Myocyte Enhancer Factor 2C, an Osteoblast Transcription Factor Identified by Dimethyl Sulfoxide (DMSO)-enhanced Mineralization

Alexandre S Stephens,Sebastien R Stephens,Carl Hobbs,Deitmar W Hutmacher,Desa Bacic-Welsh,Maria Ann Woodruff,Nigel A Morrison

doi:10.1074/jbc.m111.253518

Alexandre S Stephens, Sebastien R Stephens + Show 5 more

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https://doi.org/10.1074/jbc.m111.253518

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Abstract

Rapid mineralization of cultured osteoblasts could be a useful characteristic in stem cell-mediated therapies for fracture and other orthopedic problems. Dimethyl sulfoxide (DMSO) is a small amphipathic solvent molecule capable of stimulating cell differentiation. We report that, in primary human osteoblasts, DMSO dose-dependently enhanced the expression of osteoblast differentiation markers alkaline phosphatase activity and extracellular matrix mineralization. Furthermore, similar DMSO-mediated mineralization enhancement was observed in primary osteoblast-like cells differentiated from mouse mesenchymal cells derived from fat, a promising source of starter cells for cell-based therapy. Using a convenient mouse pre-osteoblast model cell line MC3T3-E1, we further investigated this phenomenon showing that numerous osteoblast-expressed genes were elevated in response to DMSO treatment and correlated with enhanced mineralization. Myocyte enhancer factor 2c (Mef2c) was identified as the transcription factor most induced by DMSO, among the numerous DMSO-induced genes, suggesting a role for Mef2c in osteoblast gene regulation. Immunohistochemistry confirmed expression of Mef2c in osteoblast-like cells in mouse mandible, cortical, and trabecular bone. shRNAi-mediated Mef2c gene silencing resulted in defective osteoblast differentiation, decreased alkaline phosphatase activity, and matrix mineralization and knockdown of osteoblast specific gene expression, including osteocalcin and bone sialoprotein. A flow on knockdown of bone-specific transcription factors, Runx2 and osterix by shRNAi knockdown of Mef2c, suggests that Mef2c lies upstream of these two important factors in the cascade of gene expression in osteoblasts.

Highlights

Skeletal patterning and subsequent cellular differentiation during development is a complex process involving the activation and suppression of gene regulatory programs leading to bone formation [1]
We show that Dimethyl sulfoxide (DMSO) acts as a potent enhancer of human and mouse osteoblast differentiation as demonstrated by the significant gains in alkaline phosphatase (ALP) activity and mineralization observed throughout differentiation
We show that Myocyte enhancer factor 2c (Mef2c) gene expression is dynamically regulated during osteoblast differentiation and that shRNA-mediated Mef2c gene silencing is associated with highly significant decreases in ALP activity, osteoblast gene expression, and matrix mineralization, demonstrating a critical role for Mef2c in osteoblast differentiation

Summary

Introduction

Skeletal patterning and subsequent cellular differentiation during development is a complex process involving the activation and suppression of gene regulatory programs leading to bone formation [1]. The differentiation of osteoblasts is a highly coordinated process involving a clearly defined temporal sequence of events characterized by the commitment, proliferation, and subsequent maturation of precursor cells into terminally differentiated osteoblasts [3, 4]. Osteoblast differentiation is tightly coordinated by the activities of transcriptional regulators; these include the master transcription factors RUNX2 and osterix (OSX), which govern the expression of genes involved in the developmental process [6]. RUNX2 is indispensable for skeletal development and controls osteoblast differentiation by regulating the commitment of osteoprogenitors and activating major bone matrix genes [7]. DMSO is reported to stimulate osteoblast differentiation in MC3T3-E1, a mouse pre-osteoblast cell line, through ERK activation of transcription factors Runx and osterix [12]. We show that Mef2c gene expression is dynamically regulated during osteoblast differentiation and that shRNA-mediated Mef2c gene silencing is associated with highly significant decreases in ALP activity, osteoblast gene expression, and matrix mineralization, demonstrating a critical role for Mef2c in osteoblast differentiation

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