In this study we employed an ex-vivo
passive stretch model of mechanical stimulation to determine if signaling through ERK is necessary for the mechanical activation of mTOR signaling and protein synthesis. Based on the results, it can be concluded that ERK’s contribution to these events, if any, is very limited. However, we also found that the basal level of mTOR signaling was severely impaired by ERK inhibition, which suggests that ERK is indeed a potent regulator of mTOR signaling in skeletal muscle. Hence, the seemingly negligible role of ERK in the mechanical activation of mTOR signaling is likely due, at least in part, to the relatively small increase in signaling through ERK that was induced by our model of mechanical stimulation (≈2-fold). However, a more robust increase in signaling through ERK has been observed with other types of mechanical stimulation. For example, Martineau and Gardiner 2001, demonstrated that concentric, isometric, and eccentric contractions produce a 3-, 4-, and 5-fold increase in signaling through ERK, respectively 
. Therefore, we cannot fully exclude the possibility that ERK may contribute to the activation of mTOR signaling, and protein synthesis, in other models of mechanical stimulation as previously suggested by Miyazaki et al.,
. Nevertheless, our results do clearly reveal that mechanical stimuli can induce mTOR signaling, and protein synthesis, through an ERK-independent mechanism.
To date, numerous studies have attempted to identify the molecular mechanisms that are involved in the mechanical activation of mTOR signaling 
. Based on these studies, it has been concluded that mechanical stimuli activate mTOR signaling through a unique mechanism that does not require typical candidates such as phosphoinositide 3-kinase, protein kinase B, exogenous nutrients, protein kinase C, phosphoinositide-specific phospholipase C, or changes in intracellular calcium 
. On the other hand, several lines of evidence suggest that PA may be involved 
. For example, the results of this study demonstrate that; i) mechanical stimuli can induce an increase in PA, ii) stimulating cells with PA is sufficient to induce mTOR signaling, and iii) PA can directly activate mTOR signaling in-vitro
. Furthermore, like mechanical stimuli, we found that PA induces mTOR signaling through an ERK-independent mechanism. All of these observations are consistent with a model in which mechanical stimuli induce an increase in PA, and the newly formed PA then binds and activates mTOR. Although intriguing, additional studies will be needed to test the validity of this concept.
In this study, we also used RR-mTOR mice to identify valid markers of mechanically-induced mTOR signaling. For example, the RR-mTOR mice enabled us to demonstrate that a mechanically-induced increase in p70s6k
T389 phosphorylation is fully dependent on mTOR. Conversely, we found that other commonly used markers of mTOR signaling, such as S6 S235/236 and S6 S240/244 phosphorylation, were at least partially induced via a rapamycin-insensitive/mTOR-independent mechanism. Consistent with these results, another recent study also demonstrated that rapamycin does not block mechanical overload-induced increases in S6 S235/236 and S6 S240/244 phosphorylation 
. This is particularly interesting because a previous study with p70s6k1
double knockout mice demonstrated that S6 S240/244 phosphorylation requires p70s6k
. Thus, our results might indicate that mechanical stimulation induces a partially rapamycin-resistant activation of p70s6k
, the activation of unrecognized S6 kinase(s), or an inhibition of S6 phosphatases. Furthermore, our observations illustrate that caution should be used when interpreting changes in S6 S235/236 or S6 S240/244 phosphorylation as markers of mTOR signaling.
The RR-mTOR mice also enabled us to demonstrate that mTOR is necessary for mechanical stimulation to induce an increase in 4E-BP1 S64 phosphorylation, and a decrease in total 4E-BP1. However, much to our surprise, mechanical stimulation did not alter 4E-BP1 T36/45 phosphorylation. This observation was surprising because numerous studies have shown that, when activated, the rapamycin-sensitive mTORC1 can directly phosphorylate both p70s6k
T389 and 4E-BP1 T36/45 residues 
. Furthermore, an extensively large number of studies have shown that the classical agonists of mTORC1 signaling (e.g. growth factors and nutrients) induce an increase in both p70s6k
T389 and 4E-BP1 T36/45 phosphorylation 
. Hence, observing a robust increase in p70s6k
T389 phosphorylation, in conjunction with no change in 4E-BP1 T36/45 phosphorylation, was highly unexpected. Although the reason for this disparity is not known, a recent study by Yip et al. 2010 may have revealed some important clues. Specifically, Yip et al. demonstrated that rapamycin inhibits the ability of immunopurified mTOR to phosphorylate p70s6k
T389 and 4E-BP1 T36/45 
. Furthermore, the presence of raptor was found to be necessary for mTOR to phosphorylate 4E-BP1 T36/45. However, contrary to previous assumptions, the presence of raptor was not necessary for mTOR to phosphorylate p70s6k
T389. Yet, in the absence of raptor, the ability of mTOR to phosphorylate p70s6k
T389 was still sensitive to inhibition by rapamycin. In other words, it appears that there is a rapamycin-sensitive pool of mTOR that does not involve the classical raptor-associated mTORC1 complex. Similar to the results we observed with mechanical stimulation, this uncharacterized rapamycin-sensitive pool of mTOR can phosphorylate p70s6k
T389, but not 4E-BP1 T36/45. Thus, it is very tempting to speculate that mechanical stimuli activate a unique rapamycin-sensitive pool of mTOR that is distinct from mTORC1.
As mentioned in the introduction, signaling through mTOR has been widely implicated in the regulation of protein synthesis. For example, the mTOR kinase inhibitor (Torin 1) induces a greater than 60% reduction in the rate of protein synthesis when added to wild-type mouse embryonic fibroblasts (MEF). However, Torin 1 has essentially no effect on the rate of protein synthesis when added to MEFs that are deficient of 4E-BP1 and 4E-BP2 
. Based on these observations, it has been concluded that the 4E-BPs play a key role in the mechanism through which mTOR controls protein synthesis.
In general, mTOR is thought to control the function of the 4E-BPs by inducing changes in the phosphorylation state of the protein. For example, mTOR can directly phosphorylate the 36/45 residues of 4E-BP1, and phosphorylation on these residues allows 4E-BP1 to become further phosphorylated on residues such as S64 
. When hyperphosphorylated, 4E-BP1 dissociates from eIF4E and this, in-turn, promotes the formation of the eIF4F complex and ultimately the initiation of protein synthesis 
. In addition to controlling 4E-BP1 phosphorylation, there is also emerging evidence which suggests that mTOR can regulate the abundance of 4E-BP1. For example, inducible knockout of mTOR in the adult myocardium results in an increase in total 4E-BP1 
. Furthermore, HSV-1 infection has been shown to induce a decrease in total 4E-BP1, and this effect can be prevented by rapamycin 
. Presumably, a decrease in total 4E-BP1, and hyperphosphorylation of 4E-BP1, would result in functionally equivalent effects on protein synthesis (i.e. enhanced eIF4F complex formation). Thus, we were intrigued by our results which demonstrated that mechanical stimulation induces an mTOR-dependent decrease in total 4E-BP1. Specifically, previous studies have indicated that signaling through mTOR is necessary for a mechanically-induced increase in protein synthesis but the mechanisms through which mTOR exerts this effect have not been defined. Based on our results, it would appear that a mechanically-induced decrease in total 4E-BP1 could be part of this mechanism. Furthermore, we have measured total 4E-BP1 levels in muscles that were subjected to in-vivo
eccentric contractions as described in 
, and again, we observed a decrease in total 4E-BP1 levels (data not shown). Based on these observations, it would appear that a mechanically-induced decrease in the total 4E-BP1 levels is a conserved event. Hence, in the future, it will be important to determine if the loss of total 4E-BP1 plays a significant role in the mechanism through which mechanical stimuli induce an increase in protein synthesis.
In summary, the results from this study demonstrate that mechanical stimulation induces mTOR signaling, and protein synthesis, via an ERK-independent mechanism that potentially involves a direct interaction of PA with mTOR. Since signaling through mTOR is necessary for a mechanically-induced increase in protein synthesis, and ultimately growth, these findings should help advance our understanding of how mechanical signals are converted into the molecular events that regulate skeletal muscle mass.