kg talsaclidine were injected alternately into the third cerebral ventricle and intravenously in 6 guinea pigs before and after blockade of peripheral muscarinic receptors with 1 mg/kg ipratropium bromide i.v. In another group of 6 animals the injections were given into the cisterna cerebellomedullaris using the same protocol. In both groups central administration of talsaclidine caused dose-related hypertension while intravenous injections were hypotensive. Ipratropium bromide, a peripheral antimuscarinic drug, reversed this hypotensive action of intravenous talsaclidine into hypertension, but did not inhibit the effects of central administration. In contrast, atropine, an antimuscarinic drug which passes the blood-brain barrier, abolished the effect of 0.6 mg/kg talsaclidine injected into the cisterna cerebellomedullaris of 8 guinea pigs. The hypertensive effect of a first injection of 0.6 mg/kg talsaclidine into the cisterna cerebellomedullaris of 6 guinea pigs was approximately twice as large as that of a second given 90 min after bilateral adrenalectomy. Sham operation in another 6 animals was not inhibitory. The results demonstrate that talsaclidine, a selective muscarinic M1-receptor agonist, activates central parts of the sympathetic nervous system, including central projections of the adrenals by an action

  1. Anti-kindling Effect of Bezafibrate, a Peroxisome Proliferator-activated Receptors Alpha Agonist, in Pentylenetetrazole Induced Kindling Seizure Model

    PubMed Central

    Saha, Lekha; Bhandari, Swati; Bhatia, Alka; Banerjee, Dibyajyoti; Chakrabarti, Amitava

    2014-01-01

    Background and Purpose: Studies in the animals suggested that Peroxisome proliferators activated receptors (PPARs) may be involved in seizure control and selective agonists of PPAR α or PPAR γ raise seizure thresholds. The present study was contemplated with the aim of evaluating the anti kindling effects and the mechanism of bezafibrate, a Peroxisome proliferator-activated receptors α (PPAR-α) agonist in pentylenetetrazole (PTZ) induced kindling model of seizures in rats. Methods: In a PTZ kindled Wistar rat model, different doses of bezafibrate (100 mg/kg, 200 mg/kg and 300 mg/kg) were administered intraperitoneally 30 minutes before the PTZ injection. The PTZ injection was given on alternate day till the animal became fully kindled or till 10 weeks. The parameters measured were the latency to develop kindling and incidence of kindling, histopathological study of hippocampus, hippocampal lipid peroxidation studies, serum neuron specific enolase, and hippocampal DNA fragmentation study. Results: In this study, bezafibrate significantly reduced the incidence of kindling in PTZ treated rats and exhibited a marked prolongation in the latencies to seizures. In the present study bezafibrate decreased the thiobarbituric acid-reactive substance i.e. Malondialdehyde levels, increased the reduced glutathione levels, catalase and superoxide dismutase activity in the brain. This added to its additional neuroprotective effects. Bezafibrate also reduced the neuronal damage and apoptosis in hippocampal area of the brain. Therefore bezafibrate exerted anticonvulsant properties in PTZ induced kindling model in rats. Conclusions: These findings may provide insights into the understanding of the mechanism of bezafibrate as an anti kindling agent and could offer a useful support to the basic antiepileptic therapy in preventing the development of PTZ induced seizures, suggesting its potential for therapeutic applications in temporal lobe epilepsy. PMID:25625088

  2. Α-amino-β-fluorocyclopropanecarboxylic acids as a new tool for drug development: synthesis of glutamic acid analogs and agonist activity towards metabotropic glutamate receptor 4.

    PubMed

    Lemonnier, Gérald; Lion, Cédric; Quirion, Jean-Charles; Pin, Jean-Philippe; Goudet, Cyril; Jubault, Philippe

    2012-08-01

    Herein we describe the diastereoselective synthesis of glutamic acid analogs and the evaluation of their agonist activity towards metabotropic glutamate receptor subtype 4 (mGluR4). These analogs are based on a monofluorinated cyclopropane core substituted with an α-aminoacid function. The potential of this new building block as a tool for the development of a novel class of drugs is demonstrated with racemic analog 11a that displayed the best agonist activity with an EC50 of 340 nM.

  3. Nanosecond pulse electric field (nanopulse): a novel non-ligand agonist for platelet activation.

    PubMed

    Zhang, Jue; Blackmore, Peter F; Hargrave, Barbara Y; Xiao, Shu; Beebe, Stephen J; Schoenbach, Karl H

    2008-03-15

    Nanosecond pulse stimulation of a variety of cells produces a wide range of physiological responses (e.g., apoptosis, stimulation of calcium (Ca2+) fluxes, changes in membrane potential). In this study, we investigated the effect of nanosecond pulses, which generate intense electric fields (nsPEFs), on human platelet aggregation, intracellular free Ca2+ ion concentration ([Ca2+]i) and platelet-derived growth factor release. When platelet rich plasma was pulsed with one 300ns pulse with an electric field of 30kV/cm, platelets aggregated and a platelet gel was produced. Platelet aggregation was observed with pulses as low as 7kV/cm with maximum effects seen with approximately 30kV/cm. The increases in intracellular Ca2+ release and Ca2+ influx were dose dependent on the electrical energy density and were maximally stimulated with approximately 30kV/cm. The increases in [Ca2+]i induced by nsPEF were similar to those seen with thapsigargin but not thrombin. We postulate that nsPEF caused Ca2+ to leak out of intracellular Ca2+ stores by a process involving the formation of nanopores in organelle membranes and also caused Ca2+ influx through plasma membrane nanopores. We conclude that nsPEFs dose-dependently cause platelets to rapidly aggregate, like other platelet agonists, and this is most likely initiated by the nsPEFs increasing [Ca2+]i, however by a different mechanism.

  4. Peroxisome Proliferator-Activated Receptor-γ Agonists: Potential Therapeutics for Neuropathology Associated with Fetal Alcohol Spectrum Disorders

    PubMed Central

    Drew, Paul D.; Kane, Cynthia J.M.

    2017-01-01

    Fetal alcohol spectrum disorders (FASD) result from fetal exposure to alcohol during pregnancy. These disorders present a variety of sequelae including involvement of the central nervous system (CNS) with lasting impact on cognitive function and behavior. FASD occur at an alarming rate and have significant personal and societal impact. There are currently no effective treatments for FASD. Recent studies demonstrate that ethanol induces potent neuroinflammation in many regions of the developing brain. Furthermore, anti-inflammatory agents such as peroxisome proliferator-activated receptor (PPAR)-γ agonists suppress ethanol-induced neuroinflammation and neurodegeneration. This suggests that anti-inflammatory agents may be effective in treatment of FASD. Future studies designed to determine the specific mechanisms by which alcohol induces neuroinflammation in the developing CNS may lead to targeted therapies for FASD. PMID:28203487

  5. Intrathecal alpha2 adrenoceptor agonist clonidine inhibits mechanical transmission in mouse spinal cord via activation of muscarinic M1 receptors.

    PubMed

    Honda, Kenji; Koga, Kohei; Moriyama, Tomoko; Koguchi, Masako; Takano, Yukio; Kamiya, Hiro-o

    2002-04-12

    We examined the role of the spinal muscarinic receptor subtype in the anti-nociceptive effect of intrathecal (i.t.) alpha2 adrenoceptor agonist clonidine in mice. I.t. injection of the muscarinic receptor antagonist atropine completely inhibited i.t. clonidine-induced increase in the mechanical threshold, but did not affect the increase in tail-flick latency induced by i.t. clonidine. The clonidine-induced increase in mechanical threshold was inhibited by i.t. injection of the M1 receptor antagonist pirenzepine in a dose-dependent manner, and by the M3 receptor antagonist 4-DAMP, but not by the M2 receptor antagonist methoctramine. The potency of pirenzepine was greater than that of 4-DAMP. These results suggest that the clonidine-induced increase in mechanical threshold is mediated via the activation of M1 receptors in the spinal cord.

  6. Aryl hydrocarbon receptor activation by 2,3,7,8-tetrachlorodibenzo-p-dioxin impairs human B lymphopoiesis.

    PubMed

    Li, Jinpeng; Phadnis-Moghe, Ashwini S; Crawford, Robert B; Kaminski, Norbert E

    2017-03-01

    The homeostasis of peripheral B cell compartment requires lifelong B lymphopoiesis from hematopoietic stem cells (HSC). As a result, the B cell repertoire is susceptible to disruptions of hematopoiesis. Increasing evidence, primarily from rodent models, shows that the aryl hydrocarbon receptor (AHR) regulates hematopoiesis. To study the effects of persistent AHR activation on human B cell development, a potent AHR agonist and known environmental contaminant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was utilized. An in vitro B cell development model system was established by co-culturing human cord blood-derived HSCs with irradiated human primary bone marrow stromal cells. Using this in vitro model, we found that TCDD significantly suppressed the total number of hematopoietic stem and progenitor cells (HSPC) in a concentration-dependent manner. Cell death analysis demonstrated that the decrease in cell number was not due to cytotoxicity by TCDD. In addition, TCDD markedly decreased CD34 expression on HSPCs. Structure-activity relationship studies using dioxin congeners demonstrated a correlation between the relative AHR binding affinity and the magnitude of decrease in the number of HSPCs and CD34 expression, suggesting that AHR mediates the observed TCDD-elicited changes in HSPCs. Moreover, a significant reduction in lineage committed B cell-derived from HSCs was observed in the presence of TCDD, indicating impairment of human B cell development. Similar effects of TCDD were observed regardless of the use of stromal cells in cultures indicating a direct effect of TCDD on HSCs. Collectively, we demonstrate that AHR activation by TCDD on human HSCs impairs early stages of human B lymphopoiesis.

  7. Molecular mechanism of transcriptional repression of AhR repressor involving ANKRA2, HDAC4, and HDAC5

    SciTech Connect

    Oshima, Motohiko; Mimura, Junsei; Yamamoto, Masayuki; Fujii-Kuriyama, Yoshiaki

    2007-12-14

    The Aryl hydrocarbon receptor repressor (AhRR) has been proposed to inhibit Aryl hydrocarbon receptor (AhR) activity by competing with AhR for forming a heterodimer with AhR nuclear translocator (Arnt) and subsequently binding to the xenobiotic responsive elements (XRE). However, the precise mechanism of AhRR inhibitory activity remains unknown. Analysis of the inhibitory activity of AhRR on the expression of a TK promoter-driven reporter has localized a core repressor domain in the sequence of amino acid residue 555-701. The inhibitory activity of AhRR is sensitive to a histone deacetylase (HDAC) inhibitor, trichostatin A. By using the yeast two-hybrid screening method with the C-terminal sequence of AhRR as bait, we identified a binding partner, Ankyrin-repeat protein2 (ANKRA2), a protein known to interact with HDAC4 and HDAC5. RNA interference experiments using ANKRA2 and AhRR siRNAs indicate that ANKRA2 is important for transcriptional repression by AhRR. We have found that under normal conditions, CYP1A1 gene is kept silent in MEF cells by AhRR/Arnt heterodimer, which binds to the XRE sequence in its promoter and recruits ANKRA2, HDAC4, and HDAC5 as co-repressors.

  8. Agonist activation of cytosolic Ca2+ in subfornical organ cells projecting to the supraoptic nucleus

    NASA Technical Reports Server (NTRS)

    Johnson, R. F.; Beltz, T. G.; Sharma, R. V.; Xu, Z.; Bhatty, R. A.; Johnson, A. K.

    2001-01-01

    The subfornical organ (SFO) is sensitive to both ANG II and ACh, and local application of these agents produces dipsogenic responses and vasopressin release. The present study examined the effects of cholinergic drugs, ANG II, and increased extracellular osmolarity on dissociated, cultured cells of the SFO that were retrogradely labeled from the supraoptic nucleus. The effects were measured as changes in cytosolic calcium in fura 2-loaded cells by using a calcium imaging system. Both ACh and carbachol increased intracellular ionic calcium concentration ([Ca2+]i). However, in contrast to the effects of muscarinic receptor agonists on SFO neurons, manipulation of the extracellular osmolality produced no effects, and application of ANG II produced only moderate effects on [Ca2+]i in a few retrogradely labeled cells. The cholinergic effects on [Ca2+]i could be blocked with the muscarinic receptor antagonist atropine and with the more selective muscarinic receptor antagonists pirenzepine and 4-diphenylacetoxy-N-methylpiperdine methiodide (4-DAMP). In addition, the calcium in the extracellular fluid was required for the cholinergic-induced increase in [Ca2+]i. These findings indicate that ACh acts to induce a functional cellular response in SFO neurons through action on a muscarinic receptor, probably of the M1 subtype and that the increase of [Ca2+]i, at least initially, requires the entry of extracellular Ca2+. Also, consistent with a functional role of M1 receptors in the SFO are the results of immunohistochemical preparations demonstrating M1 muscarinic receptor-like protein present within this forebrain circumventricular organ.

  9. Pharmacological profile of DA-6886, a novel 5-HT4 receptor agonist to accelerate colonic motor activity in mice.

    PubMed

    Lee, Min Jung; Cho, Kang Hun; Park, Hyun Min; Sung, Hyun Jung; Choi, Sunghak; Im, Weonbin

    2014-07-15

    DA-6886, the gastrointestinal prokinetic benzamide derivative is a novel 5-HT4 receptor agonist being developed for the treatment of constipation-predominant irritable bowel syndrome (IBS-C). The purpose of this study was to characterize in vitro and in vivo pharmacological profile of DA-6886. We used various receptor binding assay, cAMP accumulation assay, organ bath experiment and colonic transit assay in normal and chemically constipated mice. DA-6886 exhibited high affinity and selectivity to human 5-HT4 receptor splice variants, with mean pKi of 7.1, 7.5, 7.9 for the human 5-HT4a, 5-HT4b and 5-HT4d, respectively. By contrast, DA-6886 did not show significant affinity for several receptors including dopamine D2 receptor, other 5-HT receptors except for 5-HT2B receptor (pKi value of 6.2). The affinity for 5-HT4 receptor was translated into functional agonist activity in Cos-7 cells expressing 5-HT4 receptor splice variants. Furthermore, DA-6886 induced relaxation of the rat oesophagus preparation (pEC50 value of 7.4) in a 5-HT4 receptor antagonist-sensitive manner. The evaluation of DA-6886 in CHO cells expressing hERG channels revealed that it inhibited hERG channel current with an pIC50 value of 4.3, indicating that the compound was 1000-fold more selective for the 5-HT4 receptor over hERG channels. In the normal ICR mice, oral administration of DA-6886 (0.4 and 2mg/kg) resulted in marked stimulation of colonic transit. Furthermore, in the loperamide-induced constipation mouse model, 2mg/kg of DA-6886 significantly improved the delay of colonic transit, similar to 10mg/kg of tegaserod. Taken together, DA-6886 is a highly potent and selective 5-HT4 receptor agonist to accelerate colonic transit in mice, which might be therapeutic agent having a favorable safety profile in the treatment of gastrointestinal motor disorders such as IBS-C and chronic constipation.

