MEF2c control of osteoblast mineralization.

Abstract

Myocyte enhancer factor 2c (Mef2c) is a widespread transcription factor associated with cells of mesenchymal origin. Mef2c is important role in muscle differentiation as well as acting in hypertrophic chondrocytes to regulate collagen 10A1 expression. We found that Mef2c regulated differentiation of osteoblasts: in the absence of Mef2c osteoblasts could not mature and could not deposit mineralized matrix. The role of Mef2c was identified in experiments originally determined to make super-mineralizing osteoblasts for cell transplant therapy.

Surprisingly, among a group of compounds, we found that mineralization could be dramatically enhanced by the simple amphipathic solvent DMSO. Using MC3T3-E1 cells as an osteoblast model, DMSO dose-dependently increased osteoblast differentiation markers alkaline phosphatase (ALP) activity and extracellular matrix (ECM) mineralisation. The increases in ALP activity and ECM mineralisation were sustained throughout differentiation time courses indicating an anabolic function. Accordingly, numerous osteoblast marker-genes were elevated in response to DMSO treatment. DMSO caused a decrease in proliferation and increase in differentiation. Notably, the anabolic effect of DMSO was only evident in early differentiation cells which were actively proliferating. An early three day exposure to DMSO was sufficient for maximal effect. Microarray gene expression analysis of the DMSO effect revealed Mef2c as one of top DMSO super-induced genes. Mef2c expression preceded osterix, a known osteoblast differentiation factor. Knockdown by anti-Mef2c shRNA resulted in severe decreases in ALP activity and virtual elimination ECM mineralisation. Knock-in by transfection enhanced mineralization. Consistently, the expression of osteoblast-related genes was reduced in Mef2c knockdown cells, including osterix and Runx2. Furthermore, in Mef2c knock-down cell lines, Runx2 transfection as unable to relieve the block in differentiation, suggesting that Mef2c deficiency has a potent effect preventing differentiation. To conclude, our data shows a potent anabolic effect of DMSO on MC3T3-E1 osteoblast differentiation and indicates a pivotal role for Mef2c in the developmental process related to mineralization of matrix.