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Address correspondence to Jie Chen, Dept. of Cell and Structural Biology, University of Illinois at Urbana-Champaign, 601 S. Goodwin Ave., B107, Urbana, IL 61801. Tel./Fax: (217) 265-0674. email: jiechen{at}uiuc.edu
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Abstract |
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Key Words: IGF; rapamycin; skeletal muscle differentiation; PI3K; Akt
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Introduction |
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The cellular target of the bacterial macrolide rapamycin, mTOR, belongs to the phosphatidylinositol kinase (PI3K)-related family of Ser/Thr kinases and functions as a master regulator of cell growth and proliferation by regulating multiple downstream effectors (Jacinto and Hall, 2003). In cell proliferation, the best-characterized function of mTOR is the regulation of translation initiation through eIF-4E binding protein 1 (4EBP1) and S6 kinase 1 (S6K1), and the PI3K pathway acts in parallel with mTOR to regulate 4EBP1 and S6K1 (Fumagalli and Thomas, 2000; Gingras et al., 2001). The mTOR pathway is believed to mediate nutrient signals such as amino acid sufficiency (Fumagalli and Thomas, 2000; Gingras et al., 2001), as well as directly receive mitogenic signals through a lipid second messenger (Fang et al., 2001).
Implicated by the inhibitory effect of rapamycin on the differentiation of a variety of myoblasts in culture (Coolican et al., 1997; Cuenda and Cohen, 1999; Conejo et al., 2001; Erbay and Chen, 2001), mTOR's essential role in skeletal myogenesis has been demonstrated by the ability of a rapamycin-resistant mTOR mutant to rescue rapamycin-inhibited differentiation in C2C12 myoblasts (Erbay and Chen, 2001; Shu et al., 2002). Remarkably, the kinase activity of mTOR is not required for initiation of differentiation, and both S6K1 and 4EBP1 have therefore been excluded as downstream mediators of mTOR's myogenic signaling (Erbay and Chen, 2001). The molecular events regulated by mTOR in myogenesis are currently unknown. In this report, we provide strong evidence that mTOR governs myoblast differentiation by controlling the transcription of IGF-II, potentially through a nutrient-sensing pathway.
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Results and discussion |
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In addition to the mTOR pathway, the PI3K pathway and p38 MAPK are also required for skeletal myocyte differentiation (Coolican et al., 1997; Kaliman et al., 1998; Cuenda and Cohen, 1999). However, both P3 and ME activity during differentiation were insensitive to the PI3K inhibitor wortmannin and the p38 inhibitor SB202190 (Fig. 1, C and D).
mTOR is required for IGF-II mRNA production in C2C12 cells
Consistent with rapamycin's effect on P3 and ME of IGF-II, the drastic increase of IGF-II mRNA levels during differentiation was significantly blocked by rapamycin treatment of differentiating cells, as shown by the results of RNase protection assays (RPAs) in Fig. 2 A. This inhibition was not due to changes in IGF-II mRNA stability because during a 12-h window of actinomycin D treatment to suppress transcription, IGF-II mRNA remained highly stable, regardless of the presence of rapamycin (Fig. 2 B).
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Together, our data suggest that during skeletal myogenesis mTOR regulates the production of IGF-II mRNA. Moreover, this myogenic function of mTOR is independent of its kinase activity, which is consistent with our previous observations (Erbay and Chen, 2001). This conclusion contradicts that of Shu et al. (2002) who reported that a C2C12 cell line stably expressing a kinase-inactive mTOR did not differentiate in the presence of rapamycin. It is noted that a different mutation (D2338A) was used to inactivate the kinase by Shu et al. Although D2357E and D2338A are equally effective in inactivating mTOR's catalytic activity (Brown et al., 1995), it cannot be ruled out that D2338A may affect an additional biochemical property of mTOR. The possibility of clonal variation may also be considered. To avoid such a potential problem, multiple stable clones were examined in our studies as described in the previous paragraph, and transient transfection was previously used to express mTOR (Erbay and Chen, 2001).