  10. New diphenylmethane derivatives as peroxisome proliferator-activated receptor alpha/gamma dual agonists endowed with anti-proliferative effects and mitochondrial activity.

    PubMed

    Piemontese, Luca; Cerchia, Carmen; Laghezza, Antonio; Ziccardi, Pamela; Sblano, Sabina; Tortorella, Paolo; Iacobazzi, Vito; Infantino, Vittoria; Convertini, Paolo; Dal Piaz, Fabrizio; Lupo, Angelo; Colantuoni, Vittorio; Lavecchia, Antonio; Loiodice, Fulvio

    2017-02-15

    We screened a short series of new chiral diphenylmethane derivatives and identified potent dual PPARα/γ partial agonists. As both enantiomers of the most active compound 1 displayed an unexpected similar transactivation activity, we performed docking experiments to provide a molecular understanding of their similar partial agonism. We also evaluated the ability of both enantiomers of 1 and racemic 2 to inhibit colorectal cancer cells proliferation: (S)-1 displayed a more robust activity due, at least in part, to a partial inhibition of the Wnt/β-catenin signalling pathway that is upregulated in the majority of colorectal cancers. Finally, we investigated the effects of (R)-1, (S)-1 and (R,S)-2 on mitochondrial function and demonstrated that they activate the carnitine shuttle system through upregulation of carnitine/acylcarnitine carrier (CAC) and carnitine-palmitoyl-transferase 1 (CPT1) genes. Consistent with the notion that these are PPARα target genes, we tested and found that PPARα itself is regulated by a positive loop. Moreover, these compounds induced a significant mitochondrial biogenesis. In conclusion, we identified a new series of dual PPARα/γ agonists endowed with novel anti-proliferative properties associated with a strong activation of mitochondrial functions and biogenesis, a potential therapeutic target of the treatment of insulin resistance.

  11. Bitter Taste Receptor Agonists Mitigate Features of Allergic Asthma in Mice

    PubMed Central

    Sharma, Pawan; Yi, Roslyn; Nayak, Ajay P.; Wang, Nadan; Tang, Francesca; Knight, Morgan J.; Pan, Shi; Oliver, Brian; Deshpande, Deepak A.

    2017-01-01

    Asthma is characterized by airway inflammation, mucus secretion, remodeling and hyperresponsiveness (AHR). Recent research has established the bronchodilatory effect of bitter taste receptor (TAS2R) agonists in various models. Comprehensive pre-clinical studies aimed at establishing effectiveness of TAS2R agonists in disease models are lacking. Here we aimed to determine the effect of TAS2R agonists on features of asthma. Further, we elucidated a mechanism by which TAS2R agonists mitigate features of asthma. Asthma was induced in mice using intranasal house dust mite or aerosol ova-albumin challenge, and chloroquine or quinine were tested in both prophylactic and treatment models. Allergen challenge resulted in airway inflammation as evidenced by increased immune cells infiltration and release of cytokines and chemokines in the lungs, which were significantly attenuated in TAS2R agonists treated mice. TAS2R agonists attenuated features of airway remodeling including smooth muscle mass, extracellular matrix deposition and pro-fibrotic signaling, and also prevented mucus accumulation and development of AHR in mice. Mechanistic studies using human neutrophils demonstrated that inhibition of immune cell chemotaxis is a key mechanism by which TAS2R agonists blocked allergic airway inflammation and exerted anti-asthma effects. Our comprehensive studies establish the effectiveness of TAS2R agonists in mitigating multiple features of allergic asthma.

  12. Peroxisome proliferator-activated receptor-{gamma} agonists inhibit the replication of respiratory syncytial virus (RSV) in human lung epithelial cells

    SciTech Connect

    Arnold, Ralf . E-mail: ralf.arnold@medizin.uni-magdeburg.de; Koenig, Wolfgang

    2006-07-05

    We have previously shown that peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}) agonists inhibited the inflammatory response of RSV-infected human lung epithelial cells. In this study, we supply evidence that specific PPAR{gamma} agonists (15d-PGJ{sub 2}, ciglitazone, troglitazone, Fmoc-Leu) efficiently blocked the RSV-induced cytotoxicity and development of syncytia in tissue culture (A549, HEp-2). All PPAR{gamma} agonists under study markedly inhibited the cell surface expression of the viral G and F protein on RSV-infected A549 cells. This was paralleled by a reduced cellular amount of N protein-encoding mRNA determined by real-time RT-PCR. Concomitantly, a reduced release of infectious progeny virus into the cell supernatants of human lung epithelial cells (A549, normal human bronchial epithelial cells (NHBE)) was observed. Similar results were obtained regardless whether PPAR{gamma} agonists were added prior to RSV infection or thereafter, suggesting that the agonists inhibited viral gene expression and not the primary adhesion or fusion process.

  13. [Influence of GABA agonist phenibut on the neuronal activity and interaction in hippocampus and neocortex in emotionally negative situations].

    PubMed

    Ziablintseva, E A; Pavlova, I V

    2009-09-01

    The activity of individual neurons and interaction of neighboring cells in hippocampus (CA1 area) and neocortical parieto-temporal area were compared in negative emotional situations in normal and in decreased anxiety produced by systemic injection of GABA agonist: phenibut. Analysis of the autocorrelation histogram shapes showed that in both structures phenibut increased bursts of neuronal discharges, decreased the interspike intervals within the burst, increased the number of neurons with delta-frequency oscillation and decreased the number of neurons with theta-1 oscillation. In hippocampus, in addition the intensity of theta-2 frequencies increased. During phenibut action, the irritating agents evoked lesser changes in neuronal activity as compared to the norm. Analysis of the crosscorrelation histogram shapes showed that, under exposure to phenibut in both structures, there were an increase in the number of common inputs to recorded neurons and a decrease in the number of excitatory connections. In hippocampus, there was still an increase in the number of inhibitory connections. The revealed changes in neuronal activity produced by phenibut indicated a decrease in the activation level of hippocampus and neocortex, an increase of neuronal synchronization and a decrease in excitation spread among neurons, that correlated with a reduction of behavioral reactivity and anxiety of animals.

  14. Activation of latent HIV-1 expression by protein kinase C agonists. A novel therapeutic approach to eradicate HIV-1 reservoirs.

    PubMed

    Sánchez-Duffhues, Gonzalo; Vo, Minh Q; Pérez, Moisés; Calzado, Marco A; Moreno, Santiago; Appendino, Giovanni; Muñoz, Eduardo

    2011-03-01

    The persistence of latent HIV-infected cellular reservoirs represents the major hurdle to virus eradication in patients treated with highly active antiretroviral therapy. The molecular mechanisms by which integrated HIV-1 is repressed during latency have been partially identified in different models of HIV-1 latency, and the involvement of multiple processes has been demonstrated. Therefore, several molecular targets amenable to pharmacological manipulation have emerged to antagonize HIV-1 latency in the viral reservoirs. In this context, it has been suggested that successful depletion of such latent reservoirs will require a combination of therapeutic agents that can specifically and efficiently act on cells harbouring latent HIV-1 provirus. HIV-1 reactivation therapy is a potential therapeutic option to purge the viral reservoirs. The goal of this therapy is to enhance the transcriptional activity of the latent HIV-1 without inducing the polyclonal activation of non-infected cells. In this sense natural or semisynthetic protein kinase C agonists lacking tumour-promoter activities clearly fulfil this criterion, thereby opening new research avenues to purge HIV-1 reservoirs. In this review article, we have succinctly summarized the known effects of "natural products", focusing on phorboids like prostratin and ingenols, macrolides like bryostatin 1, and macrocyclic polyesters like ingols and jatrophanes. A comprehensive view on the molecular mechanisms underlying the principle of HIV-1 reactivation from latency is provided, discussing the combination of "natural products" with other experimental or conventional therapeutics.

  15. Role of calcium-activated potassium channels in the regulation of basal and agonist-elevated tones in isolated conduit arteries. Short communication.

    PubMed

    Pataricza, J; Márton, Z; Hegedus, Z; Krassói, Irén; Kun, A; Varró, A; Papp, J Gy

    2004-01-01

    Functional role of calcium-activated potassium (KCa) channels on the basal and agonist-elevated arterial tones was investigated in isolated rabbit aorta, porcine and canine coronary arteries as well as in human internal mammary artery. The vascular tones enhanced by contractile agents were increased further by preincubation of these conduit blood vessels with selective (charybdotoxin or iberiotoxin) or nonselective (tetraethylammonium) inhibitors of KCa channels. The basal tone (without an agonist) was increased only in the canine coronary artery. The results indicate a feed-back regulatory role of KCa channels counteracting the vasospasm of conduit arteries.

  16. Synthesis and structure-activity relationships of N-aryl-piperidine derivatives as potent (partial) agonists for human histamine H3 receptor.

    PubMed

    Ishikawa, Makoto; Furuuchi, Takeshi; Yamauchi, Miki; Yokoyama, Fumikazu; Kakui, Nobukazu; Sato, Yasuo

    2010-07-15

    4-((1H-imidazol-4-yl)methyl)-1-aryl-piperazine and piperidine derivatives were designed and synthesized as candidate human histamine type 3 agonists. The piperazine derivatives were found to have low (or no) affinity for human histamine H3 receptor, whereas the piperidine derivatives showed moderate to high affinity, and their agonistic activity was greatly influenced by substituents on the aromatic ring. Among the piperidine-containing compounds, 17d and 17h were potent human histamine H3 receptor agonists with high selectivity over the closely related human H4 receptor. Our results indicate that appropriate conformational restriction, that is, by the piperidine spacer moiety, favors specific binding to the human histamine H3 receptor.

  17. Differential pathway coupling efficiency of the activated insulin receptor drives signaling selectivity by XMetA, an allosteric partial agonist antibody

    Technology Transfer Automated Retrieval System (TEKTRAN)

    XMetA, an anti-insulin receptor (IR) monoclonal antibody, is an allosteric partial agonist of the IR. We have previously reported that XMetA activates the “metabolic-biased” Akt kinase signaling pathway while having little or no effect on the “mitogenic” MAPK signaling pathwayof ERK 1/2. To inves...

  18. An EP2 Agonist Facilitates NMDA-Induced Outward Currents and Inhibits Dendritic Beading through Activation of BK Channels in Mouse Cortical Neurons

    PubMed Central

    Hayashi, Yoshinori; Morinaga, Saori; Liu, Xia; Zhang, Jing; Wu, Zhou; Yokoyama, Takeshi; Nakanishi, Hiroshi

    2016-01-01

    Prostaglandin E2 (PGE2), a major metabolite of arachidonic acid produced by cyclooxygenase pathways, exerts its bioactive responses by activating four E-prostanoid receptor subtypes, EP1, EP2, EP3, and EP4. PGE2 enables modulating N-methyl-D-aspartate (NMDA) receptor-mediated responses. However, the effect of E-prostanoid receptor agonists on large-conductance Ca2+-activated K+ (BK) channels, which are functionally coupled with NMDA receptors, remains unclear. Here, we showed that EP2 receptor-mediated signaling pathways increased NMDA-induced outward currents (INMDA-OUT), which are associated with the BK channel activation. Patch-clamp recordings from the acutely dissociated mouse cortical neurons revealed that an EP2 receptor agonist activated INMDA-OUT, whereas an EP3 receptor agonist reduced it. Agonists of EP1 or EP4 receptors showed no significant effects on INMDA-OUT. A direct perfusion of 3,5′-cyclic adenosine monophosphate (cAMP) through the patch pipette facilitated INMDA-OUT, which was abolished by the presence of protein kinase A (PKA) inhibitor. Furthermore, facilitation of INMDA-OUT caused by an EP2 receptor agonist was significantly suppressed by PKA inhibitor. Finally, the activation of BK channels through EP2 receptors facilitated the recovery phase of NMDA-induced dendritic beading in the primary cultured cortical neurons. These results suggest that a direct activation of BK channels by EP2 receptor-mediated signaling pathways plays neuroprotective roles in cortical neurons. PMID:27298516

  19. A GPBAR1 (TGR5) Small Molecule Agonist Shows Specific Inhibitory Effects on Myeloid Cell Activation In Vitro and Reduces Experimental Autoimmune Encephalitis (EAE) In Vivo

    PubMed Central

    Lewis, Nuruddeen D.; Patnaude, Lori A.; Pelletier, Josephine; Souza, Donald J.; Lukas, Susan M.; King, F. James; Hill, Jonathan D.; Stefanopoulos, Dimitria E.; Ryan, Kelli; Desai, Sudha; Skow, Donna; Kauschke, Stefan G.; Broermann, Andre; Kuzmich, Daniel; Harcken, Christian; Hickey, Eugene R.; Modis, Louise K.

