1,25(OH)2D3-glycosides from Solanum glaucophyllum leaves extract induce skeletal muscle cells differentiation by p38 MAPK and Akt regulation

IRAZOQUI, P; BUITRAGO, C; GONZALEZ PARDO, V; BACHMANN, H; DE GENARO, P; RUSSO DE BOLAND, ANA

Barcelona

Workshop; The 21st Workshop on Vitamin D; 2018

Solanum glaucophyllum leaves extract (SGE) contains 1,25-dihydroxycholecalciferol glycosides which provide the organism with 1,25-dihydroxycholecalciferol due to the presence of endogenous glycosidases. We have previously shown that SGE promotes myoblast differentiation. In this work, we further studied the involvement of p38 MAPK and AKT in murine skeletal muscle cells differentiation induced by SGE. Cell cycle studies showed that SGE stimulation prompts a peak of S-phase followed by an arrest in the G0/G1-phase, events which were dependent on p38 MAPK. Time course studies showed that p38 MAPK phosphorylation statically increased at the beginning of differentiation (1 h) and remains activated for 24 hours to significantly decrease later upon SGE (10 nM) or 1α,25(OH)2D3 (1 nM) treatment. AKT phosphorylation also increases significantly during the first hour and then decreases (24-48 h) with both compounds. Furthermore, LY294002, inhibitor of PI3K pathway, suppresses myoblasts fusion into myotubes induced by SGE or synthetic 1α,25(OH)2D3, supporting the role of AKT in muscle differentiation as well as p38 MAPK. We have also confirmed preliminary studies related to gene expression of myoD1, myogenin and MCH2b during the differentiation phase (24-72 h) by qRT-PCR. myoD1 mRNA control levels remained low throughout the period of analysis and increased significantly by SGE (24-72 h) or 1α,25(OH)2D3 (24 h) treatment. mRNA expression of the differentiation marker myogenin also increased upon SGE or 1α,25(OH)2D3 treatment, however, myogenin mRNA induction by 1α,25(OH)2D3 raised later (48 h) than SGE (24 h). Finally, MHC2b mRNA expression, a late differentiation marker, increased significantly with both compounds at 72 h compared to control. Since AKT is involved in myotube formation, we investigated its participation in MHC2b expression in additional experiments. We found that MHC2b expression induced by SGE and 1α,25(OH)2D3 was blocked by LY294002. Taken together, these results suggest that SGE as well as synthetic 1α,25(OH)2D3, promote myotube formation through p38 MAPK and AKT activation.