Wnt/β-catenin Signaling Mediates HDAC Inhibitor-induced Osteoblast Differentiation

Abstract

Histone deacetylase inhibitors (HDIs) influence a broad repertoire of biological processes, including cellular proliferation, differentiation, and cell death, via inhibiting the acetylation of both histone and non-histone proteins. Currently, HDIs are used as anti-cancer and anti-epileptic drug

however, positive effects for bone regeneration have recently been reported. Mechanisms by which HDIs are believed to stimulate osteoblast differentiation include the acetylation of Runx2 and osterix

however, a further understanding of additional mechanisms is needed. In this study, to investigate additional mechanisms of HDIs in osteoblast differentiation, it was examined the role of Wnt/β-catenin signaling, one of the key positive signaling pathways for osteoblast differentiation, and Smurf1 expression as a negative regulator of osteoblast differentiation in response to HDIs. To this end, we used trichostatin A (TSA), suberoylanilide hydroxamic acid (SAHA), and MS275 as HDIs, along with cultured C2C12 cells and primary cultured mouse calvarial cells (MC cells). To elucidate the effects of HDIs on osteoblast differentiation, C2C12 cells were incubated with HDIs and a sub minimal concentration of bone morphogenetic protein 2 (BMP2, 10 ng/mL) followed by alkaline phosphatase (ALP) staining, ALP activity assays, and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) for bone marker genes. C2C12 cells were treated with only HDIs for select parts of the experiment to verify the molecular mechanisms of HDIs. Experimental results demonstrated that C2C12 cells differentiated into osteoblasts when incubated with HDIs and BMP2, but not when treated with BMP2 alone. HDIs activated Wnt/β-catenin signaling, with HDIs inducing the Wnt10A gene to the highest extent. In addition, HDIs repressed Smurf1 expression and these effects were dependent on Wnt/β-catenin signaling. Taken together, these results suggest that HDIs enhance osteoblast differentiation through the increased expression of Wnt10A and the activation of Wnt/β-catenin signaling and subsequent decrease in Smurf1 expression.