Amino acid sufficiency is required for acute activation of IGF-II transcription
Because the mTOR pathway is known to sense the availability of amino acids in proliferating cells (Fumagalli and Thomas, 2000; Gingras et al., 2001), we hypothesized that mTOR may mediate amino acid signals in the regulation of IGF-II transcription during myogenesis. Indeed, the acute activation of P3 normally seen at 3 h was abrogated when amino acids were depleted from the differentiation medium (Fig. 3). When normal concentrations of amino acids were replenished for 3 h, activation of P3 was recovered, and this reactivation was completely blocked by rapamycin, but not by wortmannin or SB202190 (Fig. 3). Similarly, the induction of ME activity at 3 h was ablated by amino acid deprivation and stimulated upon readdition of amino acids (Fig. 3). Again, the reactivation of ME by amino acids was inhibited by rapamycin and was insensitive to wortmannin and SB202190. The short duration of amino acid withdrawal ensured that the effect would be the result of affecting a signaling cascade rather than a general stoppage of protein synthesis. Indeed, the CMV-driven luciferase activity was unaffected by amino acid deprivation under similar conditions (unpublished data).
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mTOR regulates C2C12 differentiation by controlling IGF production
The regulation of IGF-II transcription by mTOR led us to hypothesize that mTOR might exert its myogenic function by controlling IGF-II secretion that is critical for initiation of the differentiation program. To probe this possibility, conditioned medium from normally differentiating "donor" C2C12 cells were transferred daily to "recipient" C2C12 cells subjected to rapamycin treatment. The recipient cells proceeded to terminally differentiate despite the presence of rapamycin, evidenced by the formation of sarcomeric myosin heavy chain (MHC)positive myotubes (Fig. 4 A, a). Thus, it appears that rapamycin's inhibitory effect on differentiation is through the elimination of a secreted factor. This factor was confirmed to be IGF-II by the observation that preincubation with an antiIGF-II antibody, but not a control rabbit IgG, completely eliminated the myogenic potency of the donor medium (Fig. 4 A, b and c). Moreover, conditioned media from rapamycin-treated RR-mTOR cells, as well as RR/KI-mTOR cells, conferred rapamycin resistance to the differentiation of the recipient cells (Fig. 4 A, d and e), further validating the requirement of mTOR and a kinase-independent mechanism. The neutralizing IGF-II antibody also abolished the rapamycin-resistant myogenic effects of the media from the recombinant mTOR-expressing cells (unpublished data).
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PI3KAkt pathway is a major mediator of mTOR's myogenic function
The PI3KAkt pathway has been shown to be critical for IGF's myogenic signaling (Coolican et al., 1997; Kaliman et al., 1998). We found that expression of a constitutively active (c.a.) Akt reversed the inhibition of C2C12 differentiation by the PI3K inhibitor LY294002 (Fig. 5 A), suggesting that Akt is the main effector for PI3K signaling in myogenesis. Remarkably, c.a.Akt also rescued differentiation from rapamycin inhibition (Fig. 5 A). The results of RPA analysis indicated that IGF-II mRNA levels were significantly inhibited by rapamycin in c.a.Akt-expressing cells (Fig. 5 C), which excluded the possibility of enhanced IGF-II production independent of mTOR in c.a.Akt cells. These results imply that the PI3KAkt pathway is sufficient to mediate IGF's myogenic signaling, and they further confirm that regulation of IGF production is a primary function for mTOR in myogenesis. It is interesting to note the contrast between the relationship of these two pathways in myogenesis and mitogenesis. In cell proliferation, the mTOR pathway and the PI3K pathway cooperate in a parallel manner to transduce growth factor signals and regulate the same downstream targets such as S6K1 (Fumagalli and Thomas, 2000); whereas in myogenesis, they regulate two sequential processes: production of IGF and subsequent IGF signaling, respectively (Fig. 5 D).
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Materials and methods |
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Plasmids
The following plasmids were gifts from various laboratories: pCEFL-HA-Akt (wild type) and pCMV6-myristoylated-HA-Akt (c.a.) were gifts from N. Ahmed and J. Blenis (Harvard Medical School, Boston, MA), mouse IGF-II cDNA was a gift from P. Rotwein (Oregon Health and Science University, Portland, OR), P3-luc reporter and basic-luc (promoterless) were gifts from A. Murrell (The Babraham Institute, Cambridge, UK; Murrell et al., 2001), and H19-luc was a gift from K. Pfeifer (National Institute of Child Health and Human Development, Bethesda, MD; Pfeifer et al., 1996). CMV-luc was as previously reported (Kim and Chen, 2000). To assemble the skeletal ME reporter H19-luc-ME, the SpeIEagI enhancer fragment (+23 kb to +27 kb) was inserted into a SpeIEagI linker present in the XhoI site 3' of the luciferase in H19-luc.
