Role Of Androgen Receptor In Myoblast Proliferation And Differentiation Regulated By Cyclic Mechanical Stretches And Its Mechanisms

Purpose The proliferation and differentiation of satellite cells and myoblasts play important roles in muscle hypertrophy,and cyclic mechanical stretch exerted on satellite cells/myoblasts in vitro is a common way used in studies to simulate exercise-induced muscle hypertrophy.Our previous study found that androgen receptor(AR)might play an important role in the pro-proliferation of 15% stretch and anti-proliferation of 20% stretch on C2C12 myoblasts.In this study,we first confirmed the role of AR in stretch-regulated proliferation of C2C12 cells using AR specific antagonist flutamide.then,compared the difference of proliferation between C2C12 and L6 myoblasts resulted from 15% and 20% cyclic mechanical stretch,and transfected AR overexpression plasmid into L6 myoblasts to verify the effects of AR on the proliferation of stretched(15% and 20% elongation)myoblasts.It has been demonstrated that the effects of AR on protein synthesis were closely related to the activation of insulin-like growth receptor-1(IGF-1)/IGF-1 receptor(IGF-1R)-phosphatidylinositol 3 kinase/protein kinase B(PI3K/Akt)and mitogen-activated protein kinases(MAPKs),however,whether the role of AR in myoblast proliferation regulated by exercise or stretch was associated with the above signal pathways remains unclear.So,we used IGF-1 neutralizing antibody,IGF-1recombinant polypeptide,as well as inhibitors of PI3K/Akt,p38 and ERK1/2 to verify if the effect of AR on mechanical stretch-regulated myoblasts proliferation was achieved by activating PI3K/Akt and MAPKs(p38 and ERK1/2)mediated by IGF-1/IGF-1R?Additionally,we also examined the effect of 15% and 20% stretch on C2C12 and L6 myoblast differentiation,and preliminary explored the roles of AR in myoblast differentiation regulated by cyclic mechanical stretch.This not only provide a new theoretical explanation for the mechanisms of cyclic mechanical stretch in regulating myoblasts proliferation and differentiation,but also a new theoretical support for the increase of skeletal muscle mass and strength by appropriate exercise and reduction by excessive exercise.Methods1.15% and 20% cyclic mechanical stretches(at 0.5 Hz duration for 6 h)exerted on C2C12 and L6 myoblasts were achieved by using Flexcell-5000 tension system.2.Roles of AR in the proliferation of myoblasts regulated by cyclic mechanical stretch were investigated through the following studies:(1)whether AR antagonist with different concentrations(20 ?M,40 ?M and 60 ?M)reduced the pro-proliferation of 15% stretch on C2C12 myoblasts;(2)the effects of 15% and20% stretches on the proliferation of L6 myoblasts,and compared with C2C12 myoblasts under the same stretch condition;(3)effects of transfection exogenous AR into L6 myoblasts on the proliferation of L6 cells subjected to 15% and 20% stretches.3.Mechanisms about the roles of AR in stretch-regulated myoblasts proliferation were explored via the following studies:(1)the effects of 15% and 20% stretches on the IGF-1 secretion(ELISA),IGF-1R expression as well as the expressions and activities of PI3K/Akt and MAPKs(p38 and ERK1/2)(by Western blot)in L6 myoblasts,and compared with C2C12 myoblasts under the same stretch condition;(2)effects of IGF-1 neutralizing antibody on the proliferation of 15% stretched C2C12 myoblasts and IGF-1 recombinant polypeptide on 15% and 20% stretched L6 myoblasts,as well as the expressions and activities of PI3K/Akt,p38 and ERK1/2;(3)effects of PI3K/Akt,p38 and ERK1/2 specific inhibitors on the proliferation of 15% stretched C2C12 and L6 myoblasts;(4)effects of exogenous AR transfection into L6 myoblasts on the above indicators of stretched L6 myoblasts.4.Effects of AR on the differentiation of myoblasts regulated by 15% and 20%stretches were detected by the following studies:(1)the formation of myotube in C2C12 and L6 myoblasts was observed(under optical microscope)on 1st,3rd,5th day after stretches(15% and 20%)finished,to evaluate if differentiation was delay,then the difference between the two myoblasts were compared;(2)the number and diameter of myotubes were examined(MHC immunofluorescence)on the 7th day after stretch finished,and the difference between the two myoblasts were compared.Results1.Roles of AR in myoblasts proliferation regulated by cyclic mechanical stretches1)Compared to 15% stretched C2C12 myoblasts,AR antagonist flutamide blocked the pro-proliferation of 15% stretch in a dose-dependent manner.2)Under resting state,the proliferation rate of L6 myoblasts was lower than C2C12 cells,and exogenous AR transfection into L6 myoblasts promoted the proliferation of L6 cells.3)Similar to C2C12 myoblasts,15% and 20% stretches promoted and inhibited the proliferation of L6 myoblasts,respectively.However,the magnitudes of the regulation on myoblast proliferation existed discrepancy,reflected by lower pro-proliferation of L6 myoblasts induced by 15% stretch than C2C12 cells(about one-third of the latter)and higher anti-proliferation of L6 myoblasts resulted from20% stretch than C2C12 cells(almost twice of the latter).4)Exogenous AR transfection into L6 cells further