Proteomic profiles of skeletal muscle cells treated with cholecalciferol, calcidiol, and calcitriol under <i>Vdr</i> gene silencing

Vitamin D insufficiency and deficiency are global prevalence that are related to lowering blood vitamin D levels. Significant lowering of blood vitamin D has been associated with a decrease in vitamin D receptor (VDR) protein expression in skeletal muscle. However, the effects of vitamin D 3 (cholecalciferol), 25(OH)D 3 (calcidiol), and 1α,25(OH) 2 D 3 (calcitriol) treatments on proteomics profiling of skeletal muscle cells under Vdr gene suppression have not been thoroughly investigated. To reveal the potential differential activation of the target proteins in responses to the treatments of vitamin D 3 and its metabolites under suppression of the Vdr gene, mouse C2C12 myoblasts were transfected with negative control siRNA or Vdr siRNA prior to inducing myogenic differentiation and then daily treated with 100 nM of cholecalciferol, calcidiol, or calcitriol for 48 h. LC-MS/MS-based proteomics analysis discovered that approximately 10% of the detected proteins (&gt;2,000 proteins) after cholecalciferol, calcidiol, or calcitriol treatment were significantly changed in Vdr siRNA-transfected cells compared to control siRNA-transfected cells. To support these findings, Gene Ontology (GO) enrichment analysis was performed using the ShinyGO web-based bioinformatics tools and revealed that cholecalciferol regulates biological process (e.g., translation, peptide biosynthesis process, and peptide metabolic process), cellular component (e.g., polysome, ribosome, and ribosomal subunit), and molecular function (e.g., porin activity, translation initiation factor binding, and peptidyl−prolyl cis−trans isomerase activity). While calcidiol regulates biological process (e.g., muscle cell differentiation, supramolecular fiber organization, and muscle structure development), cellular component (e.g., myofibril, sarcomere, contractile fiber), and molecular function (e.g., integrin binding, ribonucleoprotein complex binding, and structural constituent of ribosome). For calcitriol, its regulation involves biological process (e.g., muscle contraction, muscle structure development, and actin cytoskeleton organization), cellular component (e.g., myofilament, sarcomere, and contractile fiber), and molecular function (e.g., porin activity, structural constituent of cytoskeleton, and actin filament binding). Collectively, this study demonstrates that vitamin D 3 and its metabolites exert differential effects on the proteome of skeletal muscle cells under Vdr gene suppression. In addition, vitamin D metabolism could be a crucial process that affects vitamin D 3 action on the target proteins in skeletal muscle cells. This research project was supported by the Faculty of Science, Mahidol University, and the CIF and CNI Grant, Faculty of Science, Mahidol University (to R.S.). This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.