    2014-01-01

    GPBAR1 is a G protein-coupled receptor that is activated by certain bile acids and plays an important role in the regulation of bile acid synthesis, lipid metabolism, and energy homeostasis. Recent evidence suggests that GPBAR1 may also have important effects in reducing the inflammatory response through its expression on monocytes and macrophages. To further understand the role of GPBAR1 in inflammation, we generated a novel, selective, proprietary GPBAR1 agonist and tested its effectiveness at reducing monocyte and macrophage activation in vitro and in vivo. We have used this agonist, together with previously described agonists to study agonism of GPBAR1, and shown that they can all induce cAMP and reduce TLR activation-induced cytokine production in human monocytes and monocyte-derived macrophages in vitro. Additionally, through the usage of RNA sequencing (RNA-Seq), we identified a select set of genes that are regulated by GPBAR1 agonism during LPS activation. To further define the in vivo role of GPBAR1 in inflammation, we assessed GPBAR1 expression and found high levels on circulating mouse monocytes. Agonism of GPBAR1 reduced LPS-induced cytokine production in mouse monocytes ex vivo and serum cytokine levels in vivo. Agonism of GPBAR1 also had profound effects in the experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis, where monocytes play an important role. Mice treated with the GPBAR1 agonist exhibited a significant reduction in the EAE clinical score which correlated with reduced monocyte and microglial activation and reduced trafficking of monocytes and T cells into the CNS. These data confirm the importance of GPBAR1 in controlling monocyte and macrophage activation in vivo and support the rationale for selective agonists of GPBAR1 in the treatment of inflammatory diseases. PMID:24967665

  20. Identification of 6-octadecynoic acid from a methanol extract of Marrubium vulgare L. as a peroxisome proliferator-activated receptor γ agonist

    SciTech Connect

    Ohtera, Anna; Miyamae, Yusaku; Nakai, Naomi; Kawachi, Atsushi; Kawada, Kiyokazu; Han, Junkyu; Isoda, Hiroko; Neffati, Mohamed; Akita, Toru; Maejima, Kazuhiro; Masuda, Seiji; Kambe, Taiho; Mori, Naoki; Irie, Kazuhiro; Nagao, Masaya

    2013-10-18

    Highlights: •6-ODA, a rare fatty acid with a triple bond, was identified from Marrubium vulgare. •6-ODA was synthesized from petroselinic acid as a starting material. •6-ODA stimulated lipid accumulation in HSC-T6 and 3T3-L1 cells. •The first report of a fatty acid with a triple bond functioning as a PPARγ agonist. •This study sheds light on novel functions of a fatty acid with a triple bond. -- Abstract: 6-Octadecynoic acid (6-ODA), a fatty acid with a triple bond, was identified in the methanol extract of Marrubium vulgare L. as an agonist of peroxisome proliferator-activated receptor γ (PPARγ). Fibrogenesis caused by hepatic stellate cells is inhibited by PPARγ whose ligands are clinically used for the treatment of diabetes. Plant extracts of Marrubium vulgare L., were screened for activity to inhibit fibrosis in the hepatic stellate cell line HSC-T6 using Oil Red-O staining, which detects lipids that typically accumulate in quiescent hepatic stellate cells. A methanol extract with activity to stimulate accumulation of lipids was obtained. This extract was found to have PPARγ agonist activity using a luciferase reporter assay. After purification using several chromatographic methods, 6-ODA, a fatty acid with a triple bond, was identified as a candidate of PPARγ agonist. Synthesized 6-ODA and its derivative 9-octadecynoic acid (9-ODA), which both have a triple bond but in different positions, activated PPARγ in a luciferase reporter assay and increased lipid accumulation in 3T3-L1 adipocytes in a PPARγ-dependent manner. There is little information about the biological activity of fatty acids with a triple bond, and to our knowledge, this is the first report that 6-ODA and 9-ODA function as PPARγ agonists.

  1. Design and synthesis of downsized metastin (45-54) analogs with maintenance of high GPR54 agonistic activity.

    PubMed

    Niida, Ayumu; Wang, Zixuan; Tomita, Kenji; Oishi, Shinya; Tamamura, Hirokazu; Otaka, Akira; Navenot, Jean-Marc; Broach, James R; Peiper, Stephen C; Fujii, Nobutaka

    2006-01-01

    Metastin has been identified as a metastasis suppressor gene product that mediates its function through a G protein coupled receptor, GPR54. To refine insight into the critical pharmacophore for the activation of GPR54, we have conducted alanine and d-amino acid scanning on a biologically active metastin fragment (45-54). Based on these data and structures of peptides previously reported to activate GPR54, a series of shortened metastin (45-54) derivatives were synthesized and tested for the ability to induce GPR54 signaling. These biological experiments were performed in yeast containing human GPR54 that was coupled to the pheromone response pathway and a pheromone responsive lacZ reporter gene. Compounds 32, 33, and 39, which possess an N-terminal basic group and a C-terminal RW-amide motif, were strong agonists, similar to the level of metastin. This may provide an approach to reverse the pro-metastatic effect of metastin deletion in multiple malignant tumors.

  2. CtBP1/BARS is an activator of phospholipase D1 necessary for agonist-induced macropinocytosis.

    PubMed

    Haga, Yuki; Miwa, Noriko; Jahangeer, Saleem; Okada, Taro; Nakamura, Shun-ichi

    2009-05-06

    Vesicular trafficking such as macropinocytosis is a dynamic process that requires coordinated interactions between specialized proteins and lipids. A recent report suggests the involvement of CtBP1/BARS in epidermal growth factor (EGF)-induced macropinocytosis. Detailed mechanisms as to how lipid remodelling is regulated during macropinocytosis are still undefined. Here, we show that CtBP1/BARS is a physiological activator of PLD1 required in agonist-induced macropinocytosis. EGF-induced macropinocytosis was specifically blocked by 1-butanol but not by 2-butanol. In addition, stimulation of cells by serum or EGF resulted in the association of CtBP1/BARS with PLD1. Finally, CtBP1/BARS activated PLD1 in a synergistic manner with other PLD activators, including ADP-ribosylation factors as demonstrated by in vitro and intact cell systems. The present results shed light on the molecular basis of how the 'fission protein' CtBP1/BARS controls vesicular trafficking events including macropinocytosis.

  3. Peroxisome Proliferator-Activated Receptor (PPAR) γ and PPARα Agonists Modulate Mitochondrial Fusion-Fission Dynamics: Relevance to Reactive Oxygen Species (ROS)-Related Neurodegenerative Disorders?

    PubMed Central

    Zolezzi, Juan M.; Silva-Alvarez, Carmen; Ordenes, Daniela; Godoy, Juan A.; Carvajal, Francisco J.; Santos, Manuel J.; Inestrosa, Nibaldo C.

    2013-01-01

    Recent studies showed that the activation of the retinoid X receptor, which dimerizes with peroxisome proliferator-activated receptors (PPARs), leads to an enhanced clearance of Aβ from the brain of transgenic mice model of Alzheimer’s disease (AD), because an increased expression of apolipoprotein E and it main transporters. However, the effects observed must involve additional underlying mechanisms that have not been yet explored. Several studies conducted in our laboratory suggest that part of the effects observed for the PPARs agonist might involves mitochondrial function and, particularly, mitochondrial dynamics. In the present study we assessed the effects of oxidative stress challenge on mitochondrial morphology and mitochondrial dynamics-related proteins in hippocampal neurons. Using immunofluorescence, we evaluated the PPARγ co-activator 1α (PGC-1α), dynamin related protein 1 (DRP1), mitochondrial fission protein 1 (FIS1), and mitochondrial length, in order to determine if PPARs agonist pre-treatment is able to protect mitochondrial population from hippocampal neurons through modulation of the mitochondrial fusion-fission events. Our results suggest that both a PPARγ agonist (ciglitazone) and a PPARα agonist (WY 14.643) are able to protect neurons by modulating mitochondrial fusion and fission, leading to a better response of neurons to oxidative stress, suggesting that a PPAR based therapy could acts simultaneously in different cellular components. Additionally, our results suggest that PGC-1α and mitochondrial dynamics should be further studied in future therapy research oriented to ameliorate neurodegenerative disorders, such as AD. PMID:23675519

  4. Clinical Effects of a Topically Applied Toll-like Receptor 9 Agonist in Active Moderate-to-Severe Ulcerative Colitis

    PubMed Central

    Atreya, Raja; Bloom, Stuart; Scaldaferri, Franco; Gerardi, Viviana; Admyre, Charlotte; Karlsson, Åsa; Knittel, Thomas; Kowalski, Jan; Lukas, Milan; Löfberg, Robert; Nancey, Stephane; Petryka, Robert; Rydzewska, Grazyna; Schnabel, Robert; Seidler, Ursula; Neurath, Markus F.

    2016-01-01

    Background and Aims: Toll-like receptors [TLRs] are potential drug targets for immunomodulation. We determined the safety and efficacy of the TLR-9 agonist DNA-based immunomodulatory sequence 0150 [DIMS0150] in ulcerative colitis [UC] patients refractory to standard therapy. Methods: In this randomized, double-blind, placebo-controlled trial, 131 patients with moderate-to-severe active UC were randomized to receive two single doses of the oligonucleotide DIMS0150 [30 mg] or placebo administered topically during lower GI endoscopy at baseline and Week 4. The primary endpoint was clinical remission, defined as Clinical Activity Index [CAI] ≤4, at Week 12. Secondary endpoints included mucosal healing and symptomatic remission of key patient-reported outcomes [absence of blood in stool and weekly stool frequency <35]. Results: There was no statistical significant difference between the groups in the induction of clinical remission at Week 12, with 44.4% in the DIMS0150 group vs. 46.5% in the placebo group. However, the proportion of patients who achieved symptomatic remission was 32.1% in the DIMS0150 group vs. 14.0% in the placebo group at Week 4 [p = 0.020], and 44.4% vs. 27.9% at Week 8 [p = 0.061]. More patients on DIMS0150 compared with those on placebo had mucosal healing [34.6% vs. 18.6%; p = 0.09] and histological improvement regarding the Geboes score [30.9% vs. 9.3%; p = 0.0073] at Week 4. Significantly more patients on DIMS0150 were in clinical remission with mucosal healing at Week 4: 21% vs. 4.7% in the placebo group [p = 0.02]. DIMS0150 was well tolerated, and no safety signals compared with placebo were evident. Conclusions: Therapy with the topically applied TLR-9 agonist DIMS0150 is a promising and well-tolerated novel therapeutic option for treatment-refractory, chronic active UC patients, warranting further clinical trials. PMID:27208386

  5. Effects of α1-adrenoceptor agonist phenylephrine on swelling-activated chloride currents in human atrial myocytes.

    PubMed

    Li, Yetao; Du, Xinling

    2015-02-01

    Swelling-activated chloride currents (ICl.swell) play an important role in cardiac electrophysiology and arrhythmogenesis. However, the regulation of these currents has not been clarified to date. In this research, we focused on the function of phenylephrine, an α1-adrenoceptor agonist, in the regulation of I(Cl.swell) in human atrial myocytes. We recorded I(Cl.swell) evoked by a hypotonic bath solution with the whole-cell patch-clamp technique. We found that I(Cl.swell) increased over time, and it was difficult to achieve absolute steady state. Phenylephrine potentiated I(Cl.swell) from -1.00 ± 0.51 pA/pF at -90 mV and 2.58 ± 1.17 pA/pF at +40 mV to -1.46 ± 0.70 and 3.84 ± 1.67 pA/pF, respectively (P < 0.05, n = 6), and the upward trend in ICl.swell was slowed after washout. This effect was concentration-dependent, and the α1-adrenoceptor antagonist prazosin shifted the dose-effect curve rightward. Addition of prazosin or the protein kinase C (PKC) inhibitor bisindolylmaleimide (BIM) attenuated the effect of phenylephrine. The PKC activator phorbol 12,13-dibutyrate (PDBu) activated I(Cl.swell) from -1.69 ± 1.67 pA/pF at -90 mV and 5.58 ± 6.36 pA/pF at +40 mV to -2.41 ± 1.95 pA/pF and 7.05 ± 6.99 pA/pF, respectively (P < 0.01 at -90 mV and P < 0.05 at +40 mV; n = 6). In conclusion, the α1-adrenoceptor agonist phenylephrine augmented I(Cl.swell), a result that differs from previous reports in other animal species. The effect was attenuated by BIM and mimicked by PDBu, which indicates that phenylephrine might modulate I(Cl,swell) in a PKC-dependent manner.

  6. Peroxisome-proliferator-activated receptor-{gamma} agonists inhibit the release of proinflammatory cytokines from RSV-infected epithelial cells

    SciTech Connect

    Arnold, Ralf . E-mail: ralf.arnold@medizin.uni-magdeburg.de; Koenig, Wolfgang

    2006-03-15

    The epithelial cells of the airways are the target cells for respiratory syncytial virus (RSV) infection and the site of the majority of the inflammation associated with the disease. Recently, peroxisome-proliferator-activated receptor {gamma} (PPAR{gamma}), a member of the nuclear hormone receptor superfamily, has been shown to possess anti-inflammatory properties. Therefore, we investigated the role of PPAR{gamma} agonists (15d-PGJ{sub 2}, ciglitazone and troglitazone) on the synthesis of RSV-induced cytokine release from RSV-infected human lung epithelial cells (A549). We observed that all PPAR{gamma} ligands inhibited dose-dependently the release of TNF-{alpha}, GM-CSF, IL-1{alpha}, IL-6 and the chemokines CXCL8 (IL-8) and CCL5 (RANTES) from RSV-infected A549 cells. Concomitantly, the PPAR{gamma} ligands diminished the cellular amount of mRNA encoding for IL-6, CXCL8 and CCL5 and the RSV-induced binding activity of the transcription factors NF-{kappa}B (p65/p50) and AP-1 (c-fos), respectively. Our data presented herein suggest a potential application of PPAR{gamma} ligands in the anti-inflammatory treatment of RSV infection.

  7. Ahr2-dependence of PCB126 effects on the swim bladder in relation to expression of CYP1 and cox-2 genes in developing zebrafish.