promoted the pro-proliferative effect of 15% stretch and reversed the anti-proliferation of 20% stretch on L6 cells.The above results confirmed the roles of AR in myoblast proliferation modulated by 15% and 20% cyclic mechanical stretches.1)Roles of IGF-1/IGF-1R in myoblast proliferation regulated by stretches,and its regulation on the activities of PI3 K,p38 and ERK1/2a.15% and 20% stretches increased and decreased IGF-1 secretion and IGF-1 protein levels(our previous study results),respectively.In this study,no detectable IGF-1was secreted from L6 myoblasts regardless of unstretch or stretch(15% and 20%),but 15% and 20% stretches increased and decreased the protein expression of IGF-1R,respectively.b.IGF-1 neutralizing antibody not only inhibited the proliferation of 15% stretched C2C12 myoblasts,but also reduced the activities of PI3 K,p38 and ERK1/2 of15% stretched C2C12 cells in dose-dependent manner.c.Exogenous IGF-1 recombinant polypeptide increased the pro-proliferation of 15%stretch and reversed the anti-proliferation of 20% stretch on L6 myoblasts cells,accompanied with the increases in the protein expression of IGF-1R and the activities of PI3K/Akt,p38,and ERK1/2.2)Roles of PI3 K and MAPKs(p38 and ERK1/2)in myoblast proliferation regulated by cyclic mechanical stretches a.15% and 20% stretches increased and decreased the activities of PI3K/Akt,p38 and ERK1/2,respectively.Inhibitors of PI3 K,p38 and ERK1/2 attenuated the pro-proliferation of 15% stretch on C2C12 myoblasts(our previous study results).b.There existed discrepancy between C2C12 and L6 myoblasts in the activities of PI3K/Akt,p38 and ERK1/2 induced by 15% and 20% stretches,characteristic of(1)15% stretch had no effect on p38 activity of L6 myoblasts while increased p38 activity beyond 3 folds in C2C12 myoblasts;and the magnitude in the increase of ERK1/2 activity in L6 myoblasts was half of that in C2C12 myoblasts;as for PI3 K activity,no difference was found between the two cells.(2)The inhibitory effect of 20% stretch on p38 activity in L6 myoblasts was almost 6.7 folds compared to C2C12 myoblasts,and there was no difference in the inhibitory effect on the activities of PI3 K and ERK1/2 between C2C12 and L6 myoblasts.c.Specific inhibitors of PI3 K and ERK1/2 instead of p38 inhibitor blocked the pro-proliferation of 15% stretch on L6 myoblasts.3)Regulation of AR on IGF-1/IGF-1R,PI3 K,p38 and ERK1/2a.Exogenous AR transfected into L6 cells increased the protein expression of IGF-1R in 15% and 20% stretched L6 myoblasts despite no detectable IGF-1 secretion,accompanied with increasing pro-proliferation of 15% stretch and reversing anti-proliferation of 20% stretch.b.Exogenous AR transfection transfected into L6 cells enhanced the activities of PI3K/Akt,p38 and ERK1/2 in stretched L6 myoblasts.These results indicated that the effects of AR on myoblasts proliferation regulated by 15% and 20% cyclic mechanical stretches were mediated by IGF-1/IGF-1R,thus activating the activations of p38 and ERK1/2 in 15% stretch and inhibiting p38 activation in 20% stretch.2.Mechanisms about the roles of AR in myoblast proliferation regulated by cyclic mechanical stretches stretches1)Effects of cyclic mechanical stretches on the differentiation(differentiation delay,and the number and diameter of myotube)of C2C12 and L6 myoblasts Compared with control myoblasts,15% and 20% stretches had no effect on the beginning time of myotube formation and myotube diameter in both C2C12 and L6 cells cultured in differentiation medium.However,15% stretch increased while 20%stretch decreased the number of myotube in differentiated C2C12 and L6 myoblasts.2)Roles of AR in myoblast differentiation regulated by cyclic mechanical stretches The increase extent of myotube number induced by stretches was different between C2C12 and L6 cells,reflected by more myotube number by 15% stretch in L6 and less myotube number by 20% stretch in L6,compared to C2C12 cells.These results suggested that AR might involve in stretch-regulated myoblast differentiation,such as influencing the formed myotube number.3.Roles of AR in myoblast differentiation regulated by cyclic mechanicalConclusion1.15% stretch promoted while 20% stretch inhibited the proliferation of C2C12 myoblasts and L6 myoblasts,indicated the universality of cyclic mechanical stretches on myoblasts proliferation,but there existed a significant discrepancy in the extent of alternation between the two myoblasts.2.AR plays an important role in myoblast proliferation regulated by 15% and 20%stretches.Even in L6 myoblasts lacking AR expression,exogenous AR transfected into L6 cells also enhanced the proliferation of stretched L6 cells.3.The role of AR in the pro-proliferation of 15% stretch on myoblasts was mediated by increasing IGF-1 secretion and IGF-1R protein expression,thus activating the activations of p38 and ERK1/2,while the effect of AR in 20% stretch-induced anti-proliferation was achieved by inhibiting IGF-1/IGF-1R-p38 pathway.4.15% stretch enhanced,while 20% stretch reduced myotube number of C2C12 and L6 myoblasts,but the modulation extent was different between the two myoblasts,suggested a possible effect of AR in stretch-regulated myoblasts differentiation.