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We investigated the in vitro and in vivo osteogenic activity of licochalcone A. At low concentrations, licochalcone A stimulated the differentiation of mouse pre-osteoblastic MC3T3-E1 subclone 4 (MC4) cells and enhanced the bone morphogenetic protein (BMP)-2-induced stimulation of mouse bi-potential mesenchymal precursor C2C12 cells to commit to the osteoblast differentiation pathway. This osteogenic activity of licochalcone A was accompanied by the activation of extracellular-signal regulated kinase (ERK). The involvement of ERK was confirmed in a pharmacologic inhibition study. Additionally, noggin (a BMP antagonist) inhibited the osteogenic activity of licochalcone A in C2C12 cells. Licochalcone A also enhanced the BMP-2-stimulated expression of various BMP mRNAs. This suggested that the osteogenic action of licochalcone A in C2C12 cells could be dependent on BMP signaling and/or expression. We then tested the in vivo osteogenic activity of licochalcone A in two independent animal models. Licochalcone A accelerated the rate of skeletal development in zebrafish and enhanced woven bone formation over the periosteum of mouse calvarial bones. In summary, licochalcone A induced osteoblast differentiation with ERK activation in both MC4 and C2C12 cells and it exhibited in vivo osteogenic activity in zebrafish skeletal development and mouse calvarial bone formation. The dual action of licochalcone A in stimulating bone formation and inhibiting bone resorption, as described in a previous study, might be beneficial in treating bone-related disorders.

We found that camphoric acid significantly stimulated the differentiation of mouse osteoblastic MC3T3-E1 subclone 4 cells, as indicated by the induction of markers of osteoblastic differentiation. To elucidate the mechanism of action of camphoric acid in osteoblast differentiation, we evaluated the induction of transient receptor potential (TRP) cation channel family members and glutamate signaling molecules. TRPM7 and TRPV1 were highly expressed, but their expression was unaffected by camphoric acid. Camphoric acid is structurally similar to glutamate receptor ligands and significantly induced the expression of NMDAR1, GluR3/4, and mGluR8. However, camphoric acid exhibited weak regulatory activity toward glutamate receptors in a radioligand binding assay. Camphoric acid also significantly induced the activation of NF-kappaB and AP-1. Together, these data suggest that the stimulatory effect of camphoric acid on osteoblast differentiation was the result of its ability to induce mRNA expression of glutamate signaling molecules and to activate transcription factors.

Cell-based signal chemical genomics can profile the signalling pathway for certain cellular events by using a target-known chemical library. To ascertain its usefulness, the receptor activator of NF-kappaB ligand (RANKL)-induced osteoclastogenesis in mouse monocyte/macrophage cells RAW264.7 was used as an in vitro experimental model. Of 180 target-known inhibitors/activators formatted in a 384-well plate, 8 chemicals were shown to inhibit the osteoclast formation, but 4 chemicals enhanced this process. A variety of references support, or possibly lead one to expect the effects of these 12 chemicals on the cellular process of osteoclastogenesis in RAW264.7 cells, but several signalling pathways were newly found in this study; for example, CA-074 Me inhibiting cathepsin B and nitrendipine blocking the calcium channel could have the potential to inhibit the osteoclast formation as well as bone resorption. This is a simple but very fast and powerful method of profiling the signalling pathway of certain cellular events. Signal chemical genomics could provide invaluable information for the exploration of new target signalling processes and further target-based drug discovery strategies.