A Novel Mechanism Of Androgen Receptor Influences The Proliferation Of Myoblasts Regulated By Cyclic Mechanical Stretch-Myostatin

ObjectiveOur previous study has demonstrated that androgen receptor(AR)played an important role in the proliferation of myoblasts regulated by cyclic mechanical stretch,and this role was associated with activations of pro-proliferation signaling pathways(p38 and ERK1/2).In fact,AR’s role in the pro-proliferation of myoblasts not only achieved by activating signaling pathways that positively regulated cell proliferation,but also could realize via inhibiting signaling pathways that negatively regulated proliferation.Myostatin(MSTN),a classical negative regulator of skeletal muscle mass,has anti-proliferation and inhibition of protein synthesis.Most studies have demonstrated that decreasing MSTN expression or activity promotes satellite cells/myoblasts proliferation.However,whether AR’s role in the pro-proliferation of cyclic mechanical stretch on myoblasts was achieved through inhibition of MSTN signaling remains unknown.Phosphoinositide 3-kinase(PI3K)/protein kinase B(Akt)/mammalian target of rapamycin(mTOR)is an important signaling pathway regulating cell proliferation and growth.Our previous study demonstrated that the pro-proliferation and promotion of protein synthesis of cyclic mechanical stretch on myoblasts were related to the activation of PI3K/Akt/mTOR signaling pathway.In the present study,we aimed to confirm the regulation of AR on MSTN in myoblasts through comparing the differences of MSTN expression level between C2C12(with AR expression)and L6(without AR expression)myoblasts,determined the effects of AR antagonists treated in C2C12 myoblasts and transfection of AR overexpression plasmid into L6 myoblasts on the expression of MSTN.Then,using MSTN-neutralizing antibodies to clarify the role and mechanism of MSTN in myoblast proliferation regulated by cyclic mechanical stretch(whether achieved through the PI3K/Akt/mTOR signaling pathway),thus providing a new theoretical explanation for the role and mechanism of AR in myoblast proliferation modulated by cyclic mechanical stretch.Methods1.Under unstretched and 15% stretched conditions,whether AR’s in myoblast proliferation was related to MSTN(1)Under un-stretched condition,the protein expression of MSTN in C2C12 and L6 myoblasts was detected by Western blot,and the difference between the two cells was compared.(2)Under un-stretched condition,C2C12 cells were divided into control(CON)and AR antagonist groups(concentrations of flutamide were 20 μM,40 μM and 60μM),and L6 cells were divided into control(CON)and AR overexpression groups(AR overexpression plasmid transfection).Under 15% stretched condition,L6 cells were divided into control group(CON),AR overexpression group,15% stretch group(Str),and 15% stretch plus AR overexpression group.After treatments,cell proliferation was determined by CCK8 and the expression of MSTN was detected by Western blot.2.The role and mechanisms of MSTN in mechanical stretch-regulated myoblast proliferation(1)To determine of the optimum concentration of MSTN neutralizing antibody(Myo-AB): un-stretched C2C12 myoblasts were divided into control(CON)and MSTN neutralizing antibody administrated group,and the concentrations of MSTN neutralizing antibody were set at set at 1 μg/ml,2 μg/ml and 3 μg/ml according to manufacturer’s instructions.After treatments,cell proliferation was detected by CCK8 to determine the optimal concentration of MSTN-neutralizing antibody.(2)C2C12 myoblasts were divided into control group(CON),MSTN neutralizing antibody administration group(Myo-AB),15% stretch group(Str),and 15% stretch plus MSTN neutralizing antibody administration group(Str + Myo-AB).After stretch finished,cell proliferation was determined by CCK8 and the expressions of PI3 K,Akt,mTOR,p-PI3 K,p-Akt,and p-mTOR were examined by Western blot.Results1.Under un-stretched condition,AR’s role in myoblast proliferation was associated with MSTN(1)Under un-stretched condition,the expression level of MSTN protein was higher in C2C12 cells(with AR expression)than that in L6 cells(without AR expression)(P<0.05).(2)Under un-stretched condition,AR antagonist Flutamide inhibited the proliferation of C2C12 myoblasts(P < 0.05),and decreased the protein level of MSTN(P < 0.01).(3)Under un-stretched condition,transfection with AR overexpression plasmid into L6 myoblasts promoted the proliferation,and accompanied by the increase of MSTN expression(P<0.01).2.AR’s role in myoblast proliferation regulated by cyclic mechanical stretch was related to MSTN(1)Compared with Con group,15% stretch significantly increased AR levels in L6 myoblasts and also decreased MSTN protein levels(P < 0.05),while exogenous transfection with AR overexpression plasmid reversed the inhibition of MSTN by15% stretch(P < 0.05).(2)Exogenous transfection with AR overexpression plasmid further enhanced the proproliferative effects of 15% stretch on L6 cells and increased MSTN protein level(P<0.01).The above results indicated that AR played an important role in myoblast proliferation under un-stretched and stretched conditions,which was associated with its positive regulation on MSTN.3.MSTN regulated the proliferation of C2C12 cells by inhibiting the PI3K/Akt/mTOR signal pathway under un-stretched and stretched conditions(1)Under un-stretched condition,compared with Con group,the pro-proliferation of Myo-AB at 3 μg/ml concentration on C2C12 myoblasts was the most significant(P <0.05),thus we used 3 μg/ml concentration in the following experiments.(2)Inhibiting MSTN signaling utilizing neutralizing antibodies promoted the proliferation of un-stretched C2C12 cells(P < 0.05),and further enhanced the proproliferation of 15% stretch(P < 0.05).(3)15% stretch significantly increased PI3 K,Akt,and mTOR activities in C2C12myoblasts(P<0.01),and MSTN neutralizing antibody further enhanced the increases of PI3 K,Akt,and mTOR activities by 15% stretch(P < 0.01).The above results indicated that MSTN’s role in the proliferation of C2C12 myoblasts might be achieved through negative regulation of PI3K/Akt/mTOR pathway.ConclusionAR promoted the proliferation of unstretched and 15% stretched myoblasts,at the same time,AR inhibited the PI3K/Akt/mTOR pathway probably by positively regulating MSTN levels,thus monitoring the pro-proliferative effects of AR on myoblasts and avoiding excessive proliferation.