    PubMed

    Jönsson, Maria E; Kubota, Akira; Timme-Laragy, Alicia R; Woodin, Bruce; Stegeman, John J

    2012-12-01

    The teleost swim bladder is assumed a homolog of the tetrapod lung. Both swim bladder and lung are developmental targets of persistent aryl hydrocarbon receptor (AHR(2)) agonists; in zebrafish (Danio rerio) the swim bladder fails to inflate with exposure to 3,3',4,4',5-pentachlorobiphenyl (PCB126). The mechanism for this effect is unknown, but studies have suggested roles of cytochrome P450 1 (CYP1) and cyclooxygenase 2 (Cox-2) in some Ahr-mediated developmental effects in zebrafish. We determined relationships between swim bladder inflation and CYP1 and Cox-2 mRNA expression in PCB126-exposed zebrafish embryos. We also examined effects on β-catenin dependent transcription, histological effects, and Ahr2 dependence of the effect of PCB126 on swim bladder using morpholinos targeting ahr2. One-day-old embryos were exposed to waterborne PCB126 or carrier (DMSO) for 24h and then held in clean water until day 4, a normal time for swim bladder inflation. The effects of PCB126 were concentration-dependent with EC(50) values of 1.4 to 2.0 nM for induction of the CYP1s, 3.7 and 5.1 nM (or higher) for cox-2a and cox-2b induction, and 2.5 nM for inhibition of swim bladder inflation. Histological defects included a compaction of the developing bladder. Ahr2-morpholino treatment rescued the effect of PCB126 (5 nM) on swim bladder inflation and blocked induction of CYP1A, cox-2a, and cox-2b. With 2nM PCB126 approximately 30% of eleutheroembryos(3) failed to inflate the swim bladder, but there was no difference in CYP1 or cox-2 mRNA expression between those embryos and embryos showing inflated swim bladder. Our results indicate that PCB126 blocks swim bladder inflation via an Ahr2-mediated mechanism. This mechanism seems independent of CYP1 or cox-2 mRNA induction but may involve abnormal development of swim bladder cells.

  8. Effects of the environmental contaminants DEHP and TCDD on estradiol synthesis and aryl hydrocarbon receptor and peroxisome proliferator-activated receptor signalling in the human granulosa cell line KGN.

    PubMed

    Ernst, Jana; Jann, Johann-Christoph; Biemann, Ronald; Koch, Holger M; Fischer, Bernd

    2014-09-01

    Environmental contaminants binding to transcription factors, such as the aryl hydrocarbon receptor (AhR) and the alpha and gamma peroxisome proliferator-activated receptors (PPARs), contribute to adverse effects on the reproductive system. Expressing both the AhR and PPARs, the human granulosa cell line KGN offers the opportunity to investigate the regulatory mechanisms involved in receptor crosstalk, independent of overriding hormonal control. The aim of the present study was to investigate the impact of two environmental contaminants, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, an AhR ligand) and di-(2-ethylhexyl) phthalate (DEHP, a PPAR ligand), on gonadotrophin sensitivity and estrogen synthesis in KGN cells. Accumulation of the DEHP metabolite mono-(2-ethylhexyl) phthalate (MEHP) in DEHP-exposed cells was measured by high-performance liquid chromatography mass spectrometry, thereby demonstrating DEHP metabolism to MEHP by KGN cells. By employing TCDD ( an AhR agonist), rosiglitazone (a PPARgamma agonist) or bezafibrate (a PPARalpha agonist), the presence of a functional AhR and PPAR cascade was confirmed in KGN cells. Cytotoxicity testing revealed no effect on KGN cell proliferation for the concentrations of TCDD and DEHP used in the current study. FSH-stimulated cells were exposed to TCDD, DEHP or a mix of both and estradiol synthesis was measured by enzyme-linked immunosorbent assay and gene expression by quantitative RT-PCR. Exposure decreased estradiol synthesis (TCDD, DEHP, mix) and reduced the mRNA expression of CYP19 aromatase (DEHP, mix) and FSHR (DEHP). DEHP induced the expression of the alpha and gamma PPARs and AhR, an effect which was inhibited by selective PPAR antagonists. Studies in the human granulosa cell line KGN show that the action of endocrine-disrupting chemicals may be due to a direct activation of AhR, for example by TCDD, and by a transactivation via PPARs, for example by DEHP, inducing subsequent transcriptional changes with a broad

  9. Nortestosterone-derived synthetic progestogens do not activate the progestogen receptor of Murray-Darling rainbowfish (Melanotaenia fluviatilis) but are potent agonists of androgen receptors alpha and beta.

    PubMed

    Bain, Peter A; Kumar, Anu; Ogino, Yukiko; Iguchi, Taisen

    2015-06-01

    Synthetic progestogens derived from 19-nortestosterone can elicit a number of adverse effects in fish including decreased fecundity, altered hormone levels, disruption of normal breeding cycles, expression in females of male-specific biomarkers, development of male secondary sexual characteristics in females, and changes in the expression of steroidogenic genes. A recent in vitro study showed that a number of representatives from this class of progestins were potent agonists of fathead minnow androgen receptor (AR) and only weak agonists of progesterone receptor (PR) from the same species. This confirms that synthetic progestogens derived from 19-nortestosterone function as AR agonists in otomorphs, which express a single AR subtype. However, numerous perciformes are known to express two AR subtypes. We have recently shown that ARα and ARβ from Murray-Darling rainbowfish (Melanotaenia fluviatilis) respond differently to certain androgens and anti-androgens. The goal of the present study was to determine concentration-response profiles for selected progestins in transactivation assays driven by rainbowfish ARα, ARβ and PR in order to ascertain the relative potency of progestins against these receptors. As a means of confirming the expected activity of the progestins and reference compounds used in the study against human-derived receptors, we also established concentration-response relationships using transactivation assays driven by human PR and AR. We found that all five 19-nortestosterone-derived progestins tested were highly potent agonists of rainbowfish ARα, but that only four of the five progestins were potent agonists of rainbowfish ARβ, with norgestimate exhibiting only weak activity against rainbowfish ARβ. The spironolactone-derived progestin, drospirenone, was not an agonist of rainbowfish ARα or ARβ but was a weak agonist of rainbowfish PR. None of the 19-nortestosterone-progestins activated rainbowfish PR. These findings confirm that the

  10. Treatment with PPARα Agonist Clofibrate Inhibits the Transcription and Activation of SREBPs and Reduces Triglyceride and Cholesterol Levels in Liver of Broiler Chickens

    PubMed Central

    Zhang, Lijun; Li, Chunyan; Wang, Fang; Zhou, Shenghua; Shangguan, Mingjun; Xue, Lina; Zhang, Bianying; Ding, Fuxiang; Hui, Dequan; Liang, Aihua; He, Dongchang

    2015-01-01

    PPARα agonist clofibrate reduces cholesterol and fatty acid concentrations in rodent liver by an inhibition of SREBP-dependent gene expression. In present study we investigated the regulation mechanisms of the triglyceride- and cholesterol-lowering effect of the PPARα agonist clofibrate in broiler chickens. We observed that PPARα agonist clofibrate decreases the mRNA and protein levels of LXRα and the mRNA and both precursor and nuclear protein levels of SREBP1 and SREBP2 as well as the mRNA levels of the SREBP1 (FASN and GPAM) and SREBP2 (HMGCR and LDLR) target genes in the liver of treated broiler chickens compared to control group, whereas the mRNA level of INSIG2, which inhibits SREBP activation, was increased in the liver of treated broiler chickens compared to control group. Taken together, the effects of PPARα agonist clofibrate on lipid metabolism in liver of broiler chickens involve inhibiting transcription and activation of SREBPs and SREBP-dependent lipogenic and cholesterologenic gene expression, thereby resulting in a reduction of the triglyceride and cholesterol levels in liver of broiler chickens. PMID:26693219

  11. Down-Regulation of Ca2+-Activated K+ Channel KCa1.1 in Human Breast Cancer MDA-MB-453 Cells Treated with Vitamin D Receptor Agonists

    PubMed Central

    Khatun, Anowara; Fujimoto, Mayu; Kito, Hiroaki; Niwa, Satomi; Suzuki, Takayoshi; Ohya, Susumu

    2016-01-01

    Vitamin D (VD) reduces the risk of breast cancer and improves disease prognoses. Potential VD analogs are being developed as therapeutic agents for breast cancer treatments. The large-conductance Ca2+-activated K+ channel KCa1.1 regulates intracellular Ca2+ signaling pathways and is associated with high grade tumors and poor prognoses. In the present study, we examined the effects of treatments with VD receptor (VDR) agonists on the expression and activity of KCa1.1 in human breast cancer MDA-MB-453 cells using real-time PCR, Western blotting, flow cytometry, and voltage-sensitive dye imaging. Treatments with VDR agonists for 72 h markedly decreased the expression levels of KCa1.1 transcripts and proteins in MDA-MB-453 cells, resulting in the significant inhibition of depolarization responses induced by paxilline, a specific KCa1.1 blocker. The specific proteasome inhibitor MG132 suppressed VDR agonist-induced decreases in KCa1.1 protein expression. These results suggest that KCa1.1 is a new downstream target of VDR signaling and the down-regulation of KCa1.1 through the transcriptional repression of KCa1.1 and enhancement of KCa1.1 protein degradation contribute, at least partly, to the antiproliferative effects of VDR agonists in breast cancer cells. PMID:27973439

  12. Effects of 5-HT1A Receptor Stimulation on D1 Receptor Agonist-Induced Striatonigral Activity and Dyskinesia in Hemiparkinsonian Rats

    PubMed Central

    2013-01-01

    Accumulating evidence supports the value of 5-HT1A receptor (5-HT1AR) agonists for dyskinesias that arise with long-term L-DOPA therapy in Parkinson’s disease (PD). Yet, how 5-HT1AR stimulation directly influences the dyskinetogenic D1 receptor (D1R)-expressing striatonigral pathway remains largely unknown. To directly examine this, one cohort of hemiparkinsonian rats received systemic injections of Vehicle + Vehicle, Vehicle + the D1R agonist SKF81297 (0.8 mg/kg), or the 5-HT1AR agonist ±8-OH-DPAT (1.0 mg/kg) + SKF81297. Rats were examined for changes in abnormal involuntary movements (AIMs), rotations, striatal preprodynorphin (PPD), and glutamic acid decarboxylase (GAD; 65 and 67) mRNA via RT-PCR. In the second experiment, hemiparkinsonian rats received intrastriatal pretreatments of Vehicle (aCSF), ±8-OH-DPAT (7.5 mM), or ±8-OH-DPAT + the 5-HT1AR antagonist WAY100635 (4.6 mM), followed by systemic Vehicle or SKF81297 after which AIMs, rotations, and extracellular striatal glutamate and nigral GABA efflux were measured by in vivo microdialysis. Results revealed D1R agonist-induced AIMs were reduced by systemic and intrastriatal 5-HT1AR stimulation while rotations were enhanced. Although ±8-OH-DPAT did not modify D1R agonist-induced increases in striatal PPD mRNA, the D1R/5-HT1AR agonist combination enhanced GAD65 and GAD67 mRNA. When applied locally, ±8-OH-DPAT alone diminished striatal glutamate levels while the agonist combination increased nigral GABA efflux. Thus, presynaptic 5-HT1AR stimulation may attenuate striatal glutamate levels, resulting in diminished D1R-mediated dyskinetic behaviors, but maintain or enhance striatal postsynaptic factors ultimately increasing nigral GABA levels and rotational activity. The current findings offer a novel mechanistic explanation for previous results concerning 5-HT1AR agonists for the treatment of dyskinesia. PMID:23496922

  13. Resistance to teratogenesis by F1 and F2 embryos of PAH-adapted Fundulus heteroclitus is strongly inherited despite reduced recalcitrance of the AHR pathway.

    PubMed

    Clark, Bryan W; Bone, A J; Di Giulio, R T

    2014-12-01

    Atlantic killifish (Fundulus heteroclitus) inhabiting the Atlantic Wood Superfund site on the Elizabeth River (Portsmouth, VA, USA) are exposed to a complex mixture of polycyclic aromatic hydrocarbons (PAHs) from former creosote operations, but are resistant to the acute toxicity and cardiac teratogenesis caused by PAHs. The resistance is associated with a dramatic recalcitrance to induction of cytochrome P450 (CYP1) metabolism enzymes following exposure to aryl hydrocarbon receptor (AHR) agonists, along with an elevated antioxidant response and increased expression of several other xenobiotic metabolism and excretion enzymes. However, the heritability of the resistance in the absence of chemical stressors has been inconsistently demonstrated. Understanding the heritability of this resistance will help clarify the nature of population-level responses to chronic exposure to PAH mixtures and aid in identifying the important mechanistic components of resistance to aryl hydrocarbons. We compared the response of Atlantic Wood F1 and F2 embryos to benzo[k]fluoranthene (BkF), benzo[a]pyrene (BaP), 3,3',4,4',5-pentachlorobiphenyl (PCB-126), and a mixture of BkF and fluoranthene (Fl) to that of F1 embryos of reference site killifish. Resistance to cardiac teratogenesis and induction of CYP mRNA expression and CYP activity was determined. We found that both Atlantic Wood F1 and F2 embryos were highly resistance to cardiac teratogenesis. However, the resistance by Atlantic Wood F2 embryos to induction of CYP mRNA expression and enzyme activity was intermediate between that of Atlantic Wood F1 embryos and reference embryos. These results suggest that resistance to cardiac teratogenesis in Atlantic Wood fish is conferred by multiple factors, not all of which appear to be fully genetically heritable.

  14. Resistance to teratogenesis by F1 and F2 embryos of PAH-adapted Fundulus heteroclitus is strongly inherited despite reduced recalcitrance of the AHR pathway

    PubMed Central

    Clark, B. W.; Bone, A. J.; Di Giulio, R. T.

