BackgroundMany histone deacetylase (HDAC) inhibitors are well recognized as potential anti-cancer drugs. Inhibition of HDACs induces temporal transcription or epigenetic control, thus regulating many different biological responses. Here, we investigated the osteogenic effect of the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA; vorinostat).MethodsThe effects of SAHA on osteoblast differentiation were examined in the 6XOSE-Luc reporter assay for determination of runt-related transcription factor 2 (Runx2) activity and alkaline phosphatase (ALP) activity and in an immunoprecipitation assay to determine the Runx2 acetylation state. The osteogenic activity of SAHA in vivo was studied in and receptor activator of nuclear factor-kappa B ligand (RANKL)-induced osteoporotic mouse model.ResultsSAHA increased the transcriptional activity of Runx2 in a dose-dependent manner in the 6XOSE-Luc reporter assay. SAHA by itself was unable to induce ALP activity; however, SAHA enhanced ALP activity induced by bone morphogenetic protein-2 (BMP-2). The degree of acetylation of Runx2 was increased with SAHA treatment, which suggests that the increase in Runx2 transcriptional activity might be dependent on stabilization by acetylation. Also, SAHA successfully reversed soluble RANKL-induced osteoporotic bone loss.ConclusionsOur study shows an intriguing osteogenic potential of SAHA in a BMP-2-dependent manner and suggests that SAHA could be used at lower doses along with BMP-2 to treat osteoporosis.

Summary: Okadaic acid, a potent specific inhibitor of serine/threonine protein phosphatases type 1 and 2A, affects several gene expressions in various cells. To determine whether okadaic acid affects the expression of fibronectin in MC3T3-E1 cells, we measured mRNA level and synthesis of fibronectin by Northern blot hybridiflation and immunoprecipitation methods, respectively. Okadaic acid (10-50 ng/ml) increased both mRNA level and synthesis of fibronectin in a dose-dependent manner. The increase of fibronectin mRNA by okadaic acid was strongly attenuated by the inhibition of new protein synthesis. The results indicate that okadaic acid, inhibitor of protein phosphatases, increases fibronectin synthesis in MC3T3-E1 cells.

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TNF-α, a proinflammatory cytokine, inhibits osteoblast differentiation under diverse inflammatory conditions; however, the underlying mechanisms in terms of the TNF-α signaling pathway remain unclear. In this study, we examined the role of Msx2 in TNF-α-mediated inhibition of alkaline phosphatase (ALP) expression and the signaling pathways involved. TNF-α down-regulated ALP expression induced by bone morphogenetic protein 2 (BMP2) in C2C12 and Runx2-/calvarial cells. Over-expression of Msx2 suppressed BMP2-induced ALP expression. Furthermore, TNF-α induced Msx2 expression, and the knockdown of Msx2 by small interfering RNAs rescued ALP expression, which was inhibited by TNF-α. TNF-α activated the NF-κB and the JNK pathways. Inhibition of NF-κB or JNK activation reduced the inhibitory effect of TNF-α on ALP expression, whereas TNF-α-induced Msx2 expression was only suppressed by the inhibition of the NF-κB pathway. Taken together, these results indicate that Msx2 mediates the inhibitory action of TNF-α on BMP2-regulated osteoblast differentiation and that the TNF-α-activated NF-κB pathway is responsible for Msx2 induction.