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Insulin-like Growth Factor-1 (IGF-1) Inversely Regulates Atrophy-induced Genes via the Phosphatidylinositol 3-Kinase/Akt/Mammalian Target of Rapamycin (PI3K/Akt/mTOR) Pathway

2004; Elsevier BV; Volume: 280; Issue: 4 Linguagem: Inglês

10.1074/jbc.m407517200

1083-351X

Esther Latres, Ami R. Amini, Ashley A. Amini, Jennifer Griffiths, Francis J. Martin, Yi Wei, Hsin Chieh Lin, George D. Yancopoulos, David J. Glass,

PI3K/AKT/mTOR signaling in cancer

Skeletal muscle size is regulated by anabolic (hypertrophic) and catabolic (atrophic) processes. We first characterized molecular markers of both hypertrophy and atrophy and identified a small subset of genes that are inversely regulated in these two settings (e.g. up-regulated by an inducer of hypertrophy, insulin-like growth factor-1 (IGF-1), and down-regulated by a mediator of atrophy, dexamethasone). The genes identified as being inversely regulated by atrophy, as opposed to hypertrophy, include the E3 ubiquitin ligase MAFbx (also known as atrogin-1). We next sought to investigate the mechanism by which IGF-1 inversely regulates these markers, and found that the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway, which we had previously characterized as being critical for hypertrophy, is also required to be active in order for IGF-1-mediated transcriptional changes to occur. We had recently demonstrated that the IGF1/PI3K/Akt pathway can block dexamethasone-induced up-regulation of the atrophy-induced ubiquitin ligases MuRF1 and MAFbx by blocking nuclear translocation of a FOXO transcription factor. In the current study we demonstrate that an additional step of IGF1 transcriptional regulation occurs downstream of mTOR, which is independent of FOXO. Thus both the Akt/FOXO and the Akt/mTOR pathways are required for the transcriptional changes induced by IGF-1. Skeletal muscle size is regulated by anabolic (hypertrophic) and catabolic (atrophic) processes. We first characterized molecular markers of both hypertrophy and atrophy and identified a small subset of genes that are inversely regulated in these two settings (e.g. up-regulated by an inducer of hypertrophy, insulin-like growth factor-1 (IGF-1), and down-regulated by a mediator of atrophy, dexamethasone). The genes identified as being inversely regulated by atrophy, as opposed to hypertrophy, include the E3 ubiquitin ligase MAFbx (also known as atrogin-1). We next sought to investigate the mechanism by which IGF-1 inversely regulates these markers, and found that the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway, which we had previously characterized as being critical for hypertrophy, is also required to be active in order for IGF-1-mediated transcriptional changes to occur. We had recently demonstrated that the IGF1/PI3K/Akt pathway can block dexamethasone-induced up-regulation of the atrophy-induced ubiquitin ligases MuRF1 and MAFbx by blocking nuclear translocation of a FOXO transcription factor. In the current study we demonstrate that an additional step of IGF1 transcriptional regulation occurs downstream of mTOR, which is independent of FOXO. Thus both the Akt/FOXO and the Akt/mTOR pathways are required for the transcriptional changes induced by IGF-1. Skeletal muscle mass and fiber size is regulated in response to changes in workload, activity, conditions such as AIDS, cancer, and aging, and by cachectic glucocorticoids such as dexamethasone (1Glass D.J. Nat. Cell Biol. 2003; 5: 87-90Crossref PubMed Scopus (534) Google