|Cancer||Cell death||Cell cycle||Cytoskeleton||Exo/endocytosis||Differentiation||Division||Organelles||Signalling||Stem cells||Trafficking|
1α,25-dihydroxyvitamin D3 enhances fast-myosin heavy chain expression in differentiated C2C12 myoblasts
Hiroshi Okuno, Koshi N. Kishimoto1, Masahito Hatori and Eiji Itoi
Department of Orthopaedic Surgery, Tohoku University Graduate School of Medicine, Sendai, Japan
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.
Key words: C2C12, differentiation, myosin heavy chain, proliferation, skeletal muscle, vitamin D
Abbreviations: BCA, bicinchoninic acid, CDK, cyclin-dependent kinase, CKI, CDK inhibitor, CT, threshold cycle value, DMEM, Dulbecco's modified Eagle's medium, FBS, fetal bovine serum, GAPDH, glyceraldhyde-3-phosphate dehydrogenase, HS, horse serum, RT–PCR, reverse transcription–PCR, VD3, 1α,25-dihydroxyvitamin D3, VDR, vitamin D receptor
1To whom correspondence should be addressed (email firstname.lastname@example.org).
Vitamin D plays a major role in the regulation of calcium homoeostasis. The active form of vitamin D [VD3 (1α,25-dihydroxyvitamin D3)] is widely used in the treatment of metabolic bone diseases, such as rickets/osteomalacia, renal osteodystrophy and osteoporosis. Treatment with hydroxylated vitamin D treatment increases bone density (Papadimitropoulos et al., 2002) mainly through increased calcium uptake in the intestine (Kumar, 1986). In addition to its classical function in calcium metabolism, VD3 or its analogues regulate cell proliferation and differentiation (Miyaura et al., 1981; Walters, 1992). It has been used in the treatment of psoriasis, characterized by hyperproliferation of epidermal keratinocytes.
VD3 acts through binding in target cells to the VDR (vitamin D receptor) that belongs to the nuclear super family. VDR is expressed in various tissues including kidney, intestine, bone, parathyroid glands and skin (Minghetti and Norman, 1988; Walters, 1992; Haussler et al., 1998). Since myoblasts and myotubes have also been shown to contain VDRs in chicks (Boland et al., 1985), mice (Simpson et al., 1985) and human (Costa et al., 1986), muscle cells could respond to VD3 through VDR.
The effects of VD3 have been examined in many types of cells in different phases of differentiation. It inhibits DNA and protein synthesis dose-dependently in human myoblasts and myotubes (Costa et al., 1986). VD3 treatment in early stages of culture increases DNA synthesis and cell growth of chick embryo myoblasts in the proliferating phase in high serum. However, VD3 treatment during the subsequent stage of chick myoblast differentiation in low serum inhibits DNA synthesis (Drittanti et al., 1989). The effects of VD3 may vary with the type of cell and stage of differentiation when treatment is given.
C2C12 is a murine myogenic cell line that proliferate rapidly, and when confluent and in the presence of 2% HS (horse serum), they begin to fuse and differentiate into myotubes. When VD3 treatment began at the change to the differentiation medium, down-regulation of myosin heavy chain isoforms and myogenic transcription factors (e.g. Myf5 and myogenin) occurred (Endo et al., 2003). However, the effect of VD3 on differentiated C2C12 cells has not been investigated, which might best be done with differentiated myocytes that reflect the clinical situation.
To analyse the effect of VD3 on myoblasts and myotubes, VD3 was added in three phases of C2C12 myoblasts differentiation, proliferating, differentiating and differentiated, and cells were analysed by cell counting, colorimetry and flow cytometry. Differentiation was also examined by quantitative RT–PCR (reverse transcription–PCR), immunocytofluorescence and Western blotting for myosin heavy chain and its isoforms.
