Research Paper Volume 13, Issue 4 pp 4895—4910

Cholecalciferol (vitamin D3) has a direct protective activity against interleukin 6-induced atrophy in C2C12 myotubes

Maraiza Alves Teixeira1,2, , Marilisa De Feudis1, , Simone Reano1, , Tommaso Raiteri1, , Andrea Scircoli1, , Ivan Zaggia1, , Sara Ruga1, , Laura Salvadori1,2, , Flavia Prodam3, , Paolo Marzullo1,4, , Claudio Molinari1, , Davide Corà1,5, , Nicoletta Filigheddu1,2, ,

  • 1 Department of Translational Medicine, University of Piemonte Orientale, Novara, Italy
  • 2 Istituto Interuniversitario di Miologia (IIM), Italy
  • 3 Department of Health Sciences, University of Piemonte Orientale, Novara, Italy
  • 4 IRCCS Istituto Auxologico Italiano, Laboratory of Metabolic Research, Piancavallo (VB), Italy
  • 5 Center for Translational Research on Autoimmune and Allergic Disease (CAAD), University of Piemonte Orientale, Novara, Italy

Received: May 21, 2020       Accepted: January 13, 2021       Published: February 22, 2021
How to Cite

Copyright: © 2021 Alves Teixeira et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


We previously determined that different vitamin D metabolites can have opposite effects on C2C12 myotubes, depending on the sites of hydroxylation or doses. Specifically, 25(OH)D3 (25VD) has an anti-atrophic activity, 1,25(OH)2D3 induces atrophy, and 24,25(OH)2D3 is anti-atrophic at low concentrations and atrophic at high concentrations. This study aimed to clarify whether cholecalciferol (VD3) too, the non-hydroxylated upstream metabolite, has a direct effect on muscle cells.

Assessing the effects of VD3 treatment on mouse C2C12 skeletal muscle myotubes undergoing atrophy induced by interleukin 6 (IL6), we demonstrated that VD3 has a protective action, preserving C2C12 myotubes size, likely through promoting the differentiation and fusion of residual myoblasts and by modulating the IL6-induced autophagic flux. The lack, in C2C12 myotubes, of the hydroxylase transforming VD3 in the anti-atrophic 25VD metabolite suggests that VD3 may have a direct biological activity on the skeletal muscle. Furthermore, we found that the protective action of VD3 depended on VDR, implying that VD3 too might bind to and activate VDR. However, despite the formation of VDR-RXR heterodimers, VD3 effects do not depend on RXR activity.

In conclusion, VD3, in addition to its best-known metabolites, may directly impact on skeletal muscle homeostasis.


1,25D3-MARRS: Membrane-Associated, Rapid Response Steroid binding; 1,25VD: 1,25-dihydroxy vitamin D3, 1,25(OH)2D3, calcitriol; 1α-OHase: 1α-hydroxylase; 24,25VD: 24,25-dihydroxy vitamin D3, 24,25(OH)2D3; 25-OHases: 25-hydroxylases; 25VD: 25-hydroxycholecalciferol, 25(OH)D3; Akt: Protein kinase B; ANOVA: Analysis of variance; BCA: Bicinchoninic acid; BSA: Bovine serum albumin; CEE: Chicken embryo extract; CLQ: Chloroquine; DAPI: 4′,6-diamidino-2-phenylindole; DEXA: Dexamethasone; DM: Differentiation medium; DMEM: Dulbecco’s Modified Eagle Medium; EDL: Extensor Digitorum longus; EDTA: Ethylenediaminetetraacetic acid; EGTA: Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid; ELISA: Enzyme-linked immunosorbent assay; FBS: Fetal bovine serum; GR: Glucocorticoid Receptor; GREs: Glucocorticoid-Responsive Elements; HS: Horse serum; HEPES: 4-(2-HydroxyEthyl)-1-PiperazineEthanesulfonic Acid; IF: Immunofluorescence; IL6: Interleukin 6; IQR: Interquartile range; JAK2: Janus kinase 2; LC3: Light Chain 3; LPM: Low proliferation medium; MAFbx: Muscle Atrophy F-box; mTOR: Mechanistic target of rapamycin; MyHC: Myosin heavy chain; PBS: Phosphate-buffered saline; PCR: Polymerase chain reaction; Pdia3: Protein disulfide-isomerase A3; PFA: Paraformaldehyde; PLA: Proximity ligation assay; PVDF: Polyvinylidene difluoride; RT: Room temperature; RXR: retinoid X receptor; Q1: First quartile; Q3: Third quartile; SD: Standard deviation; SDS: Sodium Dodecyl Sulfate; SDS-PAGE: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SEM: Standard error of the mean; SRC-1: Steroid Receptor Coactivator-1; STAT3: Signal transducer and activator of transcription 3; SUnSET: SUrface SEnsing of Translation; TBS: Tris-buffered saline; VD: Vitamin D; VD3: Cholecalciferol, Vitamin D3; VDR: Vitamin D receptor; VDREs: Vitamin D-Responsive Elements; Tris: Tris(hydroxymethyl)aminomethane.