Development of an in vitro screening assay for compounds that increase bone formation

Although parathyroid hormone (PTH) has the ability to stimulate bone growth in both rats and humans, its mechanism of action is not fully understood a t the molecular level. An in vitro marker that reflects the in vivo anaboli c actions of PTH would facilitate the discovery of small-molecule compounds that stimulate bone growth. We therefore compared the patterns of gene exp ression in three cell lines treated with PTH, The levels of c-fos, collagen ase, interleukin-6 (IL-6), and collagen mRNA were determined by reverse tra nscription-polymerase chain reaction (RT-PCR) in three osteoblast-like cell lines. The most responsive marker was c-fos, which was induced 5-10-fold a fter 1 h of PTH treatment in the UMR106-01 cell line, Because it,is a Criti cal early response gene in bone growth, we investigated the possibility of using c-fos stimulation as a method to: screen for compounds that can stimu late bone formation, A highly sensitive, medium-throughput RT-PCR assay for c-fos mRNA expression was established using the Taqman(TM) Detection Syste m (Perkin Elmer, Mississauga, Ontario). Cells were treated with a series of compounds to determine the specificity of c-fos stimulation. Of the compou nds tested, only PTH, prostaglandin E-2, 8-bromo-cAMP, and forskolin induce d c-fos mRNA levels, indicating-that this assay was specific for compounds that are known to induce cAMP and stimulate bone growth. These results indi cate that a simple in vitro assay for c-fos may be a reliable method for th e screening of compounds that stimulate bone growth in vivo.