Cell culture and immunofluorescence microscopy
C2C12 myoblasts were maintained and differentiated as described previously (Erbay and Chen, 2001). Primary skeletal myoblasts were isolated from 2-wk-old mice, grown, and differentiated as previously reported (Rando and Blau, 1994). Transfections were performed using FUGENE-6 (Roche), and stable clones or pools were selected in 1 mg/ml G418. For amino acid deprivation and readdition, the cells were cultured in Dulbecco's PBS with 4.5 g/liter glucose and 2% dialyzed serum for 3 h and switched to normal differentiation medium for 3 h. For immunocytochemistry, cells were fixed in 3.7% formaldehyde and stained with MHC antibody and FITCantimouse IgG. Microscopy was performed on a microscope (model DMIL; Leica) with CPLAN 10x/0.22 NA lenses. The images were captured using a monochrome charged-couple device camera (model SPOT RT; Diagnostic Instruments), and processed as 8-bit RGB images using Adobe Photoshop 7.0.
RPA
Total RNA from C2C12 cells (in 60-mm plates) was isolated using the RNeasy mini kit (QIAGEN), and 4 µg of each RNA was applied to RPA using the RPA III kit (Ambion). Radioactive probes (used at 106 cpm/ml) were generated from linearized IGF-II cDNA construct by in vitro transcription using the MAXIscript kit (Ambion). The samples were run on 5% denaturing polyacrylamide gels and analyzed on a phosphorimager (model Cyclone; Packard Instrument Co.). To confirm loading consistency, 1-µg RNA samples were run on 0.8% agarose-formaldehyde gels, followed by ethidium bromide staining.
Reporter assays
C2C12 cells stably expressing CMV-luc, P3-luc, basic-luc, H19-luc, or H19-luc-ME were grown to 100% confluence and induced to differentiate in 2% horse serum. Various drug treatments were performed as described in the figure legends. The cells were lysed at the indicated times, and luciferase assays were performed using the Luciferase Assay Systems kit (Promega).
Kinase assays
mTOR autokinase assays and S6K1 kinase assays were performed as described previously (Erbay and Chen, 2001).
Statistical analysis
t tests were performed for all data comparisons. Unless specifically indicated in figure legends, significant difference was defined by P < 0.05.
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Acknowledgments |
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This work was supported by the National Institutes of Health grants GM58064 and AR48914 (to J. Chen) and predoctoral fellowships from the American Heart Association Midwest Affiliate (to E. Erbay and I.-H. Park).
Submitted: 28 July 2003
Accepted: 15 October 2003
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References |
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Barton-Davis, E.R., D.I. Shoturma, and H.L. Sweeney. 1999. Contribution of satellite cells to IGF-I induced hypertrophy of skeletal muscle. Acta Physiol. Scand. 167:301305.[CrossRef][Medline]
Brown, E.J., P.A. Beal, C.T. Keith, J. Chen, T.B. Shin, and S.L. Schreiber. 1995. Control of p70 s6 kinase by kinase activity of FRAP in vivo. Nature. 377:441446.[CrossRef][Medline]
Conejo, R., A.M. Valverde, M. Benito, and M. Lorenzo. 2001. Insulin produces myogenesis in C2C12 myoblasts by induction of NF-kappaB and downregulation of AP-1 activities. J. Cell. Physiol. 186:8294.[CrossRef][Medline]
Coolican, S.A., D.S. Samuel, D.Z. Ewton, F.J. McWade, and J.R. Florini. 1997. The mitogenic and myogenic actions of insulin-like growth factors utilize distinct signaling pathways. J. Biol. Chem. 272:66536662.
Cuenda, A., and P. Cohen. 1999. Stress-activated protein kinase-2/p38 and a rapamycin-sensitive pathway are required for C2C12 myogenesis. J. Biol. Chem. 274:43414346.
Erbay, E., and J. Chen. 2001. The mammalian target of rapamycin regulates C2C12 myogenesis via a kinase-independent mechanism. J. Biol. Chem. 276:3607936082.
