DNA methylation is an epigenetic mechanism regulating gene expression. In this study, we observed that DNA methyltransferase 3a (Dnmt3a) expression is decreased after muscle atrophy. We made skeletal muscle-specific Dnmt3a-knockout (Dnmt3a-KO) mice. The regeneration capacity after muscle injury was markedly decreased in Dnmt3a-KO mice. Diminished mRNA and protein expression of Dnmt3a were observed in skeletal muscles as well as in satellite cells, which are important for muscle regeneration, in Dnmt3a-KO mice. Dnmt3a-KO satellite cell showed smaller in size (length/area), suggesting suppressed myotube differentiation. Microarray analysis of satellite cells showed that expression of growth differentiation factor 5 (Gdf5) mRNA was markedly increased in Dnmt3a-KO mice. The DNA methylation level of the Gdf5 promoter was markedly decreased in Dnmt3a-KO satellite cells. In addition, DNA methylation inhibitor azacytidine treatment increased Gdf5 expression in wild-type satellite cells, suggesting Gdf5 expression is regulated by DNA methylation. Also, we observed increased inhibitor of differentiation (a target of Gdf5) mRNA expression in Dnmt3a-KO satellite cells. Thus, Dnmt3a appears to regulate satellite cell differentiation via DNA methylation. This mechanism may play a role in the decreased regeneration capacity during atrophy such as in aged sarcopenia.-Hatazawa, Y., Ono, Y., Hirose, Y., Kanai, S., Fujii, N. L., Machida, S., Nishino, I., Shimizu, T., Okano, M., Kamei, Y., Ogawa, Y. Reduced Dnmt3a increases Gdf5 expression with suppressed satellite cell differentiation and impaired skeletal muscle regeneration.

Unloading stress, such as bed rest, inhibits the regenerative potential of skeletal muscles; however, the underlying mechanisms remain largely unknown. FOXO1 expression, which induces the upregulated expression of the cell cycle inhibitors p57 and Gadd45α, is known to be increased in the skeletal muscle under unloading conditions. However, there is no report addressing FOXO1-induced inhibition of myoblast proliferation. Therefore, we induced muscle injury by cardiotoxin in transgenic mice overexpressing FOXO1 in the skeletal muscle (FOXO1-Tg mice) and observed regeneration delay in skeletal muscle mass and cross-sectional area in FOXO1-Tg mice. Increased p57 and Gadd45α mRNA levels, and decreased proliferation capacity were observed in C2C12 myoblasts expressing a tamoxifen-inducible active form of FOXO1. These results suggest that decreased proliferation capacity of myoblasts by FOXO1 disrupts skeletal muscle regeneration under FOXO1-increased conditions, such as unloading.

Summary Vitamin D is known to be effective for the prevention of muscle atrophy, such as age-related sarcopenia. However, vitamin D action in skeletal muscle tissue and muscle cells is largely unknown. We previously found that a transcription factor, FOXO1 gene expression, was induced in various muscle atrophy conditions causing muscle atrophy by upregulating atrophy-related genes, including atrogin 1 (ubiquitin ligase) and cathepsin L (lysosomal proteinase). In this study, we found that vitamin D inhibited FOXO1-mediated transcriptional activity in a reporter gene assay. Moreover, vitamin D suppressed the glucocorticoid-induced gene expression of atrogin 1 and cathepsin L in C2C12 myoblasts. Thus, vitamin D may prevent muscle atrophy via the FOXO1-mediated pathway in muscle cells. Key Words vitamin D, sarcopenia, atrophy, skeletal muscle, nuclear receptor Skeletal muscle is the largest organ in the human body, comprising approximately 40% of the total body weight, and it plays important roles in exercise, energy expenditure, and glucose/amino acid metabolism. Skeletal muscles plastically adapt to their environment, and appropriate exercise with sufficient nutrition increases muscle mass (1). Meanwhile, various life conditions, such as bedrest, aging, and other diseases, as well as a glucocorticoid treatment, cause muscle atrophy and decrease quality of life (2). In aging societies, which is a growing situation in many developed countries, the prevention/cure of atrophy is particularly important (1, 3). Understanding the molecular mechanisms underlying muscle hypertrophy/atrophy is important for developing methods to prevent muscle atrophy/dysfunction, which seriously impairs human health and quality of life.FOXO1 is a forkhead-type transcription factor that antagonizes insulin-mediated anabolic signals. We have been investigating the role of FOXO1 in metabolic regulation in skeletal muscles. Specifically, based on data that FOXO1 gene expression is induced by energy deprivation (4), we developed transgenic mice that overexpressed FOXO1 (FOXO1 Tg mice) and showed muscle atrophy (5). In addition, we showed that FOXO1 activates the genes involved in protein degradation (6, 7). FOXO1 activates the expression of atrophy-related genes such as atrogin 1 and cathepsin L in various muscle atrophyrelated conditions, including a glucocorticoid treatment (6-8). These findings suggest that FOXO1 in skeletal muscles plays an important role in muscle atrophy.Vitamin D, a fat-soluble vitamin, is well known for its role in regulating bone homeostasis. Evidence indicates that vitamin D plays roles in many other tissues including skeletal muscle (3,9). An epidemiological study suggested that a vitamin D treatment was effective for the prevention of decreased muscle mass (sarcopenia) (10). Previously, we first reported the cloning of mouse vitamin D receptor (VDR) cDNA (11). VDR is a nuclear receptor-type transcription factor, which is activated in the presence of 1,25(OH) 2 vitamin D3, the active form of vitamin D. It has been re...

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