The Effects Of Mammalian Target Of Rapamycin Signaling Pathway On Proliferation And Differentiation Of Goat Skeletal Muscle Satellite Cells

Skeletal muscle differentiation refers to mononuclear muscle cells exiting the cell cycle and fusing into multinucleated myotubes. This process is mainly regulated by the myogenic differentiation 1 (MyoD1), which regulate the myogenin expression. Moreover, myocyte enhancer factor-2 (MEF2) combines to regulating regions of skeletal muscle-specific genes to enhance the genes expression. Myoblasts proliferation and differentiation are regulated by nutrients, hormones, and self-secreted factors. The metabolism signaling caused by leucine as a nutrient, promotes mammalian target of rapamycin (mTOR) transferred to lysosome surface, and then mTOR could be activate by Ras homolog enriched in brain (Rheb). Meanwhile, insulin like growth factor 1 (IGF-1) inhibit tuberous sclerosis complex 2 (TSC2), leads to release mTOR. The activated mTOR phosphorylated substrates, such as protein phosphatases 2A (PP2A), eukaryotic initiation factor 4E-binding protein-1 (4E-BP1) and ribosomal protein S6 kinase-1 (S6K1), to stimulate protein synthesis and to promote the hypertrophy of skeletal muscle. Moreover, PP2A, 4E-BP1, and S6K1 involved in different signaling pathway to stimulate skeletal muscle development. The mechanism about mTOR activation is clear in skeletal muscle development recently, but it is unclear how to regulate MEF2 and MyoDl by downstream factors of mTOR, and then stimulate skeletal muscle-specific genes expression.In this study, goat Skeletal Satellite cells (gSSCs) have been used 2-step enzymatic digestion to isolate and differential adhesion methods to purify, and then identified the cells and induced differentiation. According to the results,0.1% type Ⅰ collagenase and 0.25% trypsin 2-step digestion method was the efficient method for the isolation of gSSCs. Primary gSSCs begin to adhere after 24 h and are adherent fully after 72 h. Immunofluorescence staining showed that gSSCs expressed Desmin, alpha-Actin, MyoD1, Myf5 and PAX7. On the first day of gSSCs induction, the cells grew together; on the third day, gSSCs fused gradually; and from the fifth to seventh day, myotubes formed and formed branches, and Immunocytochemical staining showed expression of FMM.gSSCs from above experiments, as cell model, was used to disclose the regulation of MEF2 by mTOR. When gSSCs were treated with increasing concentrations of PP242 for 3 h, S6K and MEF2C phosphorylation levels decreased; there was no change in the amounts of S6K and MEF2C in cells. S6K and MEF2C activity was greatly suppressed following 3-h treatment with 100 nM PP242. In MEF2C co-IP product, proteins obviously decreased in the 50-kD band after PP242 treated; and 8# protein ILK is an mTOR pathway component. Following 3-h treatment with 100 nM PP242, ILK protein expression was not significantly different; ILK activity increased approximate 1.7-fold (1.6816±0.061, P<0.05) compared to control (1.0±0.054, P>0.05). Moreover, MEF2C co-localized with ILK in gSSCs. ILK activity decreased gradually as the Cpd22 concentration increased, but ILK expression was not affected. ILK activity was sharply inhibited following 3-h treatment with 1000 nM Cpd22, and was approximate 1.6 times (1.5526±0.054, P<0.05). To some extent, inhibitting the ILK activity blocked mTOR signaling pathway to regulate MEF2C. ILK promotes MEF2C dephosphorylation,in vitro, and MCK expression is regulated by mTOR and ILK. According to the drug treatment results, ILK sh-RNA both up-regulated MEF2C activity and MCK expression.When 100 nM PP242 was added during induction, gSSCs differentiation was greatly inhibited, cell fusion occurred sporadically, and there were no obvious myotubes. In addition, only 0.73% of gSSCs fused and expressed MHC. S6K expression levels did not change significantly when gSSCs were induced for 24 h and mTOR was inhibited by PP242, but S6K activity was inhibited. MyoD1 expression levels and activity were not affected, but MyoG expression (0.3892 ± 0.037, P<0.05, Control:1.0034 ± 0.056, P>0.05) and mRNA levels (0.3620 ± 0.019, P<0.05, Control:1.0054 ± 0.057, P>0.05) were decreased. In MyoD1 co-IP proteins, the protein content at the 90-kDa band was significantly higher than that in the control, and then 6# protein, STAT1, and MyoD1 colocalized in the nucleus and cytoplasm, and mutual co-IP was positive. In gSSCs inducting differentiation,24-h inhibition of mTOR activity by PP242 did not affect STAT1 expression and phosphorylation levels, but STAT1 intranuclear content was increased by about 2.3-fold (Fig.2F and G: 2.3304 ± 0.049, P<0.05; Control:1.0023 ± 0.065, P>0.05); MyoDl intranuclear content did not change significantly. STAT1 expression levels were drastically reduced following STAT1 RNAi during induction (0.3016 ± 0.031, P<0.05; Control: 1.0134 ± 0.067, P>0.05). There were no significant changes in MyoD1 expression and phosphorylation levels, but MYOG mRNA levels were significantly increased (2.2901 ± 0.081, P<0.05). PP242 transcriptional regulation of MyoG was blocked following STAT1-shRNA transfection (1.765 ± 0.058, P<0.05) but was not completely suppressed.Basing on above results, in this study, gSSCs isolation, purification, cultivation, and identification had been established, and it was been disclosed that mTOR signaling pathway up-regulated MEF2 and down-regulated ILK, ILK regulated MEF2 negatively, and mTOR activated MyoD1 transcription activation via decreased STAT1 intranuclear content. The results indicate that mTOR signaling pathway regulate MEF2 activation and transcription, and MyoDl transcription activation, and further stimulate gSSCs proliferation and differentiation.