2. Materials and methods
2.1. Cell culture
C2C12, a mouse myoblast cell line obtained from Riken cell bank (Tsukuba) was maintained for growth in DMEM (Dulbecco's modified Eagle's medium: Invitrogen) supplemented with 10% FBS (fetal bovine serum) and 1% penicillin/streptomycin (Invitrogen) at 37°C in a 5% CO2 in air humidified chamber. The cells were plated at 1×105 cells per well in 6-well plates and incubated for 3 days (proliferating phase). As the cells approached confluency, the growth medium was replaced with a DMEM supplemented with 2% HS (Invitrogen) and 1% penicillin/streptomycin to initiate differentiation. The medium was changed every other day, and VD3 was added at 0, 1, 10 or 100 nM in ethanol. VD3 treatment commenced in the differentiating phase when the medium was changed to HS. VD3 treatment commenced in the fully differentiated phase was defined as from 8 days culture in HS (Figure 1A).
2.2. Cell proliferation assay
Proliferation of C2C12 myoblasts was measured by both conventional haemocytometer cell counting and the WST-1 proliferation assay (Dojindo). Cells were grown for 72 h before analysis. Six-well plates with 5×104 cells per well were used for cell counting, and 96-well plates with 5×103 cells per well were used for the WST-1 assay. WST-1 solution was added to the 96-well plates for analysis by optical densitometry after 2 h incubation.
2.3. Cell cycle analysis
To analyse the cell cycle distribution of C2C12 cells in the proliferating phase, the cells were cultured with or without VD3. After 72 h, the cells were harvested and fixed with 70% ethanol. The cells were kept at −20°C until staining. Fixed cells were treated with RNase A (Sigma) in PBS for 30 min, followed by staining with 50 mg/l propidium iodide (Sigma) in PBS. Flow cytometry was carried out with a BD FACS Calibur (Becton Dickinson). Data acquisition and analysis relied on CellQuest Pro (BD) software.
2.4. Total RNA extraction and quantitative RT–PCR
Total RNA was isolated from cell cultures using RNeasy Mini Kit (Qiagen). RT was performed using a High Capacity cDNA RT kit (Applied Biosystems). Quantitative real-time PCR assay was done with Power SYBER Green PCR Master Mix (Applied Biosystems) on the ABI PRISM 7700 Sequence Detection System (Applied Biosystems) to assess the expression of p21, p27, myogenin, myosin heavy chain neonatal, myosin heavy chain isoforms and VDR routinely in duplicate or triplicate. Each primer was designed from the GenBank® database as follows: p21, p27, myogenin, myosin heavy chain neonatal, myosin heavy chain I/β, myosin heavy chain IIa, myosin heavy chain IId/x, myosin heavy chain IIb and VDR (Table 1). The fractional cycle number at which the fluorescence passes the fixed CT (threshold cycle values) was used for quantifying by the comparative CT method. Sample values were normalized to the threshold value for GAPDH (glyceraldhyde-3-phosphate dehydrogenase) for each time-point: ΔCT = CT (experiments)−CT (GAPDH). The CT value for vehicle was used as a reference. ΔΔCT = ΔCT (VD3)−ΔCT (vehicle). The fold change in mRNA expression for each time-point was plotted using the vehicle as a reference: 2−ΔΔCT(vehicle) = 1.
Table 1 List of PCR primers used in this study
F, Forward; R, Reverse.
F, Forward; R, Reverse.
Cultured C2C12 cells were fixed in −20°C ethanol for 10 min, and permeated with PBS containing 0.1% Tween 20 (ICN Biomedicals) for 15 min at room temperature. Cells were incubated with a primary mouse monoclonal sarcomeric myosin-specific MF20 antibody [DSHB (Developmental Studies Hybridoma Bank) IA] in the PBS at 4°C overnight, followed by incubation with Alexa Fluor® 555-conjugated goat anti-mouse IgG secondary antibody (Invitrogen) in the PBS for 2 h at room temperature. Nuclei were stained with Hoechst 33342 (1:1000; Sigma).
