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. 2011 Aug;152(8):2976-86.
doi: 10.1210/en.2011-0159. Epub 2011 Jun 14.

1,25(OH)2vitamin D3 stimulates myogenic differentiation by inhibiting cell proliferation and modulating the expression of promyogenic growth factors and myostatin in C2C12 skeletal muscle cells

Leah A Garcia  1 , Keisha K King, Monica G Ferrini, Keith C Norris, Jorge N Artaza
Affiliations

1,25(OH)2vitamin D3 stimulates myogenic differentiation by inhibiting cell proliferation and modulating the expression of promyogenic growth factors and myostatin in C2C12 skeletal muscle cells

Leah A Garcia et al. Endocrinology. 2011 Aug.

Abstract

Skeletal muscle wasting is an important public health problem associated with aging, chronic disease, cancer, kidney dialysis, and HIV/AIDS. 1,25-Dihydroxyvitamin D (1,25-D3), the active form of vitamin D, is widely recognized for its regulation of calcium and phosphate homeostasis in relation to bone development and maintenance and for its calcemic effects on target organs, such as intestine, kidney, and parathyroid glands. Emerging evidence has shown that vitamin D administration improves muscle performance and reduces falls in vitamin D-deficient older adults. However, little is known of the underlying mechanism or the role 1,25-D3 plays in promoting myogenic differentiation at the cellular and/or molecular level. In this study, we examined the effect of 1,25-D3 on myoblast cell proliferation, progression, and differentiation into myotubes. C(2)C(12) myoblasts were treated with 1,25-D3 or placebo for 1, 3, 4, 7, and 10 d. Vitamin D receptor expression was analyzed by quantitative RT-PCR, Western blottings and immunofluorescence. Expression of muscle lineage, pro- and antimyogenic, and proliferation markers was assessed by immunocytochemistry, PCR arrays, quantitative RT-PCR, and Western blottings. Addition of 1,25-D3 to C(2)C(12) myoblasts 1) increased expression and nuclear translocation of the vitamin D receptor, 2) decreased cell proliferation, 3) decreased IGF-I expression, and 4) promoted myogenic differentiation by increasing IGF-II and follistatin expression and decreasing the expression of myostatin, the only known negative regulator of muscle mass, without changing growth differentiation factor 11 expression. This study identifies key vitamin D-related molecular pathways for muscle regulation and supports the rationale for vitamin D intervention studies in select muscle disorder conditions.