Fang, Y., M. Vilella-Bach, R. Bachmann, A. Flanigan, and J. Chen. 2001. Phosphatidic acid-mediated mitogenic activation of mTOR signaling. Science. 294:19421945.
Florini, J.R., D.Z. Ewton, and K.A. Magri. 1991a. Hormones, growth factors, and myogenic differentiation. Annu. Rev. Physiol. 53:201216.[CrossRef][Medline]
Florini, J.R., D.Z. Ewton, and S.L. Roof. 1991b. Insulin-like growth factor-I stimulates terminal myogenic differentiation by induction of myogenin gene expression. Mol. Endocrinol. 5:718724.[Abstract]
Florini, J.R., K.A. Magri, D.Z. Ewton, P.L. James, K. Grindstaff, and P.S. Rotwein. 1991c. "Spontaneous" differentiation of skeletal myoblasts is dependent upon autocrine secretion of insulin-like growth factor-II. J. Biol. Chem. 266:1591715923.
Fumagalli, S., and G. Thomas. 2000. S6 phosphorylation and signal transduction. Translational Control of Gene Expression. N. Sonenberg, J.W.B. Hershey, and M.B. Mathews, editors. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 695718.
Gingras, A.C., B. Raught, and N. Sonenberg. 2001. Regulation of translation initiation by FRAP/mTOR. Genes Dev. 15:807826.
Jacinto, E., and M.N. Hall. 2003. Tor signalling in bugs, brain and brawn. Nat. Rev. Mol. Cell Biol. 4:117126.[CrossRef][Medline]
Kaffer, C.R., A. Grinberg, and K. Pfeifer. 2001. Regulatory mechanisms at the mouse Igf2/H19 locus. Mol. Cell. Biol. 21:81898196.
Kaliman, P., J. Canicio, P.R. Shepherd, C.A. Beeton, X. Testar, M. Palacin, and A. Zorzano. 1998. Insulin-like growth factors require phosphatidylinositol 3-kinase to signal myogenesis: dominant negative p85 expression blocks differentiation of L6E9 muscle cells. Mol. Endocrinol. 12:6677.
Kim, J.E., and J. Chen. 2000. Cytoplasmic-nuclear shuttling of FKBP12-rapamycin-associated protein is involved in rapamycin-sensitive signaling and translation initiation. Proc. Natl. Acad. Sci. USA. 97:1434014345.
Kou, K., and P. Rotwein. 1993. Transcriptional activation of the insulin-like growth factor-II gene during myoblast differentiation. Mol. Endocrinol. 7:291302.[Abstract]
Murrell, A., S. Heeson, L. Bowden, M. Constancia, W. Dean, G. Kelsey, and W. Reik. 2001. An intragenic methylated region in the imprinted Igf2 gene augments transcription. EMBO Rep. 2:11011106.
Pfeifer, K., P.A. Leighton, and S.M. Tilghman. 1996. The structural H19 gene is required for transgene imprinting. Proc. Natl. Acad. Sci. USA. 93:1387613883.
Pilistine, S.J., A.C. Moses, and H.N. Munro. 1984. Placental lactogen administration reverses the effect of low-protein diet on maternal and fetal serum somatomedin levels in the pregnant rat. Proc. Natl. Acad. Sci. USA. 81:58535857.[Abstract]
Rando, T.A., and H.M. Blau. 1994. Primary mouse myoblast purification, characterization, and transplantation for cell-mediated gene therapy. J. Cell Biol. 125:12751287.[Abstract]
Rosenblatt, J.D., D. Yong, and D.J. Parry. 1994. Satellite cell activity is required for hypertrophy of overloaded adult rat muscle. Muscle Nerve. 17:608613.[Medline]
Shu, L., X. Zhang, and P.J. Houghton. 2002. Myogenic differentiation is dependent on both the kinase function and the N-terminal sequence of mammalian target of rapamycin. J. Biol. Chem. 277:1672616732.
Tollefsen, S.E., R. Lajara, R.H. McCusker, D.R. Clemmons, and P. Rotwein. 1989. Insulin-like growth factors (IGF) in muscle development. Expression of IGF-I, the IGF-I receptor, and an IGF binding protein during myoblast differentiation. J. Biol. Chem. 264:1381013817.