1α,25‐dihydroxyvitamin D₃ enhances fast‐myosin heavy chain expression in differentiated C2C12 myoblasts

Abstract

We investigated the effect of VD3 (1α,25-dihydroxyvitamin D3) on the proliferating, differentiating and differentiated phases of C2C12 myoblasts, a mouse skeletal muscle cell line. VD3 treatment in 10% FBS (fetal bovine serum) inhibited the proliferation and viability of the cells in a dose-dependent manner. It also dose-dependently increased the percentage of cells in the G0/G1 phase as shown by flow cytometry. In the differentiating phase, VD3 treatment inhibited the formation of myotubes and the expression of total myosin heavy chain at both the mRNA and protein levels. In the differentiated phase, treatment had no significant effect on the amount of total myosin heavy chain, as Western blot analysis with MF20 antibody [DSHB (Developmental Studies Hybridoma Bank)] showed. However, significantly greater expression of fast myosin heavy chain in 1 nM VD3 was found by Western blot analysis with MY-32 (Sigma). Thus VD3 inhibited the proliferation of myoblasts during proliferating and differentiating phases, whereas it increased the expression of the fast myosin heavy chain isoform in the differentiated phase. The data indicate that an adequate concentration of VD3 might have an anabolic effect on differentiated skeletal muscle.

Actions of 1,25(OH)2-vitamin D3 on the cellular cycle depend on VDR and p38 MAPK in skeletal muscle cells

“…In view of our previous results (Buitrago et al 2012), it was expected that a cell proliferative state would be observed at 24 h. This may be explained by the fact that now the cells were deprived of serum for 16 h, instead of 2 h as reported previously. Okuno et al (2012) Figure 7 Silencing of cyclin D3 by a specific siRNA affects VDR and myogenin expression in muscle cells. C2C12 WT cells were transfected with 50 pmol cyclin D3 siRNA or control siRNA for 6 h according to the manufacturer's specifications.…”

Vitamin D Signaling in Myogenesis: Potential for Treatment of Sarcopenia

“…Whether vitamin D has positive effects on myoblast proliferation and differentiation is currently under debate. Recent studies [27] suggest that vitamin D treatment enhances fast type (type IIa) MyHC expression in fully differentiated C2C12 myotubes. Type II muscle fibers contain type IIa MyHC [77].…”

“…However, there has been still some controversy as to whether VDR is expressed in skeletal muscle [24, 29, 32]. For example, some studies have failed to detect VDR in skeletal muscle [24, 25, 95–97]; other studies have shown that VDR protein and/or mRNA are detectable in skeletal muscle [21–23, 29, 46, 98] and myogenic cells [26, 27, 29, 31, 32, 42, 45, 46, 53, 55, 98–104]. In brief, Wang et al [105] call into question the specificity of various commercially available VDR antibodies.…”

“…They identified PKC α as main isoform correlated with the early stimulation of myoblast proliferation by 1 α ,25(OH) 2 D 3 [44]. By contrast, several studies suggest that, overall, 1 α ,25(OH) 2 D 3 or 25(OH)D 3 appears to have antiproliferative effect on myogenic cells [26, 27, 29, 32, 42]. 1 α ,25(OH) 2 D 3 (1–100 nM) inhibited proliferation of C2C12 myoblasts in a dose-dependent manner [27, 32] without inducing necrotic and apoptotic cell death [32].…”

“…By contrast, several studies suggest that, overall, 1 α ,25(OH) 2 D 3 or 25(OH)D 3 appears to have antiproliferative effect on myogenic cells [26, 27, 29, 32, 42]. 1 α ,25(OH) 2 D 3 (1–100 nM) inhibited proliferation of C2C12 myoblasts in a dose-dependent manner [27, 32] without inducing necrotic and apoptotic cell death [32]. Okuno et al [27] showed that 1 α ,25(OH) 2 D 3 arrested the cells in the G0/G1 phase concomitantly with induction of cyclin-dependent kinase (CDK) inhibitors, p21 WAF1/CIP1 that facilitates cell cycle withdrawal [123] and p27 Kip1 that inhibits a wide range of CDKs essential for cell cycle progression [124].…”