1α,25(OH)2D3-glycosides from Solanum glaucophyllum leaves extract induce myoblasts differentiation through p38 MAPK and AKT activation

Abstract

We have previously shown that Solanum glaucophyllum leaf extract (SGE) increases VDR protein levels and promotes myoblast differentiation. Here, we investigated whether p38 MAPK and AKT are involved in SGE actions. Cell-cycle studies showed that SGE prompted a peak of S-phase followed by an arrest in the G0/G1-phase through p38 MAPK. Time course studies showed that p38 MAPK and AKT phosphorylation were statistically increased by SGE (10 nM) or synthetic 1α,25(OH)₂D₃ (1 nM) treatment. Furthermore, p38 MAPK and AKT inhibitors, SB203580 and LY294002 respectively, suppressed myoblasts fusion induced by SGE or synthetic 1α,25(OH)₂D₃ We have also studied differentiation genes by qRT-PCR. myoD1 mRNA increased significantly by SGE (24-72 h) or 1α,25(OH)₂D₃ (24 h) treatment. mRNA expression of myogenin also increased upon SGE or 1α,25(OH)₂D₃ treatment. Finally, MHC2b mRNA expression, a late differentiation marker, was increased significantly by both compounds at 72 h compared to control. Taken together, these results suggest that SGE, as synthetic 1α,25(OH)₂D₃, promotes myotube formation through p38 MAPK and AKT activation.

Keywords: 1α,25(OH)2D3-glycosides; AKT; C2C12 cells; MHC2b; MyoD1; Myogenin; P38 MAPK; Proliferation.

Fig. 1.

Cell cycle progression of C2C12 skeletal muscle cells after SGE treatment: role of p38 MAPK. A. This panel shows a representative histogram and DNA quantification from three independent experiments showing the percentage of cells in G0/G1-, S- and G2/M-phases (Y-axis label) ±s.d. Data were analyzed by one way ANOVA, followed by t-test, **P<0.01. B. This panel presents a representative histogram and DNA quantification from four independent experiments expressing the percentage of cells in G0/G1-, S- and G2/M-phases (Y-axis label) ±s.d. in combined bars graphs. Different letters mean statistical differences with Tukey test.

Fig. 2.

SGE regulates AKT and p38 MAPK phosphorylation at the beginning of myogenesis. C2C12 cells were cultured in GM. To begin the differentiation, this medium was withdrawn and then replaced by DM. Treatment was started by adding 10 nM of SGE or 1 nM of synthetic 1α,25(|OH)₂D₃ (1,25D) in DM during different periods of time (1–48 h). Western blots were performed with anti p-AKT, total AKT, p-p38 MAPK, and p38α and tubulin antibodies. Tubulin was used as loading protein marker. Representative blots from three independent experiments (upper panel) and their quantification (lower panel) are shown. Protein bands quantification was done using ImageJ. Results were then represented in bar graphs as phosphorylated protein normalized with its corresponding total from treated conditions and referred to control (Fold) (lower panel). Significant differences between conditions at each time point were analyzed by Student’s t-test, **P<0.01.

Fig. 3.

SB203580 and LY294002 inhibitors prevent myoblast differentiation induced by SGE or synthetic 1α,25(OH)₂D₃. (A) Fluorescence photomicrographs show C2C12 cells undergoing differentiation for 72 h. Cells were treated with vehicle (Ctrl, control), 1 nM of 1α,25(OH)₂D₃ (1,25D), or 10 nM of SGE, or in presence of SB203580 (10 µM), or LY294002 (10 µM), as described in the text. (B) Fusion index was calculated and represented in bar graphs from three independent experiments. Significant differences between conditions were analyzed by one way ANOVA followed by Bonferroni Test. Different letters indicate statistical differences among groups for each condition (P<0.05).

Fig. 4.

Genes regulation by SGE or synthetic 1α,25(OH)₂D₃ during onset of differentiation. C2C12 cells were treated with SGE (10 nM), 1α,25(OH)₂D₃ (1,25D, 1 nM), or vehicle alone (ctrl) in DM in time course experiments (24–72 h). Total RNA was extracted and reverse transcribed and data analysis was performed for each gene expression by qRT-PCR. Bar graphs show quantitative results from three experiments performed in duplicate and expressed as a ratio between mRNA of each gene (A) myoD1, (B) myogenin, (C) MHC2b in DM under control conditions (ctrl) or treated (1,25D or SGE) previously normalized to GAPDH mRNA levels and to GM (time 0). The results were then represented in bar graphs. Significant differences between control and treated (SGE or 1,25D) conditions at each time point were analyzed by Student's t-test. ns: not significant. *P<0.05 and **P<0.01.