2.6. Western blot analysis
C2C12 cells were washed with PBS and lysed in 200 μl of lysis buffer (40 mM Tris/HCl, pH 7.5, 300 mM KCl, 1% Triton X-100 and 2 mM EDTA) mixed with protease inhibitor cocktail (1:50; Sigma) for 40 min on ice. The extracts were cleared by centrifugation at 15000 g for 30 min. Protein concentration was measured by the BCA (bicinchoninic acid) assay using a Pierce Microplate BCA Protein Assay Kit (Thermo). Equal amounts of protein in the supernatant (15 mg protein/lane) were electrophoresed on 8% SDS gels and blotted to PVDF membranes (Bio-Rad). The membranes were blocked with TBST buffer (0.01 M Tris/HCl, pH 7.5, 0.15 M NaCl and 0.05% Tween 20) containing non-fat dried skimmed milk powder for 1 h at room temperature and incubated in TBST buffer at 4°C overnight with either anti- myosin heavy chain fast (MY32) mAb (1:1000; Sigma), anti-GAPDH (6C5) mAb (1:1000; Santa Cruz) or MF20 hybridoma supernatant before being incubated with HRP (horseradish peroxidase)-conjugated secondary anti-mouse IgG (1:2500; Invitrogen) in TBST for 1 h at room temperature. Signals were visualized by chemiluminescence using an ECL® Western Blotting Detection Reagents (GE Healthcare) with a digital luminescent image analyser LAS-1000 (Fujifilm). The band intensity was analysed by ImageJ 1.37v software program (National Institutes of Health) and normalized by GAPDH.
2.7. Statistical analysis
Statistical significance between the vehicle-treated and the VD3-treated cells was judged by Welch's t test. All data were expressed as the means±S.D., with P<0.05 being considered as significant. A difference of <2-fold in the amount of the mRNA level was not considered to be significant, even when it was statistically significant.
3.1. Effects of VD3 on C2C12 myoblasts in the proliferating phase
VD3 (at 1, 10 or 100 nM) inhibited the proliferation of C2C12 myoblasts in a dose-dependent manner over 72 h. The inhibitory effects were significant at 1 nM (Figures 2A and 2B). At 72 h, cell cycle analysis showed a dose-dependent increase in the percentage of cells in G0/G1 phases (Figure 2C). Quantitative RT–PCR for VDR increased in a dose-dependent manner (Figure 2D). p21 and p27 gene expression were elevated (Figures 2E and 2F).
3.2. The effects of VD3 on C2C12 myoblasts in the differentiating phase
In the differentiating phase of C2C12 myoblasts, VD3 treatment significantly decreased mRNA expression of neonatal myosin heavy chain. At 1 and 10 nM, VD3 tended to decrease the mRNA level of myogenin, which was significantly the case at 100 nM (Figure 3A). Immunocytofluorescence for myosin heavy chain was decreased in myosin heavy chain-positive myocytes relative to the VD3 concentration (Figure 3B). Western blotting also showed that myosin heavy chain protein decreased significantly in a dose-dependent manner (Figure 3C).
3.3. Effects of VD3 on C2C12 in the differentiated phase
Myosin heavy chain isoforms were investigated at the mRNA level to follow the natural course of their expression. Differentiation of C2C12 myoblasts increased for 8 days before reaching a plateau (Figure 1B). VD3 commencing in the differentiated phase did not significantly affect neonatal myosin heavy chain at the mRNA level (Figure 4A). Myosin heavy chain-positive cells decreased in 100 nM VD3, but this was less prominent than in the differentiating phase. Myosin heavy chain-positive cells were short and round after 100-nM VD3 treatment (Figure 4B). One and 10 nM VD3 tended to increase the protein level of myosin heavy chain, but not significantly, although it did at 100 nM (Figure 4C). Myosin heavy chain type IIa mRNA level was significantly increased by 1 nM VD3, as also the myosin heavy chain type IId/x mRNA level (Figure 5A). Western blotting analysis showed that fast myosin heavy chain isoform had increased expression in 1 and 10 nM VD3, which was significant at the lower concentration (Figure 5B).