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Figures

Fig. 1.
Fig. 1.
Steady-state mRNA and protein up-regulation levels of VDR expression upon incubation of C2C12 cells with 1,25-D3. Cultures of C2C12 cells were incubated or not with 1,25-D3 (100 nm) for 1 and 4 d. Total RNA and whole-protein extracts were isolated for qRT-PCR and Western blottings, respectively. A, Mean ± sem corresponds to experiments done in triplicate; ***, P < 0.001 and Western blottings. B and C, Mean ± sem corresponds to experiments done in triplicate; ****, P < 0.0001 at 1 and 4 d, respectively. VD1 and VD2 are different pools of two samples each. In both cases, real-time PCR and Western blottings, samples and controls were normalized with GAPDH housekeeping gene. VD, Vitamin treated cells whole extracts; VDR, vitamin D receptor.
Fig. 2.
Fig. 2.
Expression and nuclear translocation of VDR upon incubation of C2C12 cells with 1,25-D3. Cultures of C2C12 cells were treated as in Fig. 1 in four-well removable chamber slides and subjected to IF using a polyclonal antibody for VDR followed by a FITC-conjugated secondary antibody (green). Cells were counterstained with DAPI (blue) to show nuclear localization. Merge pictures were done combining the green and blue pictures together to show nuclear translocation of VDR. Magnification, ×400. A, Cells incubated or not with 1,25-D3 for 1 d. B, Cells incubated or not with 1,25-D3 for 4 d.
Fig. 3.
Fig. 3.
1,25-D3 down-regulates the expression of PCNA. Cultures of C2C12 cells were treated as in Fig. 1 for 4 and 7 d. Western blottings and ICC reactions for PCNA were performed at the end of the incubation time. A, Western blots analysis was performed for whole-protein extracts at 4 and 7 d with different pools of samples (VD1 and VD2) done in triplicate with the corresponding densitometric analysis; ***, P < 0.001 and **, P < 0.01. B, Representative ICC pictures with the corresponding image analysis expressing percentage of positive cells (brown nuclear staining) for experiments done in triplicate at 4 d. C, Representative ICC pictures with the corresponding image analysis expressing percentage of positive cells (brown nuclear staining) for experiments done in triplicate at 7 d; ***, P < 0.001. Magnification, ×200 and ×400. ns, Not significant; VD, vitamin treated cells whole extracts.
Fig. 4.
Fig. 4.
1,25-D3 stimulates myogenic differentiation in C2C12 cells. Cultures of C2C12 cells were treated as in Fig. 1 for 3, 7, and 10 d. A, Representative immunocytochemistry pictures of MyoD+ cells with the corresponding image analysis expressing the ratio between MyoD+ nuclei per total nuclei field for experiments done in triplicate at 3 d. ***, P < 0.001. B, Representative ICC pictures of desmin+ cells with the corresponding image analysis expressing percentage IOD (area × intensity) for experiments done in triplicate at 7 d; ***, P < 0.001. C, Representative ICC pictures of MHC type II+ cells with the corresponding image analysis expressing mean diameter (μm) and size (width) (μm) for experiments done in triplicate at 10 d; ***, P < 0.001. Magnification, ×400.
Fig. 5.
Fig. 5.
Steady-state mRNA and protein levels modulation of IGF upon incubation of C2C12 cells with 1,25-D3. Cultures of C2C12 cells were incubated as in Fig. 1 for 4 d. Total RNA and whole-protein extracts were isolated for qRT-PCR and Western blottings, respectively. A, Mean ± sem corresponds to experiments done in triplicate for IGF-I (left panel); **, P < 0.01 and IGF-II (right panel); **, P < 0.01. B, Western blottings with the respective densitometric analysis for IGF-I; ***, P < 0.001. C, Western blottings with the respective densitometric analysis for IGF-II; ***, P < 0.001. VD1 and VD2 correspond to different pools of two samples each. In both cases, real-time PCR and Western blottings, samples and controls were normalized with GAPDH housekeeping gene. VD, Vitamin treated cells whole extracts.
Fig. 6.
Fig. 6.
1,25-D3 down-regulates the expression of Mstn in C2C12 cells. Cultures of C2C12 cells were treated as in Fig. 1 for 4 and 7 d. Total RNA and whole-cell protein extracts were isolated for qRT-PCR and Western blottings, respectively. C2C12 were also subjected to Mstn ICC. A, Mean ± sem corresponds to experiments done in triplicate for Mstn at 4 d (left panel); ***, P < 0.001 and at 7 d (right panel); ***, P < 0.001. B, Western blottings with the respective densitometric analysis for Mstn at 4 d (upper panel); **, P < 0.01 and *, P < 0.05 and for 7 d (lower panel); **, P < 0.01 and ***, P < 0.01. VD1 and VD2 are different pools of two samples each. In both cases, qRT-PCR (A) and Western blottings (B), samples and controls were normalized with GAPDH housekeeping gene. C, Corresponds to representative ICC pictures of Mstn+ cells with the corresponding image analysis expressing percentage IOD (area × intensity) for experiments done in triplicate at 7 d; ***, P < 0.001. VD, Vitamin treated cells whole extracts.
Fig. 7.
Fig. 7.
Steady-state mRNA and protein up-regulation levels of Fst expression upon incubation of C2C12 cells with 1,25-D3. Cultures of C2C12 cells were incubated as in Fig. 1 for 4 and 7 d. Total RNA and whole-protein extracts were isolated for qRT-PCR and Western blottings. VD1 and VD2 are different pools of two samples each. Mean ± sem corresponds to experiments done in triplicate; **, P < 0.01 and ***, P < 0.001. A, Cells incubated or not with 1,25-D3 for 1 d. B, Cells incubated or not with 1,25-D3 for 4 d. VD, Vitamin treated cells whole extracts; C, control cells, non-incubated with 1,25-D3. C1 and C2 correspond to different pools of two samples each.
Fig. 8.
Fig. 8.
Diagram of the role of vitamin D on muscle growth and differentiation. During development, mesodermal stem cells become committed to the myogenic fate. Muscle precursors (myoblasts) remain in a proliferative state until they exit from the cell cycle and are instructed to differentiate. Differentiation is accompanied by cell fusion, where committed myoblasts become polynucleated myotubes. 1,25-D3 decreases cell proliferation and enhances myogenic cell differentiation by modulating the expression of key pro- and antimyogenic factors, such as IGF-I, IGF-II, Fst, and Mstn.
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