The Role And Mechanisms Of Androgen Receptor On The Proliferation Of Skeletal Muscle Cells C2C12 Resulted From Cyclic Mechanical Stretch

Object:Androgens exert the promotion effects on the protein synthesis and proliferation of skeletal muscle cells as well as the muscle mass through the mediation of androgen receptor(AR). Mitogen-activated protein kinase(MAPK) pathway(such as p38,ERK1/2), Phosphatidylinositol 3-kinase(PI3K) pathway and IGF-1 also play important roles in regulating the proliferation of skeletal muscle cells. The purpose of our study was to detect the effects of AR on the proliferation of skeletal muscle cells C2C12 resulted from cyclic mechanical stretch and its mechanisms: the roles of MAPK(p38, ERK1/2), PI3 K and IGF-1.Methods:Mice skeletal muscle cell line C2C12 cells were randomly divided into five groups: control, 15% stretch for 6 h, 15% stretch for 8 h, 20% stretch for 6 h, 20%stretch for 8 h. The mechanical stretch of the C2C12 cells were completed using the Flexcell tension incubator for 15% or 20% of elongation, 6h or 8 h of duration and 0.5Hz of frequency, while control group undertook no mechanical stretch. At the end of the stretch, CCK8 and ELISA kits were used to determine the proliferation of C2C12 cells and the concentration of IGF-1 in the culture media, respectively. 24 hours after the stretching, the cells were collected and detected the expressions of AR, IGF-1R as well as the expressions and activations of p38, ERK1/2 and PI3 K by Western blot.For confirming the effects of p38, ERK1/2 and PI3 K on the AR-regulated proliferation of C2C12 cells, the specific inhibitors of p38(SB203580), ERK1/2(U0126) and PI3K(LY294002) were used to inhibit the expression or activity of p38,ERK1/2 and PI3 K, then the protein expressions of AR in C2C12 cells after 6 h of15% or 20% stretch were determined by Western blot.For clarifying the roles of IGF-1 on the AR-regulated proliferation of C2C12 cells, the cells were incubated with multiply concentrations of IGF-1 recombinant proteins before 6 h of 15% or 20% stretch, then the stretched cells were collected and the protein expressions of AR and IGF-1R as well as the expressions and activations of p38, ERK1/2 and PI3 K were determined by Western blot.Results:1. The effects of cyclic mechanical stretch on the proliferation of C2C12 cells Compared with control group, the proliferation of C2C12 cells were significantly increased by 15% stretched(p<0.05), while decreased significantly resulted from 20%stretched(p<0.05).2. The effects of cyclic mechanical stretch on the protein expressions of AR of C2C12 cells Compared with control group, 6 h of 15% stretch induced significant increases of the protein levels of AR in C2C12 cells(p<0.05), while 20% stretch for 6 h or 8 h significantly decreased the protein levels of AR in C2C12 cells(p<0.05).3. The effects of cyclic mechanical stretch on the expressions and activations of MAPK(p38, ERK1/2) and PI3 K in C2C12 cells1) Compared with control group, the activations of p38(6 h, p<0.01; 8 h, p<0.05) and ERK1/2(6 h, p<0.01) in C2C12 cells were significantly increased by 15% stretch,while the activation of ERK1/2(p<0.01) rather than p38 activation(p>0.05) was decreased significantly induced by 20% stretch for 6 h and 8 h. No detectable change in the total protein levels of p38 and ERK1/2(p>0.05) of the C2C12 cells was found by all the stretches.2) Compared with control group, the activation of PI3 K in C2C12 cell was increased significantly after 6 h of 15% stretch(p<0.01), while significant decreases were found in 20% stretch for 6 h and 8 h(p<0.01). The total protein levels of PI3 K of the C2C12 cells were unchanged in all the stretches.4. The effects of specific inhibitors of p38, ERK1/2 and PI3 K on the expressions of AR in C2C12 cells exposed to the cyclic mechanical stretch Compared with stretch control group, the protein levels of AR in the C2C12 cells exposed to 6 h of 15% or 20% stretch were decreased after inhibiting the expressions or activities of p38, ERK1/2 and PI3 K by the specific inhibitors of p38(SB203580),ERK1/2(U0126) and PI3K(LY294002)(p38, PI3K: 15%, p<0.01, 20%, p<0.05;ERK1/2, both p<0.05), which indicated that the mechanical stretch-regulated AR expressions was at least partly through the mediations of MAPK(p38, ERK1/2) and PI3 K.5. The influences of cyclic mechanical stretch on the secretion of IGF-1 in C2C12 cells Compared with control group, IGF-1 concentrations in culture media were increased significantly by 6 h of 15% stretch(p<0.01), while significant decreases of IGF-1 concentrations were induced by 8 h of 20% stretch in C2C12 cells(p<0.05).6. The effects of IGF-1 on the protein levels of AR and IGF-1R as well as the protein levels and activations of p38, ERK1/2 and PI3 K in C2C12 cells exposed to the cyclic mechanical stretch Multiply concentrations of IGF-1(26.3 m M, 65.8 m M and 131.6 m M) were incubated with C2C12 cells, then the protein levels of AR and IGF-1R as well as the protein levels and activations of p38, ERK1/2 and PI3 K in C2C12 cells were detected after 6 h of 20% stretch. 65.8 m M and 131.6 m M of IGF-1 significantly increased the protein levels of AR(p<0.01) and the activations of PI3K(p<0.05, p<0.01respectively), and 131.6 m M IGF-1 significantly increased the activations of p38 and ERK1/2(both p<0.01). All the IGF-1 caused no change on the total protein levels of p38, ERK1/2 and PI3 K.Conclusion:1. Cyclic mechanical stretch of 15% elongation promoted the proliferation of C2C12 cells, while 20% mechanical stretch inhibited the proliferation of C2C12 cells.2. The proliferation of C2C12 cells resulted from cyclic mechanical stretch was related to the stretch-induced changes of AR protein expressions.3. Cyclic mechanical stretch changed the secretion of IGF-1 to regulate the activations of MAPK(p38, ERK1/2) and PI3K/Akt, thus changing the protein expressions of AR in C2C12 cells.