    2014-01-01

    Atlantic killifish (Fundulus heteroclitus) inhabiting the Atlantic Wood Superfund site on the Elizabeth River (Portsmouth, VA, USA) are exposed to a complex mixture of polycyclic aromatic hydrocarbons (PAHs) from former creosote operations, but are resistant to the acute toxicity and cardiac teratogenesis caused by PAHs. The resistance is associated with a dramatic recalcitrance to induction of cytochrome P450 (CYP1) metabolism enzymes following exposure to aryl hydrocarbon receptor (AHR) agonists, along with an elevated antioxidant response and increased expression of several other xenobiotic metabolism and excretion enzymes. However, the heritability of the resistance in the absence of chemical stressors has been inconsistently demonstrated. Understanding the heritability of this resistance will help clarify the nature of population-level responses to chronic exposure to PAH mixtures and aid in identifying the important mechanistic components of resistance to aryl hydrocarbons. We compared the response of Atlantic Wood F1 and F2 embryos to benzo[k]fluoranthene (BkF), benzo[a]pyrene (BaP), 3,3’,4,4’,5-pentachlorobiphenyl (PCB-126), and a mixture of BkF and fluoranthene (Fl) to that of F1 embryos of reference site killifish. Resistance to cardiac teratogenesis and induction of CYP mRNA expression and CYP activity was determined. We found that both Atlantic Wood F1 and F2 embryos were highly resistance to cardiac teratogenesis. However, the resistance by Atlantic Wood F2 embryos to induction of CYP mRNA expression and enzyme activity was intermediate between that of Atlantic Wood F1 embryos and reference embryos. These results suggest that resistance to cardiac teratogenesis in Atlantic Wood fish is conferred by multiple factors, not all of which appear to be fully genetically heritable. PMID:24374617

  15. Characterization of a Novel Human-Specific STING Agonist that Elicits Antiviral Activity Against Emerging Alphaviruses

    PubMed Central

    Sali, Tina M.; Pryke, Kara M.; Abraham, Jinu; Liu, Andrew; Archer, Iris; Broeckel, Rebecca; Staverosky, Julia A.; Smith, Jessica L.; Al-Shammari, Ahmed; Amsler, Lisi; Sheridan, Kayla; Nilsen, Aaron; Streblow, Daniel N.; DeFilippis, Victor R.

    2015-01-01

    Pharmacologic stimulation of innate immune processes represents an attractive strategy to achieve multiple therapeutic outcomes including inhibition of virus replication, boosting antitumor immunity, and enhancing vaccine immunogenicity. In light of this we sought to identify small molecules capable of activating the type I interferon (IFN) response by way of the transcription factor IFN regulatory factor 3 (IRF3). A high throughput in vitro screen yielded 4-(2-chloro-6-fluorobenzyl)-N-(furan-2-ylmethyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]thiazine-6-carboxamide (referred to herein as G10), which was found to trigger IRF3/IFN-associated transcription in human fibroblasts. Further examination of the cellular response to this molecule revealed expression of multiple IRF3-dependent antiviral effector genes as well as type I and III IFN subtypes. This led to the establishment of a cellular state that prevented replication of emerging Alphavirus species including Chikungunya virus, Venezuelan Equine Encephalitis virus, and Sindbis virus. To define cellular proteins essential to elicitation of the antiviral activity by the compound we employed a reverse genetics approach that utilized genome editing via CRISPR/Cas9 technology. This allowed the identification of IRF3, the IRF3-activating adaptor molecule STING, and the IFN-associated transcription factor STAT1 as required for observed gene induction and antiviral effects. Biochemical analysis indicates that G10 does not bind to STING directly, however. Thus the compound may represent the first synthetic small molecule characterized as an indirect activator of human STING-dependent phenotypes. In vivo stimulation of STING-dependent activity by an unrelated small molecule in a mouse model of Chikungunya virus infection blocked viremia demonstrating that pharmacologic activation of this signaling pathway may represent a feasible strategy for combating emerging Alphaviruses. PMID:26646986

  16. Agonist and Antagonist Muscle EMG Activity Pattern Changes with Skill Acquisition.

    ERIC Educational Resources Information Center

    Engelhorn, Richard

    1983-01-01

    Using electromyography (EMG), researchers studied changes in the control of biceps and triceps brachii muscles that occurred as women college students learned two elbow flexion tasks. Data on EMG activity, angular kinematics, training, and angular displacement were analyzed. (Author/PP)

  17. Biostable agonists that match or exceed activity of native insect kinins on recombinant arthropod GPCRs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The multifunctional arthropod insect kinins share the evolutionarily conserved C-terminal pentapeptide motif Phe-X1-X2-Trp-Gly-NH2, where X1 = His, Asn, Ser, or Tyr and X2 = Ser, Pro, or Ala. Insect kinins regulate diuresis in many species of insects. Compounds with similar biological activity cou...

  18. Activation and desensitization of TRPV1 channels in sensory neurons by the PPARα agonist palmitoylethanolamide

    PubMed Central

    Ambrosino, Paolo; Soldovieri, Maria Virginia; Russo, Claudio; Taglialatela, Maurizio

    2013-01-01

    Background and Purpose Palmitoylethanolamide (PEA) is an endogenous fatty acid amide displaying anti-inflammatory and analgesic actions. To investigate the molecular mechanism responsible for these effects, the ability of PEA and of pain-inducing stimuli such as capsaicin (CAP) or bradykinin (BK) to influence intracellular calcium concentrations ([Ca2+]i) in peripheral sensory neurons, has been assessed in the present study. The potential involvement of the transcription factor PPARα and of TRPV1 channels in PEA-induced effects was also studied. Experimental Approach [Ca2+]i was evaluated by single-cell microfluorimetry in differentiated F11 cells. Activation of TRPV1 channels was assessed by imaging and patch-clamp techniques in CHO cells transiently-transfected with rat TRPV1 cDNA. Key Results In F11 cells, PEA (1–30 μM) dose-dependently increased [Ca2+]i. The TRPV1 antagonists capsazepine (1 μM) and SB-366791 (1 μM), as well as the PPARα antagonist GW-6471 (10 μM), inhibited PEA-induced [Ca2+]i increase; blockers of cannabinoid receptors were ineffective. PEA activated TRPV1 channels heterologously expressed in CHO cells; this effect appeared to be mediated at least in part by PPARα. When compared with CAP, PEA showed similar potency and lower efficacy, and caused stronger TRPV1 currents desensitization. Sub-effective PEA concentrations, closer to those found in vivo, counteracted CAP- and BK-induced [Ca2+]i transients, as well as CAP-induced TRPV1 activation. Conclusions and Implications Activation of PPARα and TRPV1 channels, rather than of cannabinoid receptors, largely mediate PEA-induced [Ca2+]i transients in sensory neurons. Differential TRPV1 activation and desensitization by CAP and PEA might contribute to their distinct pharmacological profile, possibly translating into potentially relevant clinical differences. PMID:23083124

  19. [Peptide-agonist of protease-activated receptor (PAR 1), similar to activated protein C, promotes proliferation in keratinocytes and wound healing of epithelial layer].

    PubMed

    Kiseleva, E V; Sidorova, M V; Gorbacheva, L R; Strukova, S M

    2014-01-01

    Activated protein C (APC) is serine protease hemostasis, independent of its anticoagulant activity, exhibits anti-inflammatory and anti-apoptotic properties that determine the possibility of the protective effects of APC in different diseases, including sepsis and chronic wound healing. APC, binding of endothelial protein C receptor (EPCR) and specifically cleaving PAR1 receptor and releasing peptide agonist PAR1 stabilizes not only endothelial cells, but also many others, including epidermal keratinocytes of the skin. We develop the hypothesis that the cytoprotective effect of APC on the cells, involved in wound healing, seem to imitate peptide - analogous of PAR1 "tethered ligand" that activate PAR1. In our work, we synthesized a peptide (AP9) - analogue of PAR1 tethered ligand, released by APC, and firstly showed that peptide AP9 (0.1-10 мM), like to APC (0.01-100 nM), stimulates the proliferative activity of human primary keratinocytes. Using a model of the formation of epithelial wounds in vitro we found that peptide AP9, as well as protease APC, accelerates wound healing. Using specific antibodies to the receptor PAR1 and EPCR was studied the receptor mechanism of AP9 action in wound healing compared with the action of APС. The necessity of both receptors - PAR1 and EPСR, for proliferative activity of agonists was revealed. Identified in our work imitation by peptide AP9 - PAR1 ligand, APC acts on keratinocytes suggests the possibility of using a peptide AP9 to stimulate tissue repair.

  20. Local release of pioglitazone (a peroxisome proliferator-activated receptor γ agonist) accelerates proliferation and remodeling phases of wound healing.

    PubMed

    Sakai, Shigeki; Sato, Keisuke; Tabata, Yasuhiko; Kishi, Kazuo

    2016-01-01

    Peroxisome proliferator-activated receptor γ (PPARγ) is a member of the nuclear receptor superfamily known for its anti-inflammatory and macrophage differentiation effects, as well as its ability to promote fat cell differentiation and reduce insulin resistance. Pioglitazone (Pio) is a PPARγ agonist used clinically as an anti-diabetic agent for improving insulin sensitivity in patients with diabetes. The objective of this study was to develop a drug delivery system (DDS) for the local release of Pio to promote wound healing. Pio of low aqueous solubility was water-solubilized by micelles formed from gelatin grafted with L-lactic acid oligomers, and incorporated into a biodegradable gelatin hydrogel. An 8-mm punch biopsy tool was used to prepare two skin wounds on either side of the midline of 8-week-old mice. Wounds were treated by the hydrogels with (Pio-hydrogel group) or without (control group) Pio, and the wound area were observed 1, 4, 7, and 14 days after treatment. In addition, a protein assay and immunohistological stain were performed to determine the effects of the Pio-hydrogel on inflammation and macrophage differentiation. The Pio-hydrogels promote wound healing. Moreover, Western blotting analysis demonstrated that treatment with Pio-hydrogels resulted in decreased levels of the cytokines MIP-2 and TGF-β, and increased levels of glucose-regulating adiponectin. It is concluded that Pio-incorporated hydrogels promote the proliferation and remodeling phases of wound healing, and may prove to be effective as wound dressings.

  1. A peroxisome proliferator-activated receptor gamma agonist influenced daily profile of energy expenditure in genetically obese diabetic rats.

    PubMed

    Yoshida, Yuki; Ichikawa, Mineko; Ohta, Minoru; Kanai, Setsuko; Kobayash, Mikako; Ichimaru, Yuhei; Shimazoe, Takao; Watanabe, Shigenori; Funakoshi, Akihiro; Miyasak, Kyoko

    2002-03-01

    Otsuka Long Evans Tokushima Fatty (OLETF) rats were developed as a model of non-insulin-dependent diabetes mellitus (NIDDM) with mild obesity. We reported that the daily profiles of energy expenditure associated with two peaks (one between 05:00 and 08:00 and the other between 20:00 and 22:00) were observed at 8 weeks of age (without NIDDM), while these two peaks disappeared at 24 weeks of age with NIDDM. As a new anti-diabetic drug, a peroxisome proliferator-activated receptor y agonist pioglitazone hydrochloride has been developed, we examined whether pioglitazone normalized daily profiles of energy expenditure at 24 weeks of age. A control diet and pioglitazone (0.1%)-containing diet were fed from 6 weeks of age. The two peaks of daily profiles of energy expenditure, which disappeared in OLETF rats with the control diet at 24 weeks of age, were reproduced by administration of pioglitazone. The respiratory quotient was lower and fat derived energy used for combustion was increased by pioglitazone at both ages. The body weight, daily food intake, plasma levels of fat, insulin, leptin and the wet weight of visceral fat were not influenced, but the levels of blood hemoglobin Alc and plasma tumor necrosis factor a were decreased by pioglitazone. Administration of pioglitazone improved daily profiles of energy expenditure via affecting glucose and fat metabolisms.

  2. Pharmacological experiments in healthy volunteers with bopindolol, a long-acting beta-adrenoceptor blocking drug with partial agonist activity.

    PubMed Central

    Aellig, W H

    1985-01-01

    Bopindolol is a potent and specific beta-adrenoceptor antagonist with partial agonist activity. In animal experiments it blocks both beta 1- and beta 2-adrenoceptors and possesses a long duration of action. In the present study in healthy volunteers bopindolol was about ten times more potent than pindolol in reducing isoprenaline-induced and exercise-induced tachycardia. In experiments on exercise-induced tachycardia an oral dose of 2 mg produced a near maximum reduction of exercise heart rate, occurring within 2 to 3 h of administration. With higher doses (up to 12 mg) the maximum effect was reached earlier (between 1 and 2 h). The long duration of action of bopindolol observed in animal studies was confirmed in man. Twenty-four hours after 4 and 10 mg bopindolol more than 2/3 of the maximum effect was still present. After 48 h 38% of the maximum effect of 4 mg and 50% of that of 12 mg remained. Even at 72 and 96 h exercise-induced tachycardia was still significantly lowered after both doses of the drug. When bopindolol was administered once daily for 5 days there was a slight increase in the maximum reduction of exercise-induced tachycardia during treatment with 1 mg/day but not with 4 mg/day, which produced a near maximum effect. PMID:2862891

  3. Discovery and Characterization of 2-Aminooxazolines as Highly Potent, Selective, and Orally Active TAAR1 Agonists

    PubMed Central

    2015-01-01

    2-Aminooxazolines were discovered as a novel structural class of TAAR1 ligands. Starting from a known adrenergic compound 1, structural modifications were made to obtain highly potent and selective TAAR1 ligands such as 12 (RO5166017), 18 (RO5256390), 36 (RO5203648), and 48 (RO5263397). These compounds exhibit drug-like physicochemical properties, have good oral bioavailability, and display in vivo activity in a variety of animal models relevant for psychiatric diseases and addiction. PMID:26985297

  4. The CYP17A1 inhibitor abiraterone exhibits estrogen receptor agonist activity in breast cancer.

    PubMed

    Capper, Cameron P; Larios, José M; Sikora, Matthew J; Johnson, Michael D; Rae, James M

    2016-05-01

    Cytochrome P450 17A1 (CYP17A1) is the requisite enzyme for synthesis of sex steroids, including estrogens and androgens. As such, inhibition of CYP17A1 is a target for inhibiting the growth of hormone-dependent cancers including prostate and breast cancer. Abiraterone, is a first in class potent and selective CYP17A1 inhibitor that has been approved for the treatment of castration-resistant prostate cancer. Given that, androgens are the precursors for estrogen production, it has been proposed that abiraterone could be an effective form of treatment for estrogen receptor (ER)-positive breast cancer, though its utility in this context has yet to be established. Abiraterone has a core steroid-like chemical structure, and so we hypothesized that it may bind to nuclear steroid receptors including ER and have estrogenic activity. We tested this hypothesis by investigating abiraterone's ability to directly modulate ER signaling in breast cancer cell line models. We show that abiraterone directly activates ER, induces ER-target gene expression, and elicits estrogen-response-element reporter activity in the ER-positive cell lines MCF-7 and T47D. Abiraterone also induced cell proliferation by ~2.5-fold over vehicle in both MCF-7 and T47D cells. Importantly, abiraterone-induced cell proliferation and ER-activity was blocked by the selective estrogen receptor downregulator (SERD) fulvestrant, confirming that abiraterone directly acts at the ER. These data suggest that abiraterone should be combined with other ER antagonists when used for the clinical management of ER-positive breast cancer.

  5. Biostable Agonists that Match or Exceed Activity of Native Insect Kinins on Recombinant Arthropod GPCRs

    DTIC Science & Technology

    2009-01-01

    diuresis in many species of insects. Compounds with similar biological activity could be exploited for the control of arthropod pest populations such as...species, insect kinins stimulate hindgut contractions, diuresis , digestive enzyme release and probably inhibit larval weight gain (Holman et al., 1990...conserved C-terminal pentapeptide motif Phe-X1-X2-Trp-Gly-NH2, where X1 = His, Asn, Ser, or Tyr and X2 = Ser, Pro, or Ala. Insect kinins regulate diuresis in

  6. Molecular level activation insights from a NR2A/NR2B agonist.

    PubMed

    Ieong Tou, Weng; Chang, Su-Sen; Wu, Dongchuan; Lai, Ted Weita; Wang, Yu Tian; Hsu, Chung Y; Chen, Calvin Yu-Chian

    2014-01-01

    N-methyl D-aspartate receptors (NMDARs), a subclass of glutamate receptors have broad actions in neural transmission for major brain functions. Overactivation of NMDARs leading to "excitotoxicity" is the underlying mechanism of neuronal death in a number of neurological diseases, especially stroke. Much research effort has been directed toward developing pharmacological agents to modulate NMDAR actions for treating neurological diseases, in particular stroke. Here, we report that Alliin, a sulfoxide in fresh garlic, exhibits affinity toward NR2A as well as NR2B receptors based on virtual screening. Biological activities of Alliin on these two receptors were confirmed in electrophysiological studies. Ligand-binding site closure, a structural change precluding ion channel opening, was observed with Alliin during 100 ns molecular dynamics simulation. Alliin interactions with NR2A and NR2B suggest that residues E/A413, H485, T690, and Y730 may play important roles in the conformation shift. Activation of NR2A and NR2B by Alliin can be differentiated from that caused by glutamate, the endogenous neurotransmitter. These characteristic molecular features in NR2A and NR2B activation provide insight into structural requirements for future development of novel drugs with selective interaction with NR2A and NR2B for treating neurological diseases, particularly stroke.

  7. Phospholipid-esterified eicosanoids are generated in agonist-activated human platelets and enhance tissue factor-dependent thrombin generation.

    PubMed

    Thomas, Christopher P; Morgan, Lloyd T; Maskrey, Benjamin H; Murphy, Robert C; Kühn, Hartmut; Hazen, Stanley L; Goodall, Alison H; Hamali, Hassan A; Collins, Peter W; O'Donnell, Valerie B

    2010-03-05

    Here, a group of specific lipids, comprising phosphatidylethanolamine (PE)- or phosphatidylcholine (PC)-esterified 12S-hydroxyeicosatetraenoic acid (12S-HETE), generated by 12-lipoxygenase was identified and characterized. 12S-HETE-PE/PCs were formed within 5 min of activation by thrombin, ionophore, or collagen. Esterified HETE levels generated in response to thrombin were 5.85 +/- 1.42 (PE) or 18.35 +/- 4.61 (PC), whereas free was 65.5 +/- 17.6 ng/4 x 10(7) cells (n = 5 separate donors, mean +/- S.E.). Their generation was stimulated by triggering protease-activated receptors-1 and -4 and signaling via Ca(2+) mobilization secretory phospholipase A2, platelet-activating factor-acetylhydrolase, src tyrosine kinases, and protein kinase C. Stable isotope labeling showed that they form predominantly by esterification that occurs on the same time scale as free acid generation. Unlike free 12S-HETE that is secreted, esterified HETEs remain cell-associated, with HETE-PEs migrating to the outside of the plasma membrane. 12-Lipoxygenase inhibition attenuated externalization of native PE and phosphatidylserine and HETE-PEs. Platelets from a patient with the bleeding disorder, Scott syndrome, did not externalize HETE-PEs, and liposomes supplemented with HETE-PC dose-dependently enhanced tissue factor-dependent thrombin generation in vitro. This suggests a role for these novel lipids in promoting coagulation. Thus, oxidized phospholipids form by receptor/agonist mechanisms, not merely as an undesirable consequence of vascular and inflammatory disease.

  8. The peroxisome proliferators activated receptor-gamma agonists as therapeutics for the treatment of Alzheimer's disease and mild-to-moderate Alzheimer's disease: a meta-analysis.

    PubMed

    Cheng, Huawei; Shang, Yuping; Jiang, Ling; Shi, Tian-lu; Wang, Lin

    2016-01-01

    Alzheimer's disease (AD) is a devastating neurodegenerative disease and there is no effective therapy for it. Peroxisome proliferators activated receptor-gamma (PPAR-γ) agonists is a promising therapeutic approach for AD and has been widely studied recently, but no consensus was available up to now. To clarify this point, a meta-analysis was performed. We searched MEDLINE, EMBASE, Cochrane Central database, PUBMED, Springer Link database, SDOS database, CBM, CNKI and Wan fang database by December 2014. Standardized mean difference (SMD), relative risk (RR) and 95% confidence interval (CI) were calculated to assess the strength of the novel therapeutics for AD and mild-to-moderate AD. A total of nine studies comprising 1314 patients and 1311 controls were included in the final meta-analysis. We found the effect of PPAR-γ agonists on Alzheimer's Disease Assessment Scale - Cognitive Subscale (ADAS-cog) scores by using STATA software. There was no evidence for obvious publication bias in the overall meta-analysis. There is insufficient evidence of statistically incognition of AD and mild-to-moderate AD patients have been improved who were treated with PPAR-γ agonists in our research. However, PPAR-γ agonists may be a promising therapeutic approach in future, especially pioglitazone, with large-scale randomized controlled trials to confirm.

  9. Agonistic induction of a covalent dimer in a mutant of natriuretic peptide receptor-A documents a juxtamembrane interaction that accompanies receptor activation.

    PubMed

    Labrecque, J; Deschênes, J; McNicoll, N; De Léan, A

    2001-03-16

    The natriuretic peptide receptor-A (NPR-A) is composed of an extracellular domain with a ligand binding site, a transmembrane-spanning domain, a kinase homology domain, and a guanylyl cyclase domain. In response to agonists (atrial natriuretic peptide (ANP) and brain natriuretic peptide), the kinase homology domain-mediated guanylate cyclase repression is removed, which allows the production of cyclic GMP. Previous work from our laboratory strongly indicated that agonists are exerting their effects through the induction of a juxtamembrane dimeric contact. However, a direct demonstration of this mechanism remains to be provided. As a tool, we are now using the properties of a new mutation, D435C. It introduces a cysteine at a position in NPR-A corresponding to a supplementary cysteine found in NPR-C6, another receptor of this family (a disulfide-linked dimer). Although this D435C mutation only leads to trace levels of NPR-A disulfide-linked dimer at basal state, covalent dimerization can be induced by a treatment with rat ANP or with other agonists. The NPR-A(D435C) mutant has not been subjected to significant structural alterations, since it shares with the wild type receptor a similar dose-response pattern of cellular guanylyl cyclase activation. However, a persistent activation accompanies NPR-A(D435C) dimer formation after the removal of the inducer agonist. On the other hand, a construction where the intracellular domain of NPR-A(D435C) has been truncated (DeltaKC(D435C)) displays a spontaneous and complete covalent dimerization. In addition, the elimination of the intracellular domain in wild type DeltaKC and DeltaKC(D435C) is associated with an increase of agonist binding affinity, this effect being more pronounced with the weak agonist pBNP. Also, a D435C secreted extracellular domain remains unlinked even after incubation with rat ANP. In summary, these results demonstrate, in a dynamic fashion, the agonistic induction of a dimeric contact in the

  10. 76 FR 80447 - Eighth Meeting: RTCA Special Committee 219: Attitude and Heading Reference Systems (AHRS)

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-12-23

    ... Special Committee 219: Attitude and Heading Reference Systems (AHRS) AGENCY: Federal Aviation...: Attitude and Heading Reference Systems (AHRS). SUMMARY: The FAA is issuing this notice to advise the public of the eighth meeting of RTCA Special Committee 219: Attitude and Heading Reference Systems...

  11. Identification of opioid ligands possessing mixed micro agonist/delta antagonist activity among pyridomorphinans derived from naloxone, oxymorphone, and hydromorphone [correction of hydropmorphone].

    PubMed

    Ananthan, Subramaniam; Khare, Naveen K; Saini, Surendra K; Seitz, Lainne E; Bartlett, Jeffrey L; Davis, Peg; Dersch, Christina M; Porreca, Frank; Rothman, Richard B; Bilsky, Edward J

    2004-03-11

    A series of pyridomorphinans derived from naloxone, oxymorphone, and hydromorphone (7a-k) were synthesized and evaluated for binding affinity at the opioid delta, micro, and kappa receptors in brain membranes using radioligand binding assays and for functional activity in vitro using [(35)S]GTP-gamma-S binding assays in brain tissues and bioassays using guinea pig ileum (GPI) and mouse vas deferens (MVD) smooth muscle preparations. The pyridine ring unsubstituted pyridomorphinans possessing the oxymorphone and hydromorphone framework displayed nearly equal binding affinity at the micro and delta receptors. Their affinities at the kappa site were nearly 10-fold less than their binding affinities at the micro and delta sites. Introduction of aryl substituents at the 5'-position on the pyridine ring improved the binding affinity at the delta site while decreasing the binding affinity at the micro site. Nearly all of the ligands possessing an N-methyl group at the17-position with or without a hydroxyl group at the 14-position of the morphinan moiety displayed agonist activity at the micro receptor with varying potencies and efficacies. In the [(35)S]GTP-gamma-S binding assays, most of these pyridomorphinans were devoid of any significant agonist activity at the delta and kappa receptors but displayed moderate to potent antagonist activity at the delta receptors. In antinociceptive evaluations using the warm-water tail-withdrawal assay in mice, the pyridomorphinans produced analgesic effects with varying potencies and efficacies when administered by the intracerebroventricular route. Among the ligands studied, the hydromorphone-derived 4-chlorophenylpyridomorphinan 7h was identified as a ligand possessing a promising profile of mixed micro agonist/delta antagonist activity in vitro and in vivo. In a repeated administration paradigm in which the standard micro agonist morphine produces significant tolerance, repeated administration of the micro agonist/delta antagonist

  12. Conformational Restriction Leading to a Selective CB2 Cannabinoid Receptor Agonist Orally Active Against Colitis

    PubMed Central

    2014-01-01

    The CB2 cannabinoid receptor has been implicated in the regulation of intestinal inflammation. Following on from the promising activity of a series of 4-oxo-1,4-dihydroquinoline-3-carboxamide, we developed constrained analogues based on a 2H-pyrazolo[4,3-c]quinolin-3(5H)-one scaffold, with improved affinity for the hCB2 receptor and had very high selectivity over the hCB1 receptor. Importantly, the lead of this series (26, hCB2: Ki = 0.39 nM, hCB1: Ki > 3000 nM) was found to protect mice against experimental colitis after oral administration. PMID:25699149

  13. Effects of artificial sweeteners on the AhR- and GR-dependent CYP1A1 expression in primary human hepatocytes and human cancer cells.

    PubMed

    Kamenickova, Alzbeta; Pecova, Michaela; Bachleda, Petr; Dvorak, Zdenek

    2013-12-01

    Food constituents may cause a phenomenon of food-drug interactions. In the current study, we examined the effects of artificial sweeteners (aspartame, acesulfame, cyclamate, saccharin) on the aryl hydrocarbon receptor (AhR) and glucocorticoid receptor (GR)-dependent expression of CYP1A1 in human hepatocytes, hepatic HepG2 and intestinal LS174T cancer cell lines. Sweeteners were tested in concentrations up to those occurring in non-alcoholic beverages. Basal and ligand-inducible AhR- and GR-dependent reporter gene activation in stably transfected HepG2 and HeLa cells, respectively, were not affected by either of the sweeteners tested after 24h of incubation. The expression of CYP1A1 mRNA and protein in primary cultures of human hepatocytes and in LS174T and HepG2 cells was not induced by any of the tested sweeteners. Overall, aspartame, acesulfame, saccharin and cyclamate had no effects on CYP1A1 expression and transcriptional activities of AhR and GR. These data imply the safety of artificial sweeteners in terms of interference with AhR, GR and CYP1A1.

  14. Effect of stretching on agonist-antagonist muscle activity and muscle force output during single and multiple joint isometric contractions.

    PubMed

    McBride, J M; Deane, R; Nimphius, S

    2007-02-01

    Eight moderately active male subjects where tested for peak force in an isometric knee extension test and peak force and rate of force development in an isometric squat test. Both tests where performed at a 100 degrees knee angle and average integrated electromyography (IEMG) was measured from the vastus medialis (VM), vastus lateralis (VL) and biceps femoris (BF) muscles. Subjects performed the two conditions, stretching (S) or control (C) in a randomized order. Subjects where tested for baseline strength measures in both the isometric knee extension and isometric squat and then either stretched or sat quietly for 10 min. Following S or C subjects where then tested at six time points. Following S peak force in the isometric knee extension was significantly (P < or = 0.05) less than C at 1, 2, 8 and 16 min post. No significant difference in peak force was found between S and C in the isometric squat. However, following S the rate of force development in the isometric squat was significantly less than C at immediately post. No significant differences where observed in IEMG of the VM or VL between S and C in either the isometric knee extension or isometric squat. However, IEMG significantly decreased in the BF at 1 min post after S in comparison with C in both the isometric knee extension and isometric squat. Stretching appears to decrease muscle force output in a single joint isometric contraction and rate of force development in a multiple joint isometric contraction. Possible changes in agonist-antagonist muscle activity patterns need to be further examined.

  15. Baclofen, an agonist at peripheral GABAB receptors, induces antinociception via activation of TEA-sensitive potassium channels

    PubMed Central

    Reis, G M L; Duarte, I D G

    2006-01-01

    Background and Purpose: Central anti-nociceptive actions of baclofen involve activation of K+ channels. Here we assessed what types of K+ channel might participate in the peripheral anti-nociception induced by baclofen. Experimental approach: Nociceptive thresholds to mechanical stimulation in rat paws treated with intraplantar prostaglandin E2.(PGE2) to induce hyperalgesia were measured 3h after PGE2 injection. Other agents were also given by intraplantar injection Key results: Baclofen elicited a dose-dependent (15 - 240 μg per paw) anti-nociceptive effect. An intermediate dose of baclofen (60 μg) did not produce antinociception in the contralateral paw, showing its peripheral site of action. The GABAB receptor antagonist saclofen (12.5 - 100 μg per paw) antagonized, in a dose-dependent manner, peripheral antinociception induced by baclofen (60 μg), suggesting a specific effect. This antinociceptive action of baclofen was unaffected by bicuculline, GABAA receptor antagonist (80 μg per paw), or by (1,2,5,6 tetrahydropyridin-4-yl) methylphosphinic acid, GABAC receptor antagonist (20 μg per paw). The peripheral antinociception induced by baclofen (60 μg) was reversed, in a dose-dependent manner, by the voltage-dependent K+ channel blockers tetraethylammonium (7.5 - 30 μg per paw) and 4-aminopyridine (2.5 - 10 μg per paw). The blockers of other K+ channels, glibenclamide (160 μg), tolbutamide (320 μg), charybdotoxin (2 μg), dequalinium (50 μg) and caesium (500 μg) had no effect. Conclusions and Implications: This study provides evidence that the peripheral antinociceptive effect of the GABAB receptor agonist baclofen results from the activation of tetraethylammonium-sensitive K+ channels. Other K+ channels appear not to be involved. PMID:17016510

  16. On the Origin of Muscle Synergies: Invariant Balance in the Co-activation of Agonist and Antagonist Muscle Pairs.

    PubMed

    Hirai, Hiroaki; Miyazaki, Fumio; Naritomi, Hiroaki; Koba, Keitaro; Oku, Takanori; Uno, Kanna; Uemura, Mitsunori; Nishi, Tomoki; Kageyama, Masayuki; Krebs, Hermano Igo

    2015-01-01

    Investigation of neural representation of movement planning has attracted the attention of neuroscientists, as it may reveal the sensorimotor transformation essential to motor control. The analysis of muscle synergies based on the activity of agonist-antagonist (AA) muscle pairs may provide insight into such transformations, especially for a reference frame in the muscle space. In this study, we examined the AA concept using the following explanatory variables: the AA ratio, which is related to the equilibrium-joint angle, and the AA sum, which is associated with joint stiffness. We formulated muscle synergies as a function of AA sums, positing that muscle synergies are composite units of mechanical impedance. The AA concept can be regarded as another form of the equilibrium-point (EP) hypothesis, and it can be extended to the concept of EP-based synergies. We introduce, here, a novel tool for analyzing the neurological and motor functions underlying human movements and review some initial insights from our results about the relationships between muscle synergies, endpoint stiffness, and virtual trajectories (time series of EP). Our results suggest that (1) muscle synergies reflect an invariant balance in the co-activation of AA muscle pairs; (2) each synergy represents the basis for the radial, tangential, and null movements of the virtual trajectory in the polar coordinates centered on the specific joint at the base of the body; and (3) the alteration of muscle synergies (for example, due to spasticity or rigidity following neurological injury) results in significant distortion of endpoint stiffness and concomitant virtual trajectories. These results indicate that muscle synergies (i.e., the balance of muscle mechanical impedance) are essential for motor control.

  17. Distinct properties of telmisartan on agonistic activities for peroxisome proliferator-activated receptor γ among clinically used angiotensin II receptor blockers: drug-target interaction analyses.

    PubMed

    Kakuta, Hirotoshi; Kurosaki, Eiji; Niimi, Tatsuya; Gato, Katsuhiko; Kawasaki, Yuko; Suwa, Akira; Honbou, Kazuya; Yamaguchi, Tomohiko; Okumura, Hiroyuki; Sanagi, Masanao; Tomura, Yuichi; Orita, Masaya; Yonemoto, Takako; Masuzaki, Hiroaki

    2014-04-01

    A proportion of angiotensin II type 1 receptor blockers (ARBs) improves glucose dyshomeostasis and insulin resistance in a clinical setting. Of these ARBs, telmisartan has the unique property of being a partial agonist for peroxisome proliferator-activated receptor γ (PPARγ). However, the detailed mechanism of how telmisartan acts on PPARγ and exerts its insulin-sensitizing effect is poorly understood. In this context, we investigated the agonistic activity of a variety of clinically available ARBs on PPARγ using isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR) system. Based on physicochemical data, we then reevaluated the metabolically beneficial effects of telmisartan in cultured murine adipocytes. ITC and SPR assays demonstrated that telmisartan exhibited the highest affinity of the ARBs tested. Distribution coefficient and parallel artificial membrane permeability assays were used to assess lipophilicity and cell permeability, for which telmisartan exhibited the highest levels of both. We next examined the effect of each ARB on insulin-mediated glucose metabolism in 3T3-L1 preadipocytes. To investigate the impact on adipogenesis, 3T3-L1 preadipocytes were differentiated with each ARB in addition to standard inducers of differentiation for adipogenesis. Telmisartan dose-dependently facilitated adipogenesis and markedly augmented the mRNA expression of adipocyte fatty acid-binding protein (aP2), accompanied by an increase in the uptake of 2-deoxyglucose and protein expression of glucose transporter 4 (GLUT4). In contrast, other ARBs showed only marginal effects in these experiments. In accordance with its highest affinity of binding for PPARγ as well as the highest cell permeability, telmisartan superbly activates PPARγ among the ARBs tested, thereby providing a fresh avenue for treating hypertensive patients with metabolic derangement.

  18. Structural Basis for Iloprost as a Dual Peroxisome Proliferator-activated Receptor [alpha/delta] Agonist

    SciTech Connect

    Jin, Lihua; Lin, Shengchen; Rong, Hui; Zheng, Songyang; Jin, Shikan; Wang, Rui; Li, Yong

    2012-03-15

    Iloprost is a prostacyclin analog that has been used to treat many vascular conditions. Peroxisome proliferator-activated receptors (PPARs) are ligand-regulated transcription factors with various important biological effects such as metabolic and cardiovascular physiology. Here, we report the crystal structures of the PPAR{alpha} ligand-binding domain and PPAR{delta} ligand-binding domain bound to iloprost, thus providing unambiguous evidence for the direct interaction between iloprost and PPARs and a structural basis for the recognition of PPAR{alpha}/{delta} by this prostacyclin analog. In addition to conserved contacts for all PPAR{alpha} ligands, iloprost also initiates several specific interactions with PPARs using its unique structural groups. Structural and functional studies of receptor-ligand interactions reveal strong functional correlations of the iloprost-PPAR{alpha}/{delta} interactions as well as the molecular basis of PPAR subtype selectivity toward iloprost ligand. As such, the structural mechanism may provide a more rational template for designing novel compounds targeting PPARs with more favorable pharmacologic impact based on existing iloprost drugs.

  19. Structural basis for iloprost as a dual peroxisome proliferator-activated receptor alpha/delta agonist.

    PubMed

    Jin, Lihua; Lin, Shengchen; Rong, Hui; Zheng, Songyang; Jin, Shikan; Wang, Rui; Li, Yong

    2011-09-09

    Iloprost is a prostacyclin analog that has been used to treat many vascular conditions. Peroxisome proliferator-activated receptors (PPARs) are ligand-regulated transcription factors with various important biological effects such as metabolic and cardiovascular physiology. Here, we report the crystal structures of the PPARα ligand-binding domain and PPARδ ligand-binding domain bound to iloprost, thus providing unambiguous evidence for the direct interaction between iloprost and PPARs and a structural basis for the recognition of PPARα/δ by this prostacyclin analog. In addition to conserved contacts for all PPARα ligands, iloprost also initiates several specific interactions with PPARs using its unique structural groups. Structural and functional studies of receptor-ligand interactions reveal strong functional correlations of the iloprost-PPARα/δ interactions as well as the molecular basis of PPAR subtype selectivity toward iloprost ligand. As such, the structural mechanism may provide a more rational template for designing novel compounds targeting PPARs with more favorable pharmacologic impact based on existing iloprost drugs.

  20. Effect of peroxisome proliferator activated receptor (PPAR)gamma agonists on prostaglandins cascade in joint cells.

    PubMed

    Moulin, David; Poleni, Paul-Emile; Kirchmeyer, Mélanie; Sebillaud, Sylvie; Koufany, Meriem; Netter, Patrick; Terlain, Bernard; Bianchi, Arnaud; Jouzeau, Jean-Yves

    2006-01-01

    In response to inflammatory cytokines, chondrocytes and synovial fibroblasts produce high amounts of prostaglandins (PG) which self-perpetuate locally the inflammatory reaction. Prostaglandins act primarily through membrane receptors coupled to G proteins but also bind to nuclear Peroxisome Proliferator-Activated Receptors (PPARs). Amongst fatty acids, the cyclopentenone metabolite of PGD2, 15-deoxy-Delta12,14PGJ2 (15d-PGJ2), was shown to be a potent ligand of the PPARgamma isotype prone to inhibit the production of inflammatory mediators. As the stimulated synthesis of PGE2 originates from the preferential coupling of inducible enzymes, cyclooxygenase-2 (COX-2) and membrane PGE synthase-1 (mPGES-1), we investigated the potency of 15d-PGJ2 to regulate prostaglandins synthesis in rat chondrocytes stimulated with interleukin-1beta (IL-1beta). We demonstrated that 15d-PGJ2, but not the high-affinity PPARgamma ligand rosiglitazone, decreased almost completely PGE2 synthesis and mPGES-1 expression. The inhibitory potency of 15d-PGJ2 was unaffected by changes in PPARgamma expression and resulted from inhibition of NF-kappaB nuclear binding and IkappaBalpha sparing, secondary to reduced phosphorylation of IKKbeta. Consistently with 15d-PGJ2 being a putative endogenous regulator of the inflammatory reaction if synthesized in sufficient amounts, the present data confirm the variable PPARgamma-dependency of its effects in joint cells while underlining possible species and cell types specificities.

  1. Activation of Relaxin Family Receptor 1 from Different Mammalian Species by Relaxin Peptide and Small-Molecule Agonist ML290

    PubMed Central

    Huang, Zaohua; Myhr, Courtney; Bathgate, Ross A. D.; Ho, Brian A.; Bueno, Amaya; Hu, Xin; Xiao, Jingbo; Southall, Noel; Barnaeva, Elena; Agoulnik, Irina U.; Marugan, Juan J.; Ferrer, Marc; Agoulnik, Alexander I.

    2015-01-01

    Relaxin peptide (RLN), which signals through the relaxin family peptide 1 (RXFP1) GPCR receptor, has shown therapeutic effects in an acute heart failure clinical trial. We have identified a small-molecule agonist of human RXFP1, ML290; however, it does not activate the mouse receptor. To find a suitable animal model for ML290 testing and to gain mechanistic insights into the interaction of various ligands with RXFP1, we have cloned rhesus macaque, pig, rabbit, and guinea pig RXFP1s and analyzed their activation by RLN and ML290. HEK293T cells expressing macaque or pig RXFP1 responded to relaxin and ML290 treatment as measured by an increase of cAMP production. Guinea pig RXFP1 responded to relaxin but had very low response to ML290 treatment only at highest concentrations used. The rabbit RXFP1 amino acid sequence was the most divergent, with a number of unique substitutions within the ectodomain and the seven-transmembrane domain (7TM). Two splice variants of rabbit RXFP1 derived through alternative splicing of the fourth exon were identified. In contrast to the other species, rabbit RXFP1s were activated by ML290, but not with human, pig, mouse, or rabbit RLNs. Using FLAG-tagged constructs, we have shown that both rabbit RXFP1 variants are expressed on the cell surface. No binding of human Eu-labeled RLN to rabbit RXFP1 was detected, suggesting that in this species, RXFP1 might be non-functional. We used chimeric rabbit–human and guinea pig–human constructs to identify regions important for RLN or ML290 receptor activation. Chimeras with the human ectodomain and rabbit 7TM domain were activated by RLN, whereas substitution of part of the guinea pig 7TM domain with the human sequence only partially restored ML290 activation, confirming the allosteric mode of action for the two ligands. Our data demonstrate that macaque and pig models can be used for ML290 testing. PMID:26347712

  2. Protein Kinase D and Gβγ Subunits Mediate Agonist-evoked Translocation of Protease-activated Receptor-2 from the Golgi Apparatus to the Plasma Membrane.

    PubMed

    Jensen, Dane D; Zhao, Peishen; Jimenez-Vargas, Nestor N; Lieu, TinaMarie; Gerges, Marina; Yeatman, Holly R; Canals, Meritxell; Vanner, Stephen J; Poole, Daniel P; Bunnett, Nigel W

    2016-05-20

    Agonist-evoked endocytosis of G protein-coupled receptors has been extensively studied. The mechanisms by which agonists stimulate mobilization and plasma membrane translocation of G protein-coupled receptors from intracellular stores are unexplored. Protease-activated receptor-2 (PAR2) traffics to lysosomes, and sustained protease signaling requires mobilization and plasma membrane trafficking of PAR2 from Golgi stores. We evaluated the contribution of protein kinase D (PKD) and Gβγ to this process. In HEK293 and KNRK cells, the PAR2 agonists trypsin and 2-furoyl-LIGRLO-NH2 activated PKD in the Golgi apparatus, where PKD regulates protein trafficking. PAR2 activation induced translocation of Gβγ, a PKD activator, to the Golgi apparatus, determined by bioluminescence resonance energy transfer between Gγ-Venus and giantin-Rluc8. Inhibitors of PKD (CRT0066101) and Gβγ (gallein) prevented PAR2-stimulated activation of PKD. CRT0066101, PKD1 siRNA, and gallein all inhibited recovery of PAR2-evoked Ca(2+) signaling. PAR2 with a photoconvertible Kaede tag was expressed in KNRK cells to examine receptor translocation from the Golgi apparatus to the plasma membrane. Irradiation of the Golgi region (405 nm) induced green-red photo-conversion of PAR2-Kaede. Trypsin depleted PAR2-Kaede from the Golgi apparatus and repleted PAR2-Kaede at the plasma membrane. CRT0066101 inhibited PAR2-Kaede translocation to the plasma membrane. CRT0066101 also inhibited sustained protease signaling to colonocytes and nociceptive neurons that naturally express PAR2 and mediate protease-evoked inflammation and nociception. Our results reveal a major role for PKD and Gβγ in agonist-evoked mobilization of intracellular PAR2 stores that is required for sustained signaling by extracellular proteases.

  3. Binding and activity of the prostacyclin receptor (IP) agonists, treprostinil and iloprost, at human prostanoid receptors: treprostinil is a potent DP1 and EP2 agonist.

    PubMed

    Whittle, Brendan J; Silverstein, Adam M; Mottola, David M; Clapp, Lucie H

    2012-07-01

    The prostacyclin analogues, iloprost and treprostinil are extensively used in treating pulmonary hypertension. Their binding profile and corresponding biochemical cellular responses on human prostanoid receptors expressed in cell lines, have now been compared. Iloprost had high binding affinity for EP1 and IP receptors (Ki 1.1 and 3.9 nM, respectively), low affinity for FP, EP3 or EP4 receptors, and very low affinity for EP2, DP1 or TP receptors. By contrast, treprostinil had high affinity for the DP1, EP2 and IP receptors (Ki 4.4, 3.6 and 32 nM, respectively), low affinity for EP1 and EP4 receptors and even lower affinity for EP3, FP and TP receptors. In functional assays, iloprost had similar high activity in elevating cyclic AMP levels in cells expressing the human IP receptor and stimulating calcium influx in cells expressing EP1 receptors (EC50 0.37 and 0.3 nM, respectively) with the rank order of activity on the other receptors comparable to the binding assays. As with binding studies, treprostinil elevated cyclic AMP with a similar high potency in cells expressing DP1, IP and EP2 receptors (EC50 0.6, 1.9 and 6.2 nM, respectively), but had low activity at the other receptors. Activation of IP, DP1 and EP2 receptors, as with treprostinil, can all result in vasodilatation of human pulmonary arteries. However, activation of EP1 receptors can provoke vasoconstriction, and hence may offset the IP-receptor mediated vasodilator effects of iloprost. Treprostinil may therefore differ from iloprost in its overall beneficial pulmonary vasorelaxant profile and other pharmacological actions, especially in diseases where the IP receptor is down-regulated.

  4. A Robotic MCF-7:WS8 Cell Proliferation Assay to Detect Agonist and Antagonist Estrogenic Activity

    PubMed Central

    Casey, Warren

    2014-01-01

    Endocrine-disrupting chemicals with estrogenic activity (EA) or anti-EA (AEA) have been extensively reported to possibly have many adverse health effects. We have developed robotized assays using MCF-7:WS8 cell proliferation (or suppression) to detect EA (or AEA) of 78 test substances supplied by the Interagency Coordinating Committee on the Validation of Alternative Methods and the National Toxicology Program’s Interagency Center for the Evaluation of Alternative Toxicological Methods for validation studies. We also assayed ICI 182,780, a strong estrogen antagonist. Chemicals to be assayed were initially examined for solubility and volatility to determine optimal assay conditions. For both EA and AEA determinations, a Range-Finder assay was conducted to determine the concentration range for testing, followed by a Comprehensive assay. Test substances with potentially positive results from an EA Comprehensive assay were subjected to an EA Confirmation assay that evaluated the ability of ICI 182,780 to reverse chemically induced MCF-7 cell proliferation. The AEA assays examined the ability of chemicals to decrease MCF-7 cell proliferation induced by nonsaturating concentrations of 17β-estradiol (E2), relative to ICI or raloxifene, also a strong estrogen antagonist. To be classified as having AEA, a saturating concentration of E2 had to significantly reverse the decrease in cell proliferation produced by the test substance in nonsaturating E2. We conclude that our robotized MCF-7 EA and AEA assays have accuracy, sensitivity, and specificity values at least equivalent to validated test methods accepted by the U.S. Environmental Protection Agency and the Organisation for Economic Co-operation and Development. PMID:24213142

  5. A robotic MCF-7:WS8 cell proliferation assay to detect agonist and antagonist estrogenic activity.

    PubMed

    Yang, Chun Z; Casey, Warren; Stoner, Matthew A; Kollessery, Gayathri J; Wong, Amy W; Bittner, George D

    2014-02-01

    Endocrine-disrupting chemicals with estrogenic activity (EA) or anti-EA (AEA) have been extensively reported to possibly have many adverse health effects. We have developed robotized assays using MCF-7:WS8 cell proliferation (or suppression) to detect EA (or AEA) of 78 test substances supplied by the Interagency Coordinating Committee on the Validation of Alternative Methods and the National Toxicology Program's Interagency Center for the Evaluation of Alternative Toxicological Methods for validation studies. We also assayed ICI 182,780, a strong estrogen antagonist. Chemicals to be assayed were initially examined for solubility and volatility to determine optimal assay conditions. For both EA and AEA determinations, a Range-Finder assay was conducted to determine the concentration range for testing, followed by a Comprehensive assay. Test substances with potentially positive results from an EA Comprehensive assay were subjected to an EA Confirmation assay that evaluated the ability of ICI 182,780 to reverse chemically induced MCF-7 cell proliferation. The AEA assays examined the ability of chemicals to decrease MCF-7 cell proliferation induced by nonsaturating concentrations of 17β-estradiol (E2), relative to ICI or raloxifene, also a strong estrogen antagonist. To be classified as having AEA, a saturating concentration of E2 had to significantly reverse the decrease in cell proliferation produced by the test substance in nonsaturating E2. We conclude that our robotized MCF-7 EA and AEA assays have accuracy, sensitivity, and specificity values at least equivalent to validated test methods accepted by the U.S. Environmental Protection Agency and the Organisation for Economic Co-operation and Development.

  6. Effect of constant administration of a gonadotropin-releasing hormone agonist on reproductive activity in mares: preliminary evidence on suppression of ovulation during the breeding season.

    PubMed

    Fitzgerald, B P; Peterson, K D; Silvia, P J

    1993-10-01

    During the breeding season, the effect of constant administration of an agonist analog of gonadotropin-releasing hormone (GnRH; goserelin acetate) on reproductive activity of mares was determined. Twenty-four mares undergoing estrous cycles were allocated at random to 6 groups (n = 4/group) and, on May 29 (day 0), received no treatment (group 1, controls), 120 micrograms (group 2), 360 micrograms (group 3), 600 micrograms (group 4), or 1,200 micrograms (group 5) of GnRH agonist/d for 28 days via a depot implanted subcutaneously. The final group of mares (group 6) was treated with 120 micrograms of GnRH agonist/d for 84 days (3 occasions at 28-day intervals). During a pretreatment period (April 19 to May 29) and for 90 days after initiation of GnRH agonist treatment, follicular development and ovulation were monitored by transrectal ultrasonography of the reproductive tract at 2- to 3-day intervals. On each occasion a blood sample was collected for determination of luteinizing hormone (LH) and progesterone. Estrous behavior was monitored by teasing of mares with a stallion. Initiation of agonist treatment was random, relative to the stage of the estrous cycle, and all mares ovulated within 11 days before or after implantation. In 3 of 4 nontreated control mares, estrous cycles were observed throughout the study, with interovulatory intervals ranging from 18 to 26 days. In the remaining mare, concentration of progesterone was high after asynchronous double ovulation during the pretreatment period, suggestive of persistent corpus luteum.(ABSTRACT TRUNCATED AT 250 WORDS)

  7. The active analog approach applied to the pharmacophore identification of benzodiazepine receptor ligands

    NASA Astrophysics Data System (ADS)

    Tebib, Souhail; Bourguignon, Jean-Jacques; Wermuth, Camille-Georges

    1987-07-01

    Applied to seven potent benzodiazepine-receptor ligands belonging to chemically different classes, the active analog approach allowed the stepwise identification of the pharmacophoric pattern associated with the recognition by the benzodiazepine receptor. A unique pharmacophore model was derived which involves six critical zones: (a) a π-electron rich aromatic (PAR) zone; (b) two electron-rich zones δ1 and δ2 placed at 5.0 and 4.5 Å respectively from the reference centroid in the PAR zone; (c) a freely rotating aromatic ring (FRA) region; (d) an out-of-plane region (OPR), strongly associated with agonist properties; and (e) an additional hydrophobic region (AHR). The model accommodates all presently known ligands of the benzodiazepine receptor, identifies sensitivity to steric hindrance close to the δ1 zone, accounts for R and S differential affinities and distinguishes requirements for agonist versus non-agonist activity profiles.

  8. Indoleamine 2,3-dioxygenase 1 (IDO1) inhibitors activate the aryl hydrocarbon receptor.

    PubMed

    Moyer, Benjamin J; Rojas, Itzel Y; Murray, Iain A; Lee, Seokwon; Hazlett, Haley F; Perdew, Gary H; Tomlinson, Craig R

    2017-03-20

    Indoleamine 2,3-dioxygenase 1 (IDO1) plays a key role in the immune system by regulating tryptophan levels and T cell differentiation. Several tumor types overexpress IDO1 to avoid immune surveillance making IDO1 of interest as a target for therapeutic intervention. As a result, several IDO1 inhibitors are currently being tested in clinical trials for cancer treatment as well as several other diseases. Many of the IDO1 inhibitors in clinical trials naturally bear structural similarities to the IDO1 substrate tryptophan, as such, they fulfill many of the structural and functional criteria as potential AHR ligands. Using mouse and human cell-based luciferase gene reporter assays, qPCR confirmation experiments, and CYP1A1 enzyme activity assays, we report that some of the promising clinical IDO1 inhibitors also act as agonists for the aryl hydrocarbon receptor (AHR), best known for its roles in xenobiotic metabolism and as another key regulator of the immune response. The dual role as IDO antagonist and AHR agonist for many of these IDO target drugs should be considered for full interrogation of their biological mechanisms and clinical outcomes.

  9. In vitro study on the agonistic and antagonistic activities of bisphenol-S and other bisphenol-A congeners and derivatives via nuclear receptors

    SciTech Connect

    Molina-Molina, José-Manuel; Amaya, Esperanza; Grimaldi, Marina; Sáenz, José-María; Real, Macarena; Fernández, Mariana F.; Balaguer, Patrick; Olea, Nicolás

    2013-10-01

    Bisphenols are a group of chemicals structurally similar to bisphenol-A (BPA) in current use as the primary raw material in the production of polycarbonate and epoxy resins. Some bisphenols are intended to replace BPA in several industrial applications. This is the case of bisphenol-S (BPS), which has an excellent stability at high temperature and resistance to sunlight. Studies on the endocrine properties of BPS have focused on its interaction with human estrogen receptor alpha (hERα), but information on its interaction with other nuclear receptors is scarce. The aim of this study was to investigate interactions of BPS, BPF, BPA and its halogenated derivatives, tetrachlorobisphenol A (TCBPA), and tetrabromobisphenol A (TBBPA), with human estrogen receptors (hERα and hERβ), androgen receptor (hAR), and pregnane X receptor (hPXR), using a panel of in vitro bioassays based on competitive binding to nuclear receptors (NRs), reporter gene expression, and cell proliferation assessment. BPS, BPF, and BPA efficiently activated both ERs, while TCBPA behaved as weak hERα agonist. Unlike BPF and BPA, BPS was more active in the hERβ versus hERα assay. BPF and BPA were full hAR antagonists (BPA > BPF), whereas BPA and BPS were weak hAR agonists. Only BPA, TCBPA, and TBBPA, were hPXR agonists (TCBPA > TBBPA > BPA). These findings provide evidence that BPA congeners and derivatives disrupt multiple NRs and may therefore interfere with the endocrine system. Hence, further research is needed to evaluate the potential endocrine-disrupting activity of putative BPA substitutes. - Highlights: • We investigated the agonist/antagonist activities of BPS, BPF, BPA, TCBPA and TBBPA. • The direct interaction of these compounds with hERα, hERβ, hAR and hPXR was studied. • BPA congeners and derivatives were found to disrupt multiple NRs. • Further evaluation of their role as endocrine-disrupting chemicals is needed.

  10. Deciphering Dimerization Modes of PAS Domains: Computational and Experimental Analyses of the AhR:ARNT Complex Reveal New Insights Into the Mechanisms of AhR Transformation

    PubMed Central

    Corrada, Dario; Soshilov, Anatoly A.; Denison, Michael S.

    2016-01-01

    The Aryl hydrocarbon Receptor (AhR) is a transcription factor that mediates the biochemical response to xenobiotics and the toxic effects of a number of environmental contaminants, including dioxins. Recently, endogenous regulatory roles for the AhR in normal physiology and development have also been reported, thus extending the interest in understanding its molecular mechanisms of activation. Since dimerization with the AhR Nuclear Translocator (ARNT) protein, occurring through the Helix-Loop-Helix (HLH) and PER-ARNT-SIM (PAS) domains, is needed to convert the AhR into its transcriptionally active form, deciphering the AhR:ARNT dimerization mode would provide insights into the mechanisms of AhR transformation. Here we present homology models of the murine AhR:ARNT PAS domain dimer developed using recently available X-ray structures of other bHLH-PAS protein dimers. Due to the different reciprocal orientation and interaction surfaces in the different template dimers, two alternative models were developed for both the PAS-A and PAS-B dimers and they were characterized by combining a number of computational evaluations. Both well-established hot spot prediction methods and new approaches to analyze individual residue and residue-pairwise contributions to the MM-GBSA binding free energies were adopted to predict residues critical for dimer stabilization. On this basis, a mutagenesis strategy for both the murine AhR and ARNT proteins was designed and ligand-dependent DNA binding ability of the AhR:ARNT heterodimer mutants was evaluated. While functional analysis disfavored the HIF2α:ARNT heterodimer-based PAS-B model, most mutants derived from the CLOCK:BMAL1-based AhR:ARNT dimer models of both the PAS-A and the PAS-B dramatically decreased the level