Epigenetics: DNA demethylation promotes skeletal myotube maturation.

Mesenchymal progenitor cells can be differentiated in vitro into myotubes that exhibit many characteristic features of primary mammalian skeletal muscle fibers. However, in general, they do not show the functional excitation-contraction coupling or the striated sarcomere arrangement typical of mature myofibers. Epigenetic modifications have been shown to play a key role in regulating the progressional changes in transcription necessary for muscle differentiation. In this study, we demonstrate that treatment of murine C2C12 mesenchymal progenitor cells with 10 µM of the DNA methylation inhibitor 5-azacytidine (5AC) promotes myogenesis, resulting in myotubes with enhanced maturity as compared to untreated myotubes. Specifically, 5AC treatment resulted in the up-regulation of muscle genes at the myoblast stage, while at later stages nearly 50% of the 5AC-treated myotubes displayed a mature, well-defined sarcomere organization, as well as spontaneous contractions that coincided with action potentials and intracellular calcium transients. Both the percentage of striated myotubes and their contractile activity could be inhibited by 20 nM TTX, 10 µM ryanodine, and 100 µM nifedipine, suggesting that action potential-induced calcium transients are responsible for these characteristics. Our data suggest that genomic demethylation induced by 5AC overcomes an epigenetic barrier that prevents untreated C2C12 myotubes from reaching full maturity.

Osteo-transcriptomics of human mesenchymal stem cells: accelerated gene expression and osteoblast differentiation induced by vitamin D reveals c-MYC as an enhancer of BMP2-induced osteogenesis.

Bone marrow-derived human mesenchymal stem cells (hMSCs) have the in vitro capacity to differentiate into osteoblasts, chondrocytes or adipocytes, depending on the applied stimulus. In order to identify novel regulators of osteogenesis in hMSCs, osteo-transcriptomics was performed whereby differentiation induced by dexamethasone (DEX), DEX+ bone morphogenetic protein 2 (BMP2), and DEX+ Vitamin D(3) (1,25(OH)(2)D(3)) was studied over a course of 12 days. Microarray analysis revealed that 2095 genes were significantly regulated by DEX+ 1,25(OH)(2)D(3), of which 961 showed accelerated expression kinetics compared to treatment by DEX alone. The majority of these genes were accelerated 24-48 h after onset of osteogenic treatment. Gene ontology (GO) analysis of these 1,25(OH)(2)D(3)-accelerated genes indicated their involvement in biological processes related to cellular differentiation and cell cycle regulation. When compared to cells treated with DEX or DEX+BMP2, treatment with DEX+ 1,25(OH)(2)D(3) clearly accelerated osteoprogenitor commitment and osteoblast maturation, as measured by alkaline phosphatase (ALP) activity and calcification of the matrix. Cell cycle progression, as observed after initial growth arrest, was not significantly accelerated by 1,25(OH)(2)D(3) and was not required for onset and progression of osteogenesis. However, expression of c-Myc was accelerated by 1,25(OH)(2)D(3), and binding sites for c-MYC were enriched in promoters of genes accelerated by 1,25(OH)(2)D(3). Lentiviral overexpression of c-MYC strongly promoted DEX+ BMP2-induced osteoblast differentiation and matrix maturation. In conclusion, our studies show for the first time that 1,25(OH)(2)D(3) strongly accelerates expression of genes involved in differentiation of hMSCs and, moreover, identify c-MYC as a novel regulator of osteogenesis.