RESUMO
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.
Assuntos
Receptores Frizzled/fisiologia , Microscopia de Fluorescência/métodos , Osteoblastos/citologia , Osteoblastos/metabolismo , Proteínas Wnt/fisiologia , Transporte Ativo do Núcleo Celular , Automação , Diferenciação Celular , Núcleo Celular/metabolismo , Células Cultivadas , Meios de Cultivo Condicionados/química , Relação Dose-Resposta a Droga , Humanos , Osteoporose/terapia , Transativadores , beta Catenina/metabolismoRESUMO
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.