The steroid hormone, VD3 inhibits the proliferation in the proliferating phase of C2C12 myoblasts and the expression of myosin heavy chain in the differentiating phase. In the differentiated phase, 1 nM VD3 tended to increase the expression of myosin heavy chain and significantly enhanced the fast myosin heavy chain isoform, which is the first demonstration to confirm the anabolic effect of the steroid on mouse skeletal muscle cell in vitro, thus indicating some of the clinical effects as previously reported.
Previous in vitro studies with squamous cell carcinoma (Hershberger et al., 1999), prostate adenocarcinoma (Getzenberg et al., 1997), cancers of the ovary (Zhang et al., 2005), breast (Colston et al., 1992) and lung (Nakagawa et al., 2005) showed that the addition of VD3 or its analogues had significant anticancer effects, and cell cycle perturbation has been analysed as an anti-proliferative activity of VD3 in tumour cells. These effects are mediated through the VDR and this regulates proliferation, apoptosis (Simboli-Campbell et al., 1996) and angiogenesis (Mantell et al., 2000). The dose-dependent increase of the VDR mRNA level we have shown suggests that VD3 induced cell-cycle arrest through VDR in proliferating phase C2C12 cells.
Cell cycle progression is regulated by cyclins, and their association with CDKs (cyclin-dependent kinases) and CKIs (CDK inhibitors). In particular, p21 (Harper et al., 1993) and p27 (Polyak et al., 1994; Toyoshima and Hunter, 1994) are inhibitors of G1 CDK. Expressions of p21 and p27 seem to be increased by VD3 treatment inducing a G0/G1 phase arrest in squamous cell carcinoma cell lines (Hager et al., 2001) and breast cancer MCF-7 cells (Verlinden et al., 1998). We found that gene expression of p21 and p27 was up-regulated by VD3 treatment in C2C12 cells (Figures 2E and 2F). G0/G1 arrest of proliferating C2C12 might be influenced by CDKs–CKIs through p21 and p27, as in other cell lines.
Gene expression of myosin heavy chain isoforms reached a plateau before 8 days of differentiation (Figure 1B), but a longer period of culture might make further differentiation into mature myotubes possible. However, prolonged culture could also result in the detachment of cells from the culture dishes by myotube contraction. Starting VD3 treatment from 8 days after differentiation seemed to prevent cell detachment.
Gene expression of neonatal myosin heavy chain in the differentiated phase was not significantly decreased by VD3 treatment, unlike in the differentiating phase. The differentiated phase contains more differentiated myotubes and fewer myoblasts than the other phases. This may more closely mimic real muscle tissue. The smaller proportion of myoblasts in the differentiated phase could be the reason for no inhibitory effect on cell proliferation being seen in this phase.
VD3 acts through two different mechanisms (Walters, 1992). VDR is widely expressed in the various tissues such as skin, parathyroid, kidney, intestine and bone (Haussler et al., 1998). It is expressed from myoblasts to myotubes in cloned human skeletal muscle cells (Costa et al., 1986). VD3 has some effects on transcription of the target gene via VDR as a nuclear receptor (Haussler et al., 1998), effects considered as genomic actions. Another is a non-genomic mechanism as VD3 exerts fast effects on calcium metabolism in muscle cell through stimulation of voltage-dependent Ca2+ channels, which is mediated by the cAMP messenger system (De Boland and Boland, 1994). VD3 can activate p38 MAPK (mitogen-activated protein kinase) and ERK1/2 (extracellular-signal-regulated kinase 1/2) in C2C12 (Buitrago et al., 2006; Ronda et al., 2007).
