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

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. C2C12 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 C2C12 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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The vitamin D receptor regulates mitochondrial function in C2C12 myoblasts

AJP Cell Physiology ◽

10.1152/ajpcell.00568.2019 ◽

2020 ◽

Vol 318 (3) ◽

pp. C536-C541 ◽

Cited By ~ 6

Author(s):

Stephen P. Ashcroft ◽

Joseph J. Bass ◽

Abid A. Kazi ◽

Philip J. Atherton ◽

Andrew Philp

Keyword(s):

Skeletal Muscle ◽

Vitamin D ◽

Protein Content ◽

Vitamin D Receptor ◽

Mitochondrial Function ◽

Mitochondrial Respiration ◽

Oxidative Capacity ◽

C2c12 Myoblasts

Vitamin D deficiency has been linked to a reduction in skeletal muscle function and oxidative capacity; however, the mechanistic bases of these impairments are poorly understood. The biological actions of vitamin D are carried out via the binding of 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) to the vitamin D receptor (VDR). Recent evidence has linked 1α,25(OH)2D3 to the regulation of skeletal muscle mitochondrial function in vitro; however, little is known with regard to the role of the VDR in this process. To examine the regulatory role of the VDR in skeletal muscle mitochondrial function, we used lentivirus-mediated shRNA silencing of the VDR in C2C12 myoblasts (VDR-KD) and examined mitochondrial respiration and protein content compared with an shRNA scrambled control. VDR protein content was reduced by ~95% in myoblasts and myotubes ( P < 0.001). VDR-KD myoblasts displayed a 30%, 30%, and 36% reduction in basal, coupled, and maximal respiration, respectively ( P < 0.05). This phenotype was maintained in VDR-KD myotubes, displaying a 34%, 33%, and 48% reduction in basal, coupled, and maximal respiration ( P < 0.05). Furthermore, ATP production derived from oxidative phosphorylation (ATPOx) was reduced by 20%, suggesting intrinsic impairments within the mitochondria following VDR-KD. However, despite the observed functional decrements, mitochondrial protein content, as well as markers of mitochondrial fission were unchanged. In summary, we highlight a direct role for the VDR in regulating skeletal muscle mitochondrial respiration in vitro, providing a potential mechanism as to how vitamin D deficiency might impact upon skeletal muscle oxidative capacity.

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The Vitamin D Receptor (VDR) Regulates Mitochondrial Function in C2C12 Myoblasts

10.1101/872127 ◽

2019 ◽

Author(s):

Stephen P. Ashcroft ◽

Joseph J. Bass ◽

Abid A. Kazi ◽

Philip J. Atherton ◽

Andrew Philp

Keyword(s):

Skeletal Muscle ◽

Vitamin D ◽

Protein Content ◽

Vitamin D Receptor ◽

Mitochondrial Function ◽

Mitochondrial Respiration ◽

Oxidative Capacity ◽

C2c12 Myoblasts

ABSTRACTVitamin D deficiency has been linked to a reduction in skeletal muscle function and oxidative capacity, however, the mechanistic basis of these impairments are poorly understood. The biological actions of vitamin D are carried out via the binding of 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) to the vitamin D receptor (VDR). Recent evidence has linked 1α,25(OH)2D3 to the regulation of skeletal muscle mitochondrial function in vitro, however, little is known with regard to the role of the VDR in this process. To examine the regulatory role of the VDR in skeletal muscle mitochondrial function, we utilised lentiviral mediated shRNA silencing of the VDR in C2C12 myoblasts (VDR-KD) and examined mitochondrial respiration and protein content compared to shRNA scrambled control. VDR protein content was reduced by ~95% in myoblasts and myotubes (P < 0.001). VDR-KD myoblasts displayed a 30%, 30% and 36% reduction in basal, coupled and maximal respiration respectively (P < 0.05). This phenotype was maintained in VDR-KD myotubes, displaying a 34%, 33% and 48% reduction in basal, coupled and maximal respiration (P < 0.05). Furthermore, ATP production derived from oxidative phosphorylation (ATPox) was reduced by 20% suggesting intrinsic impairments within the mitochondria following VDR-KD. However, despite the observed functional decrements, mitochondrial protein content as well as markers of fusion and fission were unchanged. In summary, we highlight a direct role for the VDR in regulating skeletal muscle mitochondrial respiration in vitro, providing a potential mechanism as to how vitamin D deficiency might impact upon skeletal muscle oxidative capacity.

