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Kisspeptin-10 (KP-10) acts as a tumor metastasis suppressor via its receptor, G-protein-coupled receptor 54 (GPR54). The KP-10-GPR54 system plays an important role in embryonic kidney development. However, its function in osteoblast differentiation is unknown. Osteoblast differentiation is controlled by a range of hormones and cytokines, such as bone morphogenetic protein (BMPs), and multiple transcription factors, such as Runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), and Distal-less homeobox 5 (Dlx5). In the present study, KP-10-treatment significantly increased the expression of osteogenic genes, including mRNA and protein levels of BMP2, in C3H10T1/2 cells. Moreover, KP-10 induced BMP2-luc activity and increased phosphorylation of Smad1/5/9. In addition, NFATc4 specifically mediated KP-10-induced BMP2 gene expression. However, KP-10 treatment did not induce expression of the BMP2 and Runx2 genes in GPR54−/− cells. To examine whether KP-10 induced secretion of BMP2 to the culture medium, we used the conditioned-medium (C.M) of KP-10 treated medium on C3H10T1/2 cells. Dlx5 and Runx2 expressions were higher in GPR54−/− cells treated with C.M than in those treated with KP-10. These results demonstrate that BMP2 protein has an autocrine effect upon KP-10 treatment. Taken together, these findings suggest that KP-10/GPR54 signaling induces osteoblast differentiation via NFATc4-mediated BMP2 expression.

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Alpha‐pinene (α‐pinene) is an organic compound, found in the oils of many species of coniferous trees, especially pine. α‐Pinene reportedly has antioxidant and anti‐inflammatory activities; however, its effects on osteoblasts are unknown. This study investigated the effects of α‐pinene on osteoblast differentiation and tumour necrosis factor‐alpha (TNFα)‐induced inhibition of osteogenesis. Culture in control or osteogenic medium containing α‐pinene increased osteogenic marker expression. Alkaline phosphatase staining and alizarin red S staining confirmed that α‐pinene enhanced osteoblast differentiation. Also, α‐pinene attenuated TNFα‐induced inhibition of Smad1/5/9 phosphorylation and extracellular matrix mineralization. Taken together, our findings suggest that α‐pinene enhances osteoblast differentiation and mineralization in MC3T3‐E1 pre‐osteoblasts.