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.
Suppl. Figure 1: Time course of C2C12 and H2K-mdx52 differentiation in DM. Upper panel: C2C12 differentiation in the presence of 1.5 % HS medium. Lower panel: H2K-mdx52 cell differentiation in the presence of 2.5 % HS medium. In both cases, cells were placed in DM when culture reached 80 % of confluence (day 0). After 48 hrs, early myotubes were visible and on day 5 mature myotubes were visible in the culture. Scale bar 200 µm
Suppl. Figure 2: VD3 upregulates endogenous Dtna variant gene expression resulting in isoforms 1,2 and 3 in C2C12 early myotubes. mRNA levels of endogenous Dtna transcript variants 1,2,3 and canonical in the presence or absence of VD3. Vdr and Cyp24a1 genes are used as positive controls to confirm successful VD3 delivery. C2C12 cells were treated with vehicle (100% ethanol) or VD3 (10 nM) for 48 hours in presence of differentiation medium. Canon: canonical sequence of Dtna1 (longest transcript that translates to the 84 kDa isoform in mouse). Primers designed for Dtna1 transcript variant 1-6 and 9-11 or Dtna1 transcript 2 which was specifically isolated from our C2C12 cells, gave identical results. Data are expressed as mean ± SEM. *P ≤ 0.05 **P≤ 0.01 ***P ≤ 0.001, ****P ≤ 0.0001 n = 3 individual experiments. Comparisons between groups were done using 2-way ANOVA with Sidak’s post-hoc test.
Suppl. Figure 3: DTNA upregulation in H2K-mdx52 is VDR-dependent. A) DTNA levels are mildly but not significantly elevated in H2K-mdx52 cells mock transfected with pFLAG, treated with 100 nM daily for two days and harvested 72 hrs after the first VD3 administration (day 3). B) A VD3 dose response assay in H2K-mdx52 cells mocktransfected with pFLAG elevated endogenous VDR levels up to 7 fold but failed to elevate DTNA levels. Data are expressed as mean ± SEM. *P ≤ 0.05 **P≤ 0.01 ***P ≤ 0.001. Comparisons between two groups was done by unpaired student t-tests and between four groups were performed using one-way ANOVA with Tukey post-hoc test n = 2 individual experiments
Suppl. Figure 4: Dtna-M promoter upregulation is VDR-dependent in Neuro2a cells A) Dual luciferase assay in absence of endogenous VDR expression in Neuro2a cells indicates high Dtna-B basal promoter activity that is not altered upon administration of VD3. LB activity promoter is not detectable in these cells B) Dual luciferase assay after overexpression of VDR and RXR in Neuro2a cells indicates increased Dtna-M promoter activity in the presence of VD3. Dual luciferase activity was measured in Neuro2a cell monolayers incubated in GM in the presence of vehicle or VD3 (10 nM) for 24 hours and firefly luciferase activity was normalized to renilla luciferase activity. C) Western blotting performed in Neuro2a cells transfected with 250 ng mVDR: RXR and harvested after 48 hrs of VD3 administration indicates low levels of endogenous VDR and successfully overexpressed VDR as demonstrated by quantitative analysis. Data are expressed as mean ± SEM. *P < 0.05. Comparisons between two groups was done by unpaired student t-tests and between four groups were performed using one-way ANOVA with Tukey post-hoc test n = 3 separate experiments.