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Evidence for Vitamin D Receptor Expression and Direct Effects of 1α,25(OH)2D3 in Human Skeletal Muscle Precursor Cells

Endocrinology ◽

10.1210/en.2015-1685 ◽

2016 ◽

Vol 157 (1) ◽

pp. 98-111 ◽

Cited By ~ 50

Author(s):

Karl Olsson ◽

Amarjit Saini ◽

Anna Strömberg ◽

Seher Alam ◽

Mats Lilja ◽

...

Keyword(s):

Skeletal Muscle ◽

Vitamin D ◽

Vitamin D Receptor ◽

Human Skeletal Muscle ◽

Precursor Cells ◽

Receptor Expression ◽

Direct Effects ◽

Muscle Precursor ◽

Muscle Precursor Cells

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The vitamin D receptor expression in skeletal muscle of women with distal radius fracture

Archives of Osteoporosis ◽

10.1007/s11657-018-0442-8 ◽

2018 ◽

Vol 13 (1) ◽

Cited By ~ 3

Author(s):

Kahyun Kim ◽

Hyun Sik Gong ◽

Jae-Young Lim ◽

Jong Hee Kim ◽

Goo Hyun Baek

Keyword(s):

Skeletal Muscle ◽

Vitamin D ◽

Distal Radius Fracture ◽

Distal Radius ◽

Vitamin D Receptor ◽

Receptor Expression ◽

Radius Fracture

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THU0486 The vitamin d receptor expression in skeletal muscle of women with distal radius fracture

10.1136/annrheumdis-2018-eular.4998 ◽

2018 ◽

Author(s):

H.S. Gong ◽

K. Kim

Keyword(s):

Skeletal Muscle ◽

Vitamin D ◽

Distal Radius Fracture ◽

Distal Radius ◽

Vitamin D Receptor ◽

Receptor Expression ◽

Radius Fracture

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VDR and CYP27B1 are expressed in C2C12 cells and regenerating skeletal muscle: potential role in suppression of myoblast proliferation

AJP Cell Physiology ◽

10.1152/ajpcell.00014.2012 ◽

2012 ◽

Vol 303 (4) ◽

pp. C396-C405 ◽

Cited By ~ 96

Author(s):

Ratchakrit Srikuea ◽

Xiping Zhang ◽

Ok-Kyong Park-Sarge ◽

Karyn A. Esser

Keyword(s):

Skeletal Muscle ◽

Vitamin D ◽

Cell Proliferation ◽

Potential Role ◽

Active Form ◽

C2c12 Cells ◽

Skeletal Muscle Regeneration ◽

Western Blots ◽

C2c12 Myoblasts

1α,25(OH)2D3, the active form of vitamin D3, has been reported to regulate the cell biology of skeletal muscle. However, there has been some controversy about the expression of the vitamin D receptor (VDR) and thus the potential role of vitamin D3 in skeletal muscle. In this study, we isolated and sequenced the full-length Vdr and Cyp27b1 transcripts in C2C12 myoblasts and myotubes. Western blots and immunocytochemistry confirmed protein expression in both myoblasts and myotubes clearly demonstrating that C2C12 cells express VDR and CYP27B1. To determine the vitamin D3 action, we found that C2C12 myoblasts treated with either 1α,25(OH)2D3 or 25(OH)D3 inhibited cell proliferation and this was associated with increased Vdr expression. The observation that treatment of C2C12 myoblasts with the inactive form of vitamin D3, [25(OH)D3], inhibited proliferation suggested that CYP27B1 was functionally active. We used small interfering RNA to knock down Cyp27b1 in myoblasts, and cells were treated with 25(OH)D3. The growth-suppressive effects of 25(OH)D3 were abolished, suggesting that CYP27B1 in myoblasts is necessary for the ability of 25(OH)D3 to affect cell proliferation. Finally, we analyzed expression of VDR and CYP27B1 in regenerating skeletal muscle in vivo. We found that expression of VDR and CYP27B1 increased significantly at day 7 of regeneration, and these results confirm the expression of Vdr and Cyp27b1 in vivo and suggest a potential role for vitamin D3 in skeletal muscle regeneration following injury.

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