Under un-stretched state, AR deficiency slows down the proliferation rate of myoblasts
We first confirmed that AR expressed in C2C12 myoblasts whereas no detectable AR was examined in L6 myoblasts (Fig. 1A), consistent with previous studies. Then the proliferation of C2C12 and L6 cells under un-stretched state were compared, and observed that the proliferation rate of L6 cells was significantly lower than that in C2C12 cells, as evidenced by the number of L6 myoblasts were about 80.6% and 72.5% of C2C12 myoblasts at 24 h and 48 h after seeding, respectively. To further clarify AR’s roles in myoblasts proliferation, we explored the impacts of exogenous transfection AR into L6 myoblasts and AR knockdown in C2C12 myoblasts on their proliferation, and found that the proliferation was promoted in L6 myoblasts by transfection with AR overexpression plasmid (Fig. 1B) while inhibited in C2C12 myoblasts by AR knockdown utilizing siRNA AR (Fig. 1C). These results demonstrated that AR played an important role in myoblasts proliferation under un-stretched state.
The degrees of 15% stretch-induced pro-proliferation and 20% stretch-induced anti-proliferation in L6 myoblasts are different from that in C2C12 myoblasts
Our previous work has reported that 15% and 20% stretches promoted and inhibited the proliferation of C2C12 myoblasts (with AR expression), respectively [17]. In the current study, the proliferation of L6 myoblasts (without AR expression) undertaken 15% or 20% stretch was detected, and we were surprised to found that 15% and 20% stretches promoted and inhibited (rather than no influence) the proliferation of L6 myoblasts, respectively; but compared to C2C12 myoblasts, 15% stretch-induced pro-proliferative effect on L6 myoblasts was much lower (less than half of C2C12 cells), while 20% stretch-induced anti-proliferation on L6 myoblasts was obvious higher (above 2-fold of C2C12 cells) (Fig. 2), which indicated an important role of AR in stretch-modulated proliferation of myoblasts.
AR overexpression promotes 15% stretch-induced pro-proliferation, and reverses 20% stretch-induced anti-proliferation in L6 myoblasts
AR protein could be obviously detected in L6 myoblasts after transfection with AR overexpression plasmid (Fig. 3). AR overexpression further enhanced the proliferation of 15% stretched L6 myoblasts by approximately 50% (Fig. 3A), reaching similar pro-proliferation degree of 15% stretched C2C12 myoblasts (Fig. 3B), and reversed the anti-proliferation of 20% stretch on L6 myoblasts (Fig. 4).
AR's role in 15% stretch-induced pro-proliferation is fulfilled via activating p38 and ERK1/2 rather than PI3K/Akt
To clarify relevant signal pathways involved in AR's roles in 15% stretch-induced pro-proliferation, the increased degrees of activities of PI3K/Akt, ERK1/2 and p38 were compared between C2C12 and L6 myoblasts subjected to 15% stretch, and we found that there was no difference in PI3K activity between the two cells but the increment of ERK1/2 activity in L6 myoblasts was lower (almost half) than that in C2C12 myoblasts, and what’s more interesting was that p38 activity had no change in 15% stretched L6 cells but about 2 folds increase in 15% stretched C2C12 myoblasts (Fig. 5A). These results indicated that activated ERK1/2 and p38 (especially p38) but not PI3K were associated with AR's pro-proliferative effect on 15% stretched myoblasts.
Furthermore, the activations of those above molecules in L6 cells were detected after AR overexpression plasmid transfection, and we found that accompanied with proliferation increase of L6 cells (Fig. 3), the activities of p38 and ERK1/2, especially p38 activity were remarkable increased (Fig. 5C), demonstrating AR's role in promoting the proliferation of 15% stretched myoblasts was via activating p38 and ERK1/2. In addition, although there was no difference in 15% stretch-induced increase in PI3K/Akt activations between C2C12 and L6 myoblasts, exogenous AR could further increase the activities of PI3K/Akt in 15% stretched L6 myoblasts (Fig. 5B).
Effect of decreased AR on 20% stretch-induced anti-proliferation is mediated by inhibiting p38 instead of ERK1/2 and PI3K/Akt
To identify relevant signal molecules involved in decreased AR's roles in 20% stretch-induced anti-proliferation on myoblasts, we compared the discrepancy between C2C12 and L6 myoblasts subjected to 20% stretch in the activities of PI3K, p38 and ERK1/2, and found that the attenuated degree of p38 activity by 20% stretch was higher in L6 myoblasts than that in C2C12 myoblasts (approximately 5.7 folds), while the decreases of PI3K and ERK's activities resulted from 20% stretch were similar to that in C2C12 cells (Fig. 6A), which indicated that the activation of p38 but not PI3K and ERK was related to decreased AR's anti-proliferation on 20% stretched myoblasts.
Furthermore, transfection with AR overexpression plasmid into 20% stretched L6 myoblasts led to an enormous enhancement in p38 activity while mild increases in the activations of PI3K, Akt and ERK1/2 (Fig. 6B and 6C), demonstrated that the anti-proliferative effect of decreased AR on 20% stretched myoblasts was via inhibiting p38 activation (instead of ERK1/2 and PI3K's activations).
AR overexpression up-regulates IGF-1R levels but not IGF-1 secretion of L6 myoblasts subjected to 15% or 20% stretch
IGF-1 secretion from L6 myoblasts undertaken 15% or 20% stretch were determined after transfection with AR overexpression plasmid, and still no detectable IGF-1 was secreted. For IGF-1R, significant increase and decrease of IGF-1R protein levels were observed in L6 myoblasts subjected to 15% (Fig. 7A) and 20% (Fig. 7B) stretches, respectively. AR overexpression enhanced the IGF-1R levels in both 15% and 20% stretched L6 myoblasts (Fig. 7).
Exogenous IGF-1 recombinant polypeptide further increases IGF-1R protein level and the activities of PI3K/Akt and MAPKs (p38 and ERK1/2) in 15% stretched L6 myoblasts, accompanied with the enhanced proliferation.
Exogenous IGF-1 recombinant polypeptide with various concentrations (200, 500, and 1000 ng/ml) were incubated with cells to verify the regulation of IGF-1 on the proliferation as well as PI3K/Akt, p38 and ERK1/2 signals in 15% stretch myoblasts. As shown in Fig.8, IGF-1 recombinant polypeptide further promoted the proliferation of 15% stretched L6 myoblasts in a dose-dependent manner (Fig. 8A), accompanied with dose-dependent increases in protein level of IGF-1R (Fig. 8B) and in activities of PI3K, Akt, p38 and ERK1/2 (increased phosphorylated protein levels of PI3K, Akt, p38 and ERK1/2) (Fig. 8C and 8D).