Yoshizawa et al. (1997) have generated VDR gene-deleted mice (VDR−/−) as an animal model of type II vitamin D-dependent rickets. These mice have a unique feature in growing normally with no bone or metabolic anomalies for 3 weeks from birth until they are weaned. This feature is presumably due to the high calcium content or other critical nutrients in the breast milk. Further analysis showed that the diameters of skeletal muscle fibre at 3 weeks old were significantly decreased (∼20%) on average compared with wild-type mice (Endo et al., 2003). Our in vitro results may be more consistent with the muscle phenotype of the wild-type mice than the VDR−/− mice. Moreover, skeletal muscle may be a direct physiological target of VDR actions.
Skeletal muscle fibres are classified as slow-twitch (type I) and fast-twitch (type II) fibres on the basis of the contractile properties and oxidative capacity. Fast-twitch (type II) fibres have 4 subtypes (IIA, IIB, IIC and IID/X). They are classified on the basis of myosin heavy chain isoforms into types I, IIa, IId/x and IIb by histochemical and immunohistochemical staining of myosin heavy chain (Billeter et al., 1981; Staron and Pette, 1986; Termin et al., 1989; Schiaffino and Reggiani, 1996).
Many studies suggest that the expression of myosin heavy chain or myosin heavy chain subtypes, which we used as differentiation markers, is regulated by multiple signalling pathways and transcription factors rather than by a single signalling pathway (Spangenburg and Booth, 2003). In this and previous studies, we found that VD3 plays an important role in cell proliferation and differentiation in C2C12. VD3 treatment enhanced fast myosin heavy chain expression in the differentiated C2C12 cells (Figure 5B). However, the molecular mechanisms of the VD3 action on fast myosin heavy chain synthesis remain unexplained.
Analysis of cells from VDR−/− or 1α-hydroxylase−/− mice may be useful for clarification of the VD3 mechanisms. Alternatively, knock-down of up-regulated genes by VD3 in skeletal muscle cells could provide important information about the relationship between VD3 treatment and the expression of fast myosin heavy chain.
We have shown that 100 nM VD3 inhibited cell proliferation in the proliferating phase, decreased myosin heavy chain synthesis and the deformed cell shapes in the differentiating and differentiated phases, which might be due to some toxic effects of VD3.
A recent meta-analysis (Bischoff-Ferrari et al., 2004) based on several randomized clinical trials (Graafmans et al., 1996; Pfeifer et al., 2000; Gallagher et al., 2001; Bischoff et al., 2003; Dukas et al., 2004) found that vitamin D supplementation reduced the risk of falls. The mechanisms by which vitamin D reduces this risk have not been clarified. Clinically, it is known that vitamin D supplementation improves muscle strength in patients with vitamin D deficiency (Eastwood et al., 1977). The effect of vitamin D in reducing falls may be due to increasing muscle strength by its direct effects on muscle tissue. Fast-twitch (type II) muscle has the features of fast contraction and easy fatigability. Increase of fast-twitch (type II) muscle fibre may contribute in maintaining physical balance in momentary postural change, reducing falls. Vitamin D treatment accounted for a 59% reduction in falls, increased the relative number and size of type II muscle fibres, and improved muscle strength compared with placebo (Sato et al., 2005). Our study suggests that the elevated expression of fast myosin heavy chain may explain how vitamin D supplementation results in these clinical responses, although there could be many other factors that contribute.
Hiroshi Okuno and Koshi Kishimoto conceived the experiments. Hiroshi Okuno performed the experiments, and analysed data together with Koshi Kishimoto. Hiroshi Okuno and Koshi Kishimoto co-wrote the paper. Masahito Hatori and Eiji Itoi provided valuable help on the preparing the manuscript. All authors discussed the results and commented on the paper.
We thank Mr Katsuyoshi Shoji and Ms Michiko Fukuyama for the technical assistance.
This work was supported by the
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Received 1 November 2010/22 May 2011; accepted 26 January 2012
Published as Cell Biology International Immediate Publication 26 January 2012, doi:10.1042/CBI20100782
© The Author(s) Journal compilation © 2012 International Federation for Cell Biology
ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)