25-hydroxyvitamin D upregulates L6 muscle cell differentiation induced by mononuclear cells via the Notch signaling pathway

Abstract During muscle regeneration, myoblasts engage in cross-talk with immune cells to achieve optimal proliferation and differentiation. In this process, cytokines secreted by immune cells are described to modulate the kinetic of muscle differentiation. Taking into account that immune and muscle cells are both targets of vitamin D, we investigated in vitro the impact of 25-hydroxyvitamin D (25(OH)D) on the transcriptional response of muscle cells in presence of mononuclear cells. To address this objective, an in vitro model of co-culture using L6 myogenic cell line and peripheral blood mononuclear cell (PBMC) isolated from rat was used and compared to L6 cultured alone. Cells were treated with 25(OH)D (125 nM) during the 6 days of differentiation. Gene expression of 25(OH)D metabolism actors, muscle differentiation and metabolism markers, and of Notch signaling pathway effectors were studied in L6 cells by qPCR. In mono-cultured L6 cells, a 25(OH)D treatment induced a 3-fold increase ( p < 0.05) in VDR mRNA expression at 24 h while no change in mRNA expression of the muscle differentiation markers i.e. Myog , Myh2 and Des was observed. In the presence of PBMCs, the mRNA expression of these markers was enhanced (27.5 times for myogenin, p < 0.05) resulting in an overexpression of the Notch pathway effectors ( Dll : 6.8-fold and Hes1 : x3.8-fold, p < 0.05). The 25(OH)D counteracted these effects of the PBMCs on L6 gene expression with the exception of the interleukin 6 transcript and protein. In the present study, our in vitro approach demonstrates the importance of immune cells in stimulating muscle cell differentiation. Taken as a whole, the data show that 25(OH)D attenuates in vitro the Notch pathway-dependent effects of immune cells on muscle cell differentiation and energy metabolism.