1,25‐Dihydroxyvitamin D3 Mediates L6 Myoblast Differentiation via Vitamin D Receptor (VDR)

Abstract

In our previous study, it was demonstrated that skeletal muscle fiber type composition was changed by dietary supplementation of 25‐hydroxyvitamin D3 in growing rats. Potential mechanisms of 25‐hydroxyvitamin D3, a precursor of 1,25‐dihydroxyvitamin D3 (active form), have been suggested in previous studies that VDR was involved with mouse C2C12 myoblasts. However, it has not been explained in rat L6 myoblasts. In this study, we examined the role of VDR in skeletal muscle differentiation and fiber type‐specific gene expression by using short hairpin RNA (shRNA) for VDR knockdown in rat L6 myoblast. L6 myoblasts transfected either with scrambled or shVDR vector were treated with vehicle (CON), 1 pM, 10 pM, or 100 pM 1,25‐dihydroxyvitamin D3 (VitD) for 8 days of differentiation. VDR knockdown was confirmed by Western blot. And myogenic regulatory markers and fiber type‐specific gene expression levels were determined by qPCR. VDR protein was increased by VitD at 10 pM and 100 pM compared to that of CON (1.46 and 1.89 folds, respectively, p<0.05). However, shVDR was effective in suppressing VDR up to 10 pM as VDR protein levels were decreased to 34 %, 24 %, and 39 % (CON, 1 pM, and 10 pM, respectively, p<0.05) compared to scrambled vector. And at 100 pM VitD, VDR was not repressed by shVDR compared to that of scrambled vector. Relative gene expression levels of both MyoD and Myogenin were downregulated by VDR knockdown in CON (0.56 and 0.36 folds, respectively, p<0.05), however, VitD treatment abolished the effects. Fiber type‐specific genes were also modulated by VDR knockdown. Gene expression level of Myh7, dominantly expressed gene in slow‐twitch fiber, was suppressed to 37 %, 29 %, and 43 % (CON, 1 pM, and 10 pM, respectively, p<0.05) by VDR knockdown compared to scrambled vector, but was restored at 100 pM. Myh2, which consists of fast‐twitch fiber, was also downregulated by VDR knockdown at CON (0.57 folds, p<0.05), but its level was restored by VitD treatment. The present study demonstrates that VDR affects mRNA levels of the genes involved in L6 myoblast differentiation and fiber type specificity. Also, the VDR knockdown effect was partially modulated by VitD. It could suggest that the effects of VitD on myogenic differentiation might be partially mediated through VDR.