Scholar, 2Glass D.J. Trends Mol. Med. 2003; 9: 344-350Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar, 3Jagoe R.T. Goldberg A.L. Curr. Opin. Clin. Nutr. Metab. Care. 2001; 4: 183-190Crossref PubMed Scopus (328) Google Scholar). An increase in adult muscle mass and fiber size is called "hypertrophy" and is associated with increased protein synthesis (4Goldspink D.F. Garlick P.J. McNurlan M.A. Biochem. J. 1983; 210: 89-98Crossref PubMed Scopus (101) Google Scholar). A decrease in mass, called "atrophy," is characterized by enhanced protein degradation (3Jagoe R.T. Goldberg A.L. Curr. Opin. 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Hypertrophy in adult skeletal muscle is accompanied by the increased expression of insulin-like growth factor-1 (IGF-1) 1The abbreviations used are: IGF-1, insulin-like growth factor-1; PI3K, phosphatidylinositol 3-kinase; mTOR, mammalian target of rapamycin; DEX, dexamethasone; D2 and D3, day 2 and day 3, respectively; RAP, rapamycin; MT, metallothionein; PLF, proliferin; PV, parvalbumin; LY, LY294002.1The abbreviations used are: IGF-1, insulin-like growth factor-1; PI3K, phosphatidylinositol 3-kinase; mTOR, mammalian target of rapamycin; DEX, dexamethasone; D2 and D3, day 2 and day 3, respectively; RAP, rapamycin; MT, metallothionein; PLF, proliferin; PV, parvalbumin; LY, LY294002. (4Goldspink D.F. Garlick P.J. McNurlan M.A. Biochem. J. 1983; 210: 89-98Crossref PubMed Scopus (101) Google Scholar, 7DeVol D.L. Rotwein P. Sadow J.L. Novakofski J. Bechtel P.J. Am. J. Physiol. 1990; 259: E89-E95PubMed Google Scholar). When IGF-1 was overexpressed in the skeletal muscle of transgenic mice an increase in muscle size resulted (8Coleman M.E. DeMayo F. Yin K.C. Lee H.M. Geske R. Montgomery C. Schwartz R.J. J. Biol. Chem. 1995; 270: 12109-12116Abstract Full Text Full Text PDF PubMed Scopus (536) Google Scholar, 9Musaro A. McCullagh K. Paul A. Houghton L. Dobrowolny G. Molinaro M. Barton E.R. Sweeney H.L. Rosenthal N. Nat. Genet. 2001; 27: 195-200Crossref PubMed Scopus (887) Google Scholar). Furthermore, addition of IGF-1 in vitro to differentiated muscle cells promotes myotube hypertrophy (10Florini J.R. Ewton D.Z. Coolican S.A. Endocr. Rev. 1996; 17: 481-517PubMed Google Scholar, 11Rommel C. Clarke B.A. Zimmermann S. Nunez L. Rossman R. Reid K. Moelling K. Yancopoulos G.D. Glass D.J. Science. 1999; 286: 1738-1741Crossref PubMed Scopus (661) Google Scholar, 12Rommel C. Bodine S.C. Clarke B.A. Rossman R. Nunez L. Stitt T.N. Yancopoulos G.D. Glass D.J. Nat. Cell Biol. 2001; 3: 1009-1013Crossref PubMed Scopus (1208) Google Scholar), supporting the idea that IGF-1 is sufficient to induce hypertrophy. The binding of IGF-1 to its receptor triggers the activation of phosphatidylinositol 3-kinase (PI3K). PI3K phosphorylates the membrane phospholipid phosphatidylinositol 4,5-bisphosphate to produce phosphatidylinositol 3,4,5-trisphosphate (13Matsui T. Nagoshi T. Rosenzweig A. Cell Cycle. 2003; 2: 220-223Crossref PubMed Scopus (168) Google Scholar, 14Vivanco I. Sawyers C.L. Nat. Rev. Cancer. 2002; 2: 489-501Crossref PubMed Scopus (5130) Google Scholar), creating a lipid binding site on the cell membrane for the serine/threonine kinase Akt (also called Akt1 and PKB, for protein kinase B) (15Alessi D.R. James S.R. Downes C.P. Holmes A.B. Gaffney P.R. Reese C.B. Cohen P. Curr. Biol. 1997; 7: 261-269Abstract Full Text Full Text PDF PubMed Google Scholar, 16Andjelkovic M. Alessi D.R. Meier R. Fernandez A. Lamb N.J. Frech M. Cron P. Cohen P. 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Cancer. 