Department of Molecular Therapy, National Institute of Neuroscience, National Centre of Neurology and Psychiatry, Tokyo, Japan.
Fujii Memorial Institute of Medical Sciences, Tokushima University, Tokushima, Japan.
J Mol Endocrinol. 2020 Apr;64(3):195-208. doi: 10.1530/JME-19-0229.
The biologically active metabolite of vitamin D, 1,25-dihydroxyvitamin D3 (VD3), exerts its tissue-specific actions through binding to its intracellular vitamin D receptor (VDR) which functions as a heterodimer with retinoid X receptor (RXR) to recognize vitamin D response elements (VDRE) and activate target genes. Upregulation of VDR in murine skeletal muscle cells occurs concomitantly with transcriptional regulation of key myogenic factors upon VD3 administration, reinforcing the notion that VD3 exerts beneficial effects on muscle. Herein we elucidated the regulatory role of VD3/VDR axis on the expression of dystrobrevin alpha (DTNA), a member of dystrophin-associated protein complex (DAPC). In C2C12 cells, Dtna and VDR gene and protein expression were upregulated by 1-50 nM of VD3 during all stages of myogenic differentiation. In the dystrophic-derived H2K-mdx52 cells, upregulation of DTNA by VD3 occurred upon co-transfection of VDR and RXR expression vectors. Silencing of MyoD1, an E-box binding myogenic transcription factor, did not alter the VD3-mediated Dtna induction, but Vdr silencing abolished this effect. We also demonstrated that VD3 administration enhanced the muscle-specific Dtna promoter activity in presence of VDR/RXR only. Through site-directed mutagenesis and chromatin immunoprecipitation assays, we have validated a VDRE site in Dtna promoter in myogenic cells. We have thus proved that the positive regulation of Dtna by VD3 observed during in vitro murine myogenic differentiation is VDR mediated and specific. The current study reveals a novel mechanism of VDR-mediated regulation for Dtna, which may be positively explored in treatments aiming to stabilize the DAPC in musculoskeletal diseases.
维生素 D 的生物活性代谢物 1,25-二羟维生素 D3（VD3）通过与其细胞内维生素 D 受体（VDR）结合发挥其组织特异性作用，VDR 与维甲酸 X 受体（RXR）形成异二聚体，以识别维生素 D 反应元件（VDRE）并激活靶基因。VD3 给药后，鼠骨骼肌细胞中的 VDR 上调与关键成肌因子的转录调节同时发生，这强化了 VD3 对肌肉有益的观点。在此，我们阐明了 VD3/VDR 轴对肌营养不良蛋白相关蛋白复合物（DAPC）成员肌营养不良蛋白α（DTNA）表达的调节作用。在 C2C12 细胞中，在肌生成分化的所有阶段，1-50 nM 的 VD3 均可上调 Dtna 和 VDR 基因和蛋白表达。在源自 DMD 的 H2K-mdx52 细胞中，VDR 和 RXR 表达载体共转染时，VD3 上调 DTNA 的表达。MyoD1（E 盒结合的成肌转录因子）沉默不会改变 VD3 介导的 Dtna 诱导，但 Vdr 沉默消除了这种效应。我们还证明，仅在存在 VDR/RXR 的情况下，VD3 给药可增强肌肉特异性 Dtna 启动子活性。通过定点诱变和染色质免疫沉淀分析，我们已经在成肌细胞中验证了 Dtna 启动子中的 VDRE 位点。因此，我们证明了在体外鼠肌生成分化过程中观察到的 VD3 对 Dtna 的正向调节是由 VDR 介导和特异性的。本研究揭示了 VDR 介导的 Dtna 调节的新机制，这可能在旨在稳定肌肉骨骼疾病中 DAPC 的治疗中得到积极探索。
