Zingerone is a non-volatile compound found mainly in dried ginger. Zingerone increases the expression of osteogenic markers and has antioxidant effects. A previous study showed that zingerone accelerated osteoblast differentiation by suppressing the expression of Smad7, a member of the inhibitory Smad (I-Smad) family. However, it is not known if zingerone can induce osteoblast differentiation by regulating Smad1/5/9, a member of the receptor-regulated Smad (R-Smad) family. In addition, osteoblast differentiation induced by Smad1/5/9 mediated increases in the expression of heme oxygenase 1 (HO-1) has not been reported. This study investigated the effects of zingerone on osteoblast differentiation and confirmed the relationship between Smad1/5/9 and HO-1. Zingerone increased the expression of osteogenic genes including runt-related transcription factor 2 (Runx2), distal-less homeobox (Dlx5) and osteocalcin (OC) and also promoted Smad1/5/9 phosphorylation. Interestingly, HO-1 expression was also elevated by zingerone, and an inhibitor of HO-1 (Sn[IV] protoporphyrin IX dichloride [SnPP]) suppressed the zingeroneinduced increase in HO-1 expression and expression of osteogenic marker genes such as Dlx5, Runx2 and OC. Protein phosphatase 2A Cα (PP2A Cα, an inhibitor of Smad1/5/9) suppressed the zingerone-induced increase in HO-1 expression and expression of osteogenic marker genes. The zingerone-induced increase in HO-1 luciferase activity was suppressed by PP2A Cα. Taken together; our data demonstrate that zingerone promotes osteoblast differentiation by increasing Smad1/5/9 mediated HO-1 expression.
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