Vitamin D signaling regulates proliferation, differentiation, and myotube size in C2C12 skeletal muscle cells
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Updated: May 5, 2026
Modeling Myotonic Dystrophy 1 in C2C12 Myoblast Cells
Published on: July 29, 2016
Christian M Girgis1, Roderick J Clifton-Bligh, Nancy Mokbel
1Garvan Institute of Medical Research (C.M.G., N.M., K.C., J.E.G.), Sydney, New South Wales 2010, Australia; Faculty of Medicine (C.M.G., R.J.C.-B., J.E.G.), University of Sydney, Sydney, New South Wales 2008, Australia; The Kolling Institute of Medical Research (R.J.C.-B.) and Royal North Shore Hospital (R.J.C.-B.), Sydney, New South Wales 2065, Australia; Department of Endocrinology and Diabetes (J.E.G.), Westmead Hospital, Sydney, New South Wales 2145, Australia; and St Vincent's Clinical School (J.E.G.), University of New South Wales, Sydney, New South Wales 2052, Australia.
Abstract:
Vitamin D deficiency is linked to a range of muscle disorders including myalgia, muscle weakness, and falls. Humans with severe vitamin D deficiency and mice with transgenic vitamin D receptor (VDR) ablation have muscle fiber atrophy. However, molecular mechanisms by which vitamin D influences muscle function and fiber size remain unclear. A central question is whether VDR is expressed in skeletal muscle and is able to regulate transcription at this site. To address this, we examined key molecular and morphologic changes in C2C12 cells treated with 25-hydroxyvitamin D (25OHD) and 1,25-dihydroxyvitamin D (1,25(OH)(2)D). As well as stimulating VDR expression, 25(OH)D and 1,25(OH)(2)D dose-dependently increased expression of the classic vitamin D target cytochrome P450, family 24, subfamily A, polypeptide 1 (CYP24A1), demonstrating the presence of an autoregulatory vitamin D-endocrine system in these cells. Luciferase reporter studies demonstrated that cytochrome P450, family 27, subfamily B, polypeptide 1 (CYP27B1) was functional in these cells. Both 25OHD and 1,25(OH)(2)D altered C2C12 proliferation and differentiation. These effects were related to the increased expression of genes involved in G(0)/G(1) arrest (retinoblastoma protein [Rb], 1.3-fold; ATM, 1.5-fold, both P < .05), downregulation of mRNAs involved in G(1)/S transition, including myc and cyclin-D1 (0.7- and 0.8-fold, both P < .05) and reduced phosphorylation of Rb protein (0.3-fold, P < .005). After serum depletion, 1,25(OH)(2)D (100nM) suppressed myotube formation with decreased mRNAs for key myogenic regulatory factors (myogenin, 0.5-fold; myf5, 0.4-fold, P < .005) but led to a 1.8-fold increase in cross-sectional size of individual myotubes associated with markedly decreased myostatin expression (0.2-fold, P < .005). These data show that vitamin D signaling alters gene expression in C2C12 cells, with effects on proliferation, differentiation, and myotube size.
Insights
Vitamin D influences muscle health by altering gene expression in C2C12 cells, affecting proliferation, differentiation, and myotube size. This study clarifies the molecular mechanisms of vitamin D
Area of Science:
- Muscle physiology and molecular biology
- Endocrinology and cellular signaling
Background:
- Vitamin D deficiency is associated with muscle disorders like myalgia and weakness.
- The precise molecular mechanisms by which vitamin D impacts muscle function and fiber size are not fully understood.
- Investigating the role of the vitamin D receptor (VDR) in skeletal muscle is crucial.
Purpose of the Study:
- To determine if the vitamin D receptor (VDR) is expressed in skeletal muscle cells.
- To investigate the molecular and morphological effects of vitamin D metabolites on C2C12 muscle cells.
- To elucidate the role of vitamin D signaling in regulating skeletal muscle gene expression, proliferation, differentiation, and myotube size.
Main Methods:
- Treatment of C2C12 cells with 25-hydroxyvitamin D (25OHD) and 1,25-dihydroxyvitamin D (1,25(OH)(2)D).
- Analysis of VDR and target gene expression (e.g., CYP24A1, CYP27B1) using molecular assays.
- Luciferase reporter assays to assess gene regulation.
- Evaluation of cell proliferation, differentiation, cell cycle gene expression (Rb, ATM, myc, cyclin-D1), and myotube formation and size.
Main Results:
- Vitamin D metabolites (25OHD and 1,25(OH)(2)D) stimulated VDR expression and activated the vitamin D endocrine system in C2C12 cells.
- Vitamin D signaling altered C2C12 cell proliferation and differentiation by modulating cell cycle gene expression.
- 1,25(OH)(2)D suppressed myotube formation but increased myotube size, associated with decreased expression of myogenic regulatory factors and myostatin.
Conclusions:
- Vitamin D signaling pathways are active in skeletal muscle cells (C2C12).
- Vitamin D influences key cellular processes including proliferation, differentiation, and myotube hypertrophy.
- These findings provide molecular insights into how vitamin D affects skeletal muscle size and function.
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