2002; 2: 489-501Crossref PubMed Scopus (5130) Google Scholar, 19Datta S.R. Brunet A. Greenberg M.E. Genes Dev. 1999; 13: 2905-2927Crossref PubMed Scopus (3718) Google Scholar). Direct and indirect targets downstream of Akt include the mammalian target of rapamycin (mTOR), p70S6K, and PHAS-1 (4EBP-1), key regulatory proteins involved in translation and protein synthesis (12Rommel C. Bodine S.C. Clarke B.A. Rossman R. Nunez L. Stitt T.N. Yancopoulos G.D. Glass D.J. Nat. Cell Biol. 2001; 3: 1009-1013Crossref PubMed Scopus (1208) Google Scholar, 20Cross D.A. Alessi D.R. Cohen P. Andjelkovich M. Hemmings B.A. Nature. 1995; 378: 785-789Crossref PubMed Scopus (4364) Google Scholar, 21Nave B.T. Ouwens M. Withers D.J. Alessi D.R. Shepherd P.R. Biochem. J. 1999; 344: 427-431Crossref PubMed Scopus (780) Google Scholar, 22Scott P.H. Lawrence Jr., J.C. J. Biol. Chem. 1998; 273: 34496-34501Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). The PI3K/Akt pathway is a crucial intracellular signaling mechanism underlying muscle hypertrophy (1Glass D.J. Nat. Cell Biol. 2003; 5: 87-90Crossref PubMed Scopus (534) Google Scholar). In vivo activation of the PI3K pathway by introduction of a mutant form of Ras, competent only to activate PI3K, caused hypertrophy of regenerating muscle (23Murgia M. Serrano A.L. Calabria E. Pallafacchina G. Lomo T. Schiaffino S. Nat. Cell Biol. 2000; 2: 142-147Crossref PubMed Scopus (189) Google Scholar), and pharmacological blockade of PI3K activity with the drug LY294002 (LY) blocks IGF-1-induced hypertrophy in vitro (12Rommel C. Bodine S.C. Clarke B.A. Rossman R. Nunez L. Stitt T.N. Yancopoulos G.D. Glass D.J. Nat. Cell Biol. 2001; 3: 1009-1013Crossref PubMed Scopus (1208) Google Scholar). During adaptive hypertrophy in adult muscle and in IGF-1-induced myotube hypertrophy, Akt is phosphorylated and activated, as is the Akt substrate mTOR (12Rommel C. Bodine S.C. Clarke B.A. Rossman R. Nunez L. Stitt T.N. Yancopoulos G.D. Glass D.J. Nat. Cell Biol. 2001; 3: 1009-1013Crossref PubMed Scopus (1208) Google Scholar, 24Bodine S.C. Stitt T.N. Gonzalez M. Kline W.O. Stover G.L. Bauerlein R. Zlotchenko E. Scrimgeour A. Lawrence J.C. Glass D.J. Yancopoulos G.D. Nat. Cell Biol. 2001; 3: 1014-1019Crossref PubMed Scopus (1916) Google Scholar, 25Pallafacchina G. Calabria E. Serrano A.L. Kalhovde J.M. Schiaffino S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9213-9218Crossref PubMed Scopus (308) Google Scholar). Additionally, hypertrophy elicits the phosphorylation of two known regulators of protein synthesis downstream of mTOR signaling, p70S6K and PHAS-1, thereby promoting increased translation (12Rommel C. Bodine S.C. Clarke B.A. Rossman R. Nunez L. Stitt T.N. Yancopoulos G.D. Glass D.J. Nat. Cell Biol. 2001; 3: 1009-1013Crossref PubMed Scopus (1208) Google Scholar, 24Bodine S.C. Stitt T.N. Gonzalez M. Kline W.O. Stover G.L. Bauerlein R. Zlotchenko E. Scrimgeour A. Lawrence J.C. Glass D.J. Yancopoulos G.D. Nat. Cell Biol. 2001; 3: 1014-1019Crossref PubMed Scopus (1916) Google Scholar, 26Reynolds T.H. Bodine S.C. Lawrence Jr., J.C. J. Biol. Chem. 2002; 277: 17657-17662Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar). The requirement for mTOR-mediated signaling in hypertrophy has been demonstrated pharmacologically; blockade with the mTOR inhibitor rapamycin decreases muscle hypertrophy in vivo and in vitro and blunts the hypertrophy-associated phosphorylation of p70S6K and PHAS-1 (12Rommel C. Bodine S.C. Clarke B.A. Rossman R. Nunez L. Stitt T.N. Yancopoulos G.D. Glass D.J. Nat. Cell Biol. 2001; 3: 1009-1013Crossref PubMed Scopus (1208) Google Scholar, 24Bodine S.C. Stitt T.N. Gonzalez M. Kline W.O. Stover G.L. Bauerlein R. Zlotchenko E. Scrimgeour A. Lawrence J.C. Glass D.J. Yancopoulos G.D. Nat. Cell Biol. 2001; 3: 1014-1019Crossref PubMed Scopus (1916) Google Scholar). Genetic approaches have provided further evidence for the role of the PI3K/Akt pathway in hypertrophy. Expression of constructs encoding constitutively active forms of either PI3K or Akt induced muscle hypertrophy both in vivo (24Bodine S.C. Stitt T.N. Gonzalez M. Kline W.O. Stover G.L. Bauerlein R. Zlotchenko E. Scrimgeour A. Lawrence J.C. Glass D.J. Yancopoulos G.D. Nat. Cell Biol. 2001; 3: 1014-1019Crossref PubMed Scopus (1916) Google Scholar, 25Pallafacchina G. Calabria E. Serrano A.L. Kalhovde J.M. Schiaffino S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9213-9218Crossref PubMed Scopus (308) Google Scholar, 27Lai K-M. Gonzalez M. Poueymirou W.T. Kline W.O. Na E. Zlotchenko E. Stitt T.N. Economides A. Yancopoulos G.D. Glass D.J. Mol. Cell. Biol. 2004; 24: 9295-9304Crossref PubMed Scopus (332) Google Scholar) and in vitro (11Rommel C. Clarke B.A. Zimmermann S. Nunez L. Rossman R. Reid K. Moelling K. Yancopoulos G.D. Glass D.J. Science. 1999; 286: 1738-1741Crossref PubMed Scopus (661) Google Scholar, 12Rommel C. Bodine S.C. Clarke B.A. Rossman R. Nunez L. Stitt T.N. Yancopoulos G.D. Glass D.J. Nat. Cell Biol. 2001; 3: 1009-1013Crossref PubMed Scopus (1208) Google Scholar). Skeletal muscle atrophy, denoted by a decrease in muscle mass and fiber size, can be driven by such disparate stimuli as denervation, immobilization, sepsis, cachexia, or glucocorticoid treatment (3Jagoe R.T. Goldberg A.L. Curr. Opin. Clin. Nutr. Metab. Care. 2001; 4: 183-190Crossref PubMed Scopus (328) Google Scholar, 28Jackman R.W. Kandarian S.C. Am. J. Physiol. 2004; 287: C834-C843Crossref PubMed Scopus (704) Google Scholar). Atrophy is characterized by increases in protein degradation processes, particularly the ATP-dependent proteolytic ubiquitin-proteasome pathway (3Jagoe R.T. Goldberg A.L. Curr. Opin. Clin. Nutr. Metab. Care. 2001; 4: 183-190Crossref PubMed Scopus (328) Google Scholar). During atrophy, there is an increase in ubiquitin-protein conjugates and increased transcription of components of the ubiquitin degradation pathway (3Jagoe R.T. Goldberg A.L. Curr. Opin. Clin. Nutr. Metab. Care. 2001; 4: 183-190Crossref PubMed Scopus (328) Google Scholar, 5Hasselgren P.O. Curr. Opin. Clin. Nutr. Metab. Care. 1999; 2: 201-205Crossref PubMed Scopus (258) Google Scholar). A screen for genetic markers of atrophy identified two genes that are up-regulated rapidly in multiple models of muscle atrophy in vivo, including dexamethasone-induced wasting, which also show highly muscle-specific expression (29Gomes M.D. Lecker S.H. Jagoe R.T. Navon A. Goldberg A.L. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14440-14445Crossref PubMed Scopus (1358) Google Scholar, 30Bodine S.C. Latres E. Baumhueter S. Lai V.K. Nunez L. Clarke B.A. Poueymirou W.T. Panaro F.J. Na E. Dharmarajan K. Pan Z.Q. Valenzuela D.M. DeChiara T.M. Stitt T.N. Yancopoulos G.D. Glass D.J. Science. 2001; 294: 1704-1708Crossref PubMed Scopus (2633) Google Scholar). These genes, MAFbx (muscle atrophy F-box, also called atrogin-1) and MuRF1 (muscle RING finger 1), both encode ubiquitin ligases, which function to conjugate ubiquitin to protein substrates (29Gomes M.D. Lecker S.H. Jagoe R.T. Navon A. Goldberg A.L. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14440-14445Crossref PubMed Scopus (1358) Google Scholar, 30Bodine S.C. Latres E. Baumhueter S. Lai V.K. Nunez L. Clarke B.A. Poueymirou W.T. Panaro F.J. Na E. Dharmarajan K. Pan Z.Q. Valenzuela D.M. DeChiara T.M. Stitt T.N. Yancopoulos G.D. Glass D.J. Science. 2001; 294: 1704-1708Crossref PubMed Scopus (2633) Google Scholar). The functional importance of these gene products in atrophy processes was demonstrated by the generation of MAFbx–/– and MuRF1–/– mice. Mice lacking either gene show sparing of muscle mass following denervation (30Bodine S.C. Latres E. Baumhueter S. Lai V.K. Nunez L. Clarke B.A. Poueymirou W.T. Panaro F.J. Na E. Dharmarajan K. Pan Z.Q. Valenzuela D.M. DeChiara T.M. Stitt T.N. Yancopoulos G.D. Glass D.J. Science. 2001; 294: 1704-1708Crossref PubMed Scopus (2633) Google Scholar). Furthermore, MuRF1 and MAFbx have been shown to be up-regulated in a number of other models of atrophy, indicating that these genes are highly faithful markers of the atrophy process (29Gomes M.D. Lecker S.H. Jagoe R.T. Navon A. Goldberg A.L. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14440-14445Crossref PubMed Scopus (1358) Google Scholar, 30Bodine S.C. Latres E. Baumhueter S. Lai V.K. Nunez L. Clarke B.A. Poueymirou W.T. Panaro F.J. Na E. Dharmarajan K. Pan Z.Q. Valenzuela D.M. DeChiara T.M. Stitt T.N. Yancopoulos G.D. Glass D.J. Science. 2001; 294: 1704-1708Crossref PubMed Scopus (2633) Google Scholar, 31Wray C.J. Mammen J.M. Hershko D.D. Hasselgren P.O. Int. J. Biochem. Cell Biol. 2003; 35: 698-705Crossref PubMed Scopus (183) Google Scholar, 32Li Y.-P. Chen Y. Li A.S. Reid M.B. Am. J. Physiol. Cell Physiol. 2003; 285: C806Crossref PubMed Scopus (262) Google Scholar). In recent studies it was shown that the hypertrophy-inducing PI3K/Akt pathway could dominantly block the atrophy-inducing effects of dexamethasone (33Sandri M. Sandri C. Gilbert A. Skurk C. Calabria E. Picard A. Walsh K. Schiaffino S. Lecker S.H. Goldberg A.L. Cell. 2004; 117: 399-412Abstract Full Text Full Text PDF PubMed Scopus (2195) Google Scholar, 34Stitt T.N. Drujan D. Clarke B.A. Panaro F.J. Timofeyva Y. Kline W.O. Gonzalez M. Yancopoulos G.D. Glass D.J. Mol. Cell. 2004; 14: 395-403Abstract Full Text Full Text PDF PubMed Scopus (1472) Google Scholar), via Akt-mediated phosphorylation and subsequent inhibition of the FOXO family of transcription factors (33Sandri M. Sandri C. Gilbert A. Skurk C. Calabria E. Picard A. Walsh K. Schiaffino S. Lecker S.H. Goldberg A.L. Cell. 2004; 117: 399-412Abstract Full Text Full Text PDF PubMed Scopus (2195) Google Scholar, 34Stitt T.N. Drujan D. Clarke B.A. Panaro F.J. Timofeyva Y. Kline W.O. Gonzalez M. Yancopoulos G.D. Glass D.J. Mol. Cell. 2004; 14: 395-403Abstract Full Text Full Text PDF PubMed Scopus (1472) Google Scholar); FOXO was shown to be necessary for the induction of both MuRF1 (34Stitt T.N. Drujan D. Clarke B.A. Panaro F.J. Timofeyva Y. Kline W.O. Gonzalez M. Yancopoulos G.D. Glass D.J. Mol. Cell. 2004; 14: 395-403Abstract Full Text Full Text PDF PubMed Scopus (1472) Google Scholar) and MAFbx/atrogin-1 (33Sandri M. Sandri C. Gilbert A. Skurk C. Calabria E. Picard A. Walsh K. Schiaffino S. Lecker S.H. Goldberg A.L. Cell. 2004; 117: 399-412Abstract Full Text Full Text PDF PubMed Scopus (2195) Google Scholar, 34Stitt T.N. Drujan D. Clarke B.A. Panaro F.J. Timofeyva Y. Kline W.O. Gonzalez M. Yancopoulos G.D. Glass D.J. Mol. Cell. 2004; 14: 395-403Abstract Full Text Full Text PDF PubMed Scopus (1472) Google Scholar). Thereby by blocking FOXO, Akt blocked the induction of atrophy signaling. In an effort to further characterize hypertrophy/atrophy interactions, we sought to define genes that were induced in one direction by IGF-1 and inversely regulated by the atrophy-inducing glucocorticoid dexamethasone (DEX). These inversely regulated genes would comprise a set of markers of both hypertrophy and atrophy and would therefore function as barometers for the growth state of the muscle. We further sought to determine whether the IGF-1/PI3K/Akt pathway was dominant in regulating these markers (as it was in the case of MuRF1 and MAFbx) and, if so, which branches of the Akt pathway were involved in this instance of IGF-1-mediated regulation. Cell Culture—C2C12 myoblasts (American Type Culture Collection (ATCC), Rockville, MD) were maintained in define media as described previously (12Rommel C. Bodine S.C. Clarke B.A. Rossman R. Nunez L. Stitt T.N. Yancopoulos G.D. Glass D.J. Nat. Cell Biol. 2001; 3: 1009-1013Crossref PubMed Scopus (1208) Google Scholar). The myoblasts were fused into myotubes at confluence, by shifting the proliferation medium (Dulbecco's modified Eagle's medium/10% fetal bovine serum) to differentiation media (Dulbecco's modified Eagle's medium/2% horse serum) and altering the atmospheric conditions from 5 to 7.5% CO2. The time point at which differentiation is induced is referred to as day 0 (D0). The concentration of chemicals used was 10 ng/ml IGF-1 ("long-R3-IGF-1," Sigma), 100 μm DEX (Sigma), 10 μm LY294002 (Calbiochem), and 20 ng/ml rapamycin (RAP) (Calbiochem). IGF-1 and the pharmacological inhibitors LY and RAP were administered either on day two (D2) or day three (D3) post-fusion, while DEX treatments were administered on D3. Cultures were fixed and photographed by glutaraldehyde-induced autofluorescence, as described (34Stitt T.N. Drujan D. Clarke B.A. Panaro F.J. Timofeyva Y. Kline W.O. Gonzalez M. Yancopoulos G.D. Glass D.J. Mol. Cell. 2004; 14: 395-403Abstract Full Text Full Text PDF PubMed Scopus (1472) Google Scholar). Myotube diameters were measured at the end of each treatment using SPOT RT v3.2 for Windows software. The results are expressed as means ± S.E.; statistical significance was assessed using a t test for paired samples. For FOXO1 immunolocalization, adenoviral infections were performed as described previously (34Stitt T.N. Drujan D. Clarke B.A. Panaro F.J. Timofeyva Y. Kline W.O. Gonzalez M. Yancopoulos G.D. Glass D.J. Mol. Cell. 2004; 14: 395-403Abstract Full Text Full Text PDF PubMed Scopus (1472) Google Scholar). Briefly, myotube cultures in 6-well dishes were infected with myc-tagged wild-type (wt) or mutant, constitutively active FOXO1 adenovirus stocks, serum-starved for 20 h beginning on day 3 post-differentiation, and then treated with either 10 μm LY294002 or 20 ng/ml RAP for 3 h prior to fixation in 3.2% paraformaldehyde. Myotubes were permeabilized with 0.1% Nonidet P-40 and incubated with 9E10 anti-myc monoclonal antibody overnight, followed by 2 h of staining with Cy3-conjugated goat anti-mouse antibody (Sigma). Immunoblot Analysis—Cells were lysed in Triton buffer (0.5% Triton X-100, 250 mm NaCl, 50 mm Tris, pH 7.5, 1 mm EDTA, 50 mm NaF) with various protease inhibitors including 1 mm sodium orthovanadate, 1 μg/ml aprotinin, 100 nm okadaic acid, 5 μg/ml tosyl lysyl chloromethyl ketone, 10 μg/ml tosyl-l-phenylalanine chloromethyl ketone, 10 μg/ml soybean trypsin, and 1 mm phenylmethylsulfonyl fluoride and cleared by centrifugation at 16,000 × g for 15 min at 4 °C. Protein concentration was quantified using Pierce bicinchoninic acid (BCA®) assay kit. For immunoblot analysis, soluble fractions were separated in 10% Invitrogen precast tris-glycine gels using SDS-PAGE. Phosphorylation of Akt was determined with rabbit polyclonal anti-Akt and phospho-Akt (Ser473) antibodies (Cell Signaling). A rabbit polyclonal anti-peptide polyclonal antibody against MAFbx was generated (Open Biosystems), against amino acids 19–63 of rat MAFbx protein sequence. Other antibodies used in the study, including rabbit anti-p70S6K polyclonal antibody (Santa Cruz Biotechnology) and anti-phospho p70S6K (Thr421/Ser424) polyclonal antibody (Cell Signaling), were used. Northern Analysis—Northern blot analyses were performed as described previously (34Stitt T.N. Drujan D. Clarke B.A. Panaro F.J. Timofeyva Y. Kline W.O. Gonzalez M. Yancopoulos G.D. Glass D.J. Mol. Cell. 2004; 14: 395-403Abstract Full Text Full Text PDF PubMed Scopus (1472) Google Scholar). The isolation of the total RNA was completed using Qiagen's Rneasy® midi kit. Microarray Analysis—The integrity of the total RNA was determined using Agilent Technologies 2100 Bioanalyzer and the RNA 6000 Lab-Chip® kit. Total RNA concentration was measured by light absorption characteristics using a Beckman DU®520 general purpose UV-visible spectrophotometer. Gene expression was first determined using Agilent's mouse cDNA microarray kit (catalog number G4104A). In a second microarray experiment, Agilent's mouse 22k oligonucleotide microarray kit (catalog number G4121A) was used. Chips were scanned using the Agilent DNA microarray scanner. Analysis of the scanned chips was carried out using Cluster/Treeview by Michael Eisen at Stanford University. TaqMan® Real-time Quantitative Reverse Transcriptase PCR—The cDNA sequences for MAFbx/atrogin-1, MuRF1, metallothionein-1 (MT-1), metallothionein-2 (MT-2), metallothionein-3 (MT-3), and proliferin (PLF), and parvalbumin (PV) are obtainable from GenBank™. PCR primers and TaqMan® fluorogenic probes were designed from the corresponding cDNA sequences using the Primer Express 1.5 software program (Applied Biosystems), and synthesis was performed by Applied Biosystems. PCR was performed with 25 ng of cDNA using TaqMan® RT reagents kit and TaqMan® PCR core reagents kit (Applied Biosystems). Each RNA sample had a control reaction without reverse transcriptase, to evaluate any genomic DNA contamination. A standard curve generated from known amounts of genomic DNA was used to determine the amount of each RNA. A glyceraldehyde-3-phosphate dehydrogenase control RNA served to determine that equal amounts of cDNA were used in the analysis. Units used in Figs. 3 and 6B are arbitrary, based on a standard curve for RNA level using genomic DNA; for example 20 = the level of signal obtained with the sample gene from 20 ng of genomic DNA.Fig. 6A, requirement of the PI3K/Akt/mTOR pathway for IGF-1 mediated gene changes in PLF, PLF-2, PLF-3, PV, MT-2, and MAFbx. Red signifies up-regulation, and green signifies down-regulation. B, TaqMan® real-time reverse transcriptase PCR analysis of MT-1, MT-2, MAFbx, PV, and PLF after the indicated treatments. Each bar represents the standard deviation of triplicates of each sample. Units are arbitrary, based on a standard curve for RNA level using genomic DNA; for example 20 = the level of signal obtained with the sample gene from 20 ng of genomic DNA.View Large Image Figure ViewerDownload Hi-res image Download (PPT) IGF-1 Causes Hypertrophy, and Dexamethasone Induces Atrophy, in C2C12 Myotubes—We have previously validated the in vitro C2C12 muscle cell line as a system to delineate the signaling pathways mediating IGF-1-induced myotube hypertrophy and demonstrated that such hypertrophy is induced by the activation of the PI3K/Akt pathway (12Rommel C. Bodine S.C. Clarke B.A. Rossman R. Nunez L. Stitt T.N. Yancopoulos G.D. Glass D.J. Nat. Cell Biol. 2001; 3: 1009-1013Crossref PubMed Scopus (1208) Google Scholar), a mechanism subsequently implicated in hypertrophy in vivo (24Bodine S.C. Stitt T.N. Gonzalez M. Kline W.O. Stover G.L. Bauerlein R. Zlotchenko E. Scrimgeour A. Lawrence J.C. Glass D.J. Yancopoulos G.D. Nat. Cell Biol. 2001; 3: 1014-1019Crossref PubMed Scopus (1916) Google Scholar). Furthermore, we have established an analogous in vitro model of skeletal muscle atrophy, utilizing the cachectic glucocorticoid DEX (34Stitt T.N. Drujan D. Clarke B.A. Panaro F.J. Timofeyva Y. Kline W.O. Gonzalez M. Yancopoulos G.D. Glass D.J. Mol. Cell. 2004; 14: 395-403Abstract Full Text Full Text PDF PubMed Scopus (1472) Google Scholar), which induces muscle wasting in vivo by inducing the ATP-dependent proteasome pathway (35Wing S.S. Goldberg A.L. Am. J. Physiol. 1993; 264: E668-E676PubMed Google Scholar, 36Hong D.H. Forsberg N.E. Mol. Cell. Endocrinol. 1995; 108: 199-209Crossref PubMed Scopus (73) Google Scholar) and which also causes decreased protein production in vitro (34Stitt T.N. Drujan D. Clarke B.A. Panaro F.J. Timofeyva Y. Kline W.O. Gonzalez M. Yancopoulos G.D. Glass D.J. Mol. Cell. 2004; 14: 395-403Abstract Full Text Full Text PDF PubMed Scopus (1472) Google Scholar, 36Hong D.H. Forsberg N.E. Mol. Cell. Endocrinol. 1995; 108: 199-209Crossref PubMed Scopus (73) Google Scholar). Our initial goal was to determine the set of genes whose expression was perturbed at least 2-fold upon treatment with sufficient levels of IGF-1 to induce hypertrophy or sufficient concentrations of DEX so as to induce phenotypic atrophy. To induce hypertrophy, C2C12 myotubes were differentiated for 2 days (D2 myotubes) and treated with IGF-1 for 24 or 48 h. Analysis of myotubes after 48 h of IGF-1 treatment demonstrated an increase in myotube diameters of 226% (0.86 versus 0.38 relative units) (Fig. 1A). Treatment with IGF-1 also induced phosphorylation of Akt (Fig. 1B), as had been previously demonstrated in myotubes (11Rommel C. Clarke B.A. Zimmermann S. Nunez L. Rossman R. Reid K. Moelling K. Yancopoulos G.D. Glass D.J. Science. 1999; 286: 1738-1741Crossref PubMed Scopus (661) Google Scholar, 12Rommel C. Bodine S.C. Clarke B.A. Rossman R. Nunez L. Stitt T.N. Yancopoulos G.D. Glass D.J. Nat. Cell Biol. 2001; 3: 1009-1013Crossref PubMed Scopus (1208) Google Scholar); such biochemical activation served as a positive control, establishing that the PI3K/Akt pathway had been activated in the myotubes that were later assessed for mRNA changes. To induce atrophy, D3 myotubes were treated with DEX for either 8 or 24 h. The myotubes were subsequently fixed and assayed for changes in myotube diameters. Addition of DEX resulted in a distinct atrophic phenotype (Fig. 1A), with a decrease in myotube diameter of 37% (0.24 versus 0.38 relative units) (Fig. 1B). As a biochemical control, protein levels of the E3 ubiquitin ligase MAFbx were assessed (Fig. 1C), a previously validated marker of skeletal muscle atrophy (29Gomes M.D. Lecker S.H. Jagoe R.T. Navon A. Goldberg A.L. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14440-14445Crossref PubMed Scopus (1358) Google Scholar, 30Bodine S.C. Latres E. Baumhueter S. Lai V.K. Nunez L. Clarