Development of a highly sensitive, high-throughput, mass spectrometry-based assay for rat procollagen type-I N-terminal propeptide (PINP) to measure bone formation activity.

Type-I procollagen aminoterminal propeptide (PINP) is a useful biomarker for bone formation activity that is used to monitor treatment of bone disorders including osteoporosis. Studies with human patients under long-term therapy for osteoporosis by daily injection of parathyroid hormone (PTH) demonstrated that the circulating level of PINP at 3 months of treatment, measured by radioimmunoassay, was a good predictor for bone mineral density (BMD) at 18 months. It is important to have PINP assays for other species to elucidate processes of bone formation and for the development of new therapeutic options that can enhance bone formation activity. Currently, only a human PINP radioimmunoassay is commercially available for clinical use, which may not be cross reactive with PINP from other species. For example, rat PINP has little amino acid sequence homology to human PINP. Therefore, we developed a new, highly sensitive, high-throughput mass spectrometry-based assay for PINP from rat plasma or serum that does not rely on antibody reagents. Circulating levels of PINP showed age-dependent changes in rats. Prednisolone treatment, which is known to retard bone formation activity, led to a significant decrease in PINP, whereas PTH treatment dose-dependently increased PINP. The throughput of the assay parallels that of most antibody-based assays so that it can handle a large number of samples that are generated from preclinical animal studies. PINP in rats may serve as a biomarker for bone formation activity, and this assay could be instrumental in studying bone physiology in rat experimental models.

Screening for activators of the wingless type/Frizzled pathway by automated fluorescent microscopy.

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.

High-content screening assay for activators of the Wnt/Fzd pathway in primary human cells.

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.