The kinase mTOR functions as a master regulator of cell growth, proliferation, and various types of cellular differentiation by sensing nutrient availability and cellular energy levels (20
). For myogenesis in cultured cells, mTOR regulates IGF-II transcription in response to amino acid signaling, and this is critical for initiation of myogenic differentiation (11
). Up to now, little is known about how the mTOR-IGF-II pathway is regulated during myogenesis. Here we show that the NE protein NET39 associates with mTOR and represses mTOR-IGF-II signaling during myoblast differentiation. The NET39-mTOR interaction is robust, since we detected it in cell lysates after solubilization under stringent conditions (i.e., nonionic detergent and 250 mM NaCl, which dissociates the mTORC components raptor and rictor from mTOR [25
]), and we found that the association persisted in 2 M salt and 2 M urea. Using the kinase activity of mTOR as a proxy for its functional state, we found that knockdown of NET39 enhanced the activity of mTOR at the onset of C2C12 cell differentiation, as indicated by p70S6K phosphorylation, and accelerated the production and secretion of the critical autocrine/paracrine factor IGF-II. Conversely, overexpression of NET39 repressed mTOR activity upon a shift of cells to differentiation conditions.
It should be pointed out that the function of mTOR during myoblast differentiation as a signaling factor upstream of IGF-II transcription (11
) is at least partially distinct from its functions in mature myotubes, where it can mediate IGF-I-induced hypertrophy by serving as a key downstream kinase (37
). The mTOR-IGF-II axis serves as a distinctive initiator of myogenic differentiation that plays a crucial role only in the early stage of myogenesis, since inactivation of mTOR by rapamycin at DM2 is not able to abolish myogenesis (35
). These results are very similar to our finding that NET39 influences myogenesis only at an early stage of differentiation. Intriguingly, the kinase activity of mTOR is reported not to be required for mTOR-regulated myoblast differentiation, although the exact mechanism by which mTOR regulates transcription of IGF-II needs to be determined (10
). Since mTOR is localized mainly in the nucleus in C2C12 myoblasts (51
), NET39 may recruit mTOR to the INM. This may serve as a physical sequestration mechanism to limit the availability of mTOR in control of IGF-II transcription (11
), and the upregulation of NET39 may therefore impose negative feedback for a precisely tuned regulation of myogenesis. Indeed, physical sequestration of transcriptional regulators at the NE as a means of inhibiting their activity has been described previously (18
). It is plausible that the NET39-mTOR interaction is regulated by specific posttranslational modifications (on NET39 and/or mTOR) which appear or function only during the period of cell cycle exit.
NET39 is the only known member of the LPP family that is localized at the NE. We could not detect phosphatase activity of NET39 toward common phospholipid substrates. These results, combined with the observation that NET39 contains nonconservative substitutions in presumptive key catalytic site residues, suggest that NET39 may be a phosphatase-dead variant. In a similar vein, NET39 also has been proposed to be a candidate type 2 sphingomyelin synthase, but a recent study indicated that it does not have this enzyme activity (22
). The LPP family member most closely related to NET39 that shows LPP activity, Ppapdc2, contains intact phosphatase motifs (17
). Nonetheless, we cannot exclude the possibility that NET39 serves as a phosphatase for an unknown lipid substrate and regulates lipid signaling in the nucleus. Interestingly, LPP3 has been reported to repress β-catenin-mediated transcription and axis duplication in a phosphatase-independent manner (12
). This report, together with our findings, implies that some LPP family members, including NET39, exert some of their functions in a manner independent of lipid signaling.
The strong transcriptional upregulation of NET39 during myogenesis likely is controlled by early myogenic regulatory transcription factors, such as MyoD or MEF2. Consistent with this notion, the upregulation of NET39 parallels that of myogenin, another MyoD/MEF2 target. MEF2, which is selectively active in brain as well as in muscle, also may control the expression of NET39 that is observed in the former tissue. Interestingly, HDAC9 was recently identified as an MEF2 target gene (19
). Since HDAC9 serves as a corepressor of MEF2, upregulation of HDAC9 during myogenesis might serve as a negative feedback mechanism to limit myogenic gene expression (19
). NET39 may be part of an additional negative feedback mechanism, because it has the ability to inhibit expression of myogenic genes by the mTOR-IGF2 pathway, and potentially by other pathways (see the following paragraph).
Although we have focused mainly on regulation of the mTOR-IGF-II pathway by NET39 in this study, other NET39-binding partners identified in our proteomic screen also may contribute to NET39 regulation of myogenesis. Among the binding partners we identified, we currently are analyzing β-catenin and TIP120B, which were recently reported to activate MyoD and myogenin, respectively (24
). Moreover, β-catenin is known to be required for early myogenic differentiation (4
In this study, we observed a potent modulation of myogenic differentiation by silencing or overexpression of NET39 in myoblasts, where it normally is expressed at only low levels. NET39 is expressed at much higher levels after myoblast differentiation and in mature muscle. We predict that NET39 will engage in conceptually similar physiological regulation in the latter biological contexts. In this case, we propose that NET39 functions are linked to muscle homeostasis in both normal and pathological muscle regeneration. Such functions could be particularly important in the heart, where pathological hypertrophy is a major human health issue. It is conceivable that genetic defects in human NET39 might be manifest as pathological cardiac hypertrophy. We are constructing a mouse model for conditional disruption of NET39 to directly test this hypothesis.
A close involvement of NET39 in muscle homeostasis is supported by data from a recently published study on muscle regeneration in the mouse (29
). It was found that the mRNA of NET39 in experimentally damaged mouse muscle was strongly diminished 3 days after cardiotoxin administration, whereas the expression of mTOR was not affected. The decreased expression of NET39 correlated with transcriptional upregulation of IGF-II, the mTOR-regulated autocrine gene, as well as with upregulation of MyoD and myogenin. This animal study supports our observations with cultured cells showing that NET39 negatively regulates myogenesis by counteracting the mTOR-IGF-II pathway. As outlined above, it is plausible, if not likely, that NET39 regulates additional homeostatic pathways in muscle. The list of potential NET39 targets we identified by proteomics in this study, which include structural/contractile proteins, ER membrane proteins, and proteins involved in apoptosis, will provide a valuable framework for future studies addressing this question.