RESUMEN
Development of means to screen primary human cells rather than established cell lines is important in improving the predictive value of cellular assays in drug discovery. We describe a method of using automated fluorescent microscopy to detect activators of the wingless type/Frizzled (Wnt/Fzd) pathway in primary human preosteoblasts. This technique relies on detection of endogenous beta-catenin translocation to the nucleus as an indicator of pathway activation, requires only a limited number of primary cells, and is robust enough for automation and high-content, high-throughput screening. Identification of activators of the Wnt/Fzd pathway in human preosteoblasts may be useful in providing lead compounds for the treatment of osteoporosis.
Asunto(s)
Receptores Frizzled/fisiología , Microscopía Fluorescente/métodos , Osteoblastos/citología , Osteoblastos/metabolismo , Proteínas Wnt/fisiología , Transporte Activo de Núcleo Celular , Automatización , Diferenciación Celular , Núcleo Celular/metabolismo , Células Cultivadas , Medios de Cultivo Condicionados/química , Relación Dosis-Respuesta a Droga , Humanos , Osteoporosis/terapia , Transactivadores , beta Catenina/metabolismoRESUMEN
Hypercholesterolemia is a major risk factor for coronary artery disease. Oxysterols are known to inhibit cholesterol biosynthesis and have been explored as potential antihypercholesterolemic agents. The ability of 3beta-hydroxy-5alpha-cholest-8(14)-en-15-one (15-ketosterol) to lower non-HDL cholesterol has been demonstrated in rodent and primate models, but the mechanisms of action remain poorly understood. Here we show in a coactivator recruitment assay and cotransfection assays that the 15-ketosterol is a partial agonist for liver X receptor-alpha and -beta (LXRalpha and LXRbeta). The binding affinity for the LXRs was comparable to those of native oxysterols. In a macrophage cell line of human origin, the 15-ketosterol elevated ATP binding cassette transporter ABCA1 mRNA in a concentration-dependent fashion with a potency similar to those of other oxysterols. We further found that in human embryonic kidney HEK 293 cells, the 15-ketosterol suppressed sterol-responsive element binding protein processing activity and thus inhibited mRNA expression of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, LDL receptor, and PCSK9. Our data thus provide a molecular basis for the hypocholesterolemic activity of the 15-ketosterol and further suggest its potential antiatherosclerotic benefit as an LXR agonist.
Asunto(s)
Colestenonas/farmacología , Proteínas de Unión al ADN/metabolismo , Hidroximetilglutaril-CoA Reductasas/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Proteína 2 de Unión a Elementos Reguladores de Esteroles/antagonistas & inhibidores , Transportador 1 de Casete de Unión a ATP , Transportadoras de Casetes de Unión a ATP/biosíntesis , Células Cultivadas , Proteínas de Unión al ADN/agonistas , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Receptores X del Hígado , Receptores Nucleares Huérfanos , Proproteína Convertasa 9 , Proproteína Convertasas , Receptores Citoplasmáticos y Nucleares/agonistas , Serina Endopeptidasas/biosíntesisRESUMEN
We have developed a high-content screening (HCS) assay to find activators of Wnt/Frizzled (Wnt/Fzd), a pathway known to be important in bone formation. Utilizing primary human preosteoblasts as a model, activation of the Wnt/Fzd pathway was detected by monitoring the stabilization and translocation of the transcription factor beta-catenin from cytoplasm to the nucleus. Endogenous beta-catenin was detected in preosteoblasts by immunofluorescent staining, and subcellular localization was determined by HCS using the Cellomics (Pittsburgh, PA) ArrayScan IV. Positive controls, including Wnt3A-conditioned medium and inhibitors of glycogen synthase kinase-3beta, resulted in increased nuclear beta-catenin. The assay had a Z'-factor of 0.6 and was conducive to automation for high-throughput screening/HCS. By combining standard immunofluorescence technology with automated fluorescence microscopy, we demonstrate the capability of screening cell-signaling pathways in primary human cells.
Asunto(s)
Bioensayo/métodos , Proteínas del Citoesqueleto/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Proteínas/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Transactivadores/metabolismo , Células Cultivadas , Técnica del Anticuerpo Fluorescente Directa/métodos , Receptores Frizzled , Humanos , Microscopía Fluorescente/métodos , Osteoblastos/citología , Preparaciones Farmacéuticas , Proteínas Wnt , beta CateninaRESUMEN
Forskolin and cAMP have been shown to have paradoxical effects in the regulation of expression levels of mRNA of cytochrome P450 3A (CYP3A) family members. We demonstrate in this study that forskolin upregulated the promoter for CYP3A4 independent of its ability to increase cAMP levels. This activity was explained showing forskolin directly activated the pregnane-X-receptor, a known regulator of CYP3A genes.
Asunto(s)
Colforsina/farmacología , AMP Cíclico/fisiología , Sistema Enzimático del Citocromo P-450/biosíntesis , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Línea Celular , Citocromo P-450 CYP3A , Sistema Enzimático del Citocromo P-450/genética , Activación Enzimática , Histona Acetiltransferasas , Humanos , Ligandos , Coactivador 1 de Receptor Nuclear , Receptor X de Pregnano , ARN Mensajero/biosíntesis , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de Esteroides/metabolismo , Factores de Transcripción/metabolismoRESUMEN
We characterize the ability of the liver X receptor (LXRalpha [NR1H3] and LXRbeta [NR1H2]) agonist, T0901317, to activate the farnesoid X receptor (FXR [NR4H4]). Although T0901317 is a much more potent activator of LXR than FXR, this ligand actually activates FXR more potently than a natural bile acid FXR ligand, chenodeoxycholic acid. Thus, the FXR activity of T0901317 must be considered when utilizing this agonist as a pharmacological tool to investigate LXR function.
Asunto(s)
Anticolesterolemiantes/farmacología , Proteínas de Unión al ADN/agonistas , Receptores Citoplasmáticos y Nucleares/agonistas , Factores de Transcripción/agonistas , Miembro 11 de la Subfamilia B de Transportador de Casetes de Unión al ATP , Transportadoras de Casetes de Unión a ATP/efectos de los fármacos , Transportadoras de Casetes de Unión a ATP/genética , Células Cultivadas , Ácido Quenodesoxicólico/farmacología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Hidrocarburos Fluorados , Ligandos , Receptores X del Hígado , Receptores Nucleares Huérfanos , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Sulfonamidas , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Natural products have been identified as ligands for a number of members of the nuclear hormone receptor (NHR) superfamily. Often these natural products are used as dietary supplements to treat myriad ailments ranging from perimenopausal hot flashes to hypercholesterolemia and reduced cognitive function. Examples of some natural product ligands for NHRs include genestein (estrogen receptors NR3A1 and NR3A2), guggulsterone (farnesoid X receptor NR1H4), and St. John's wort (pregnane X receptor, NR1I2). In this study, we identified the first nonoxysterol natural product that functions as a ligand for the liver X receptor (LXRalpha and LXRbeta; NR1H3, NR1H2), a NHR that acts as the receptor for oxysterols and plays a key role in regulation of cholesterol metabolism and transport as well as glucose metabolism. We show that paxilline, a fungal metabolite, is an efficacious agonist of both LXRalpha and LXRbeta in biochemical and in vitro cell-based assays. Paxilline binds directly to both receptors and is an activator of LXR-dependent transcription in cell-based reporter assays. We also demonstrate that paxilline binding to the receptors results in efficient activation of transcription of two physiological LXR target genes, ABCA1 and SREBP. The discovery of paxilline, the first reported nonoxysterol natural product ligand of the LXRs, may provide insight into the mechanism of ligand recognition by these receptors and reaffirms the utility of examining natural product libraries for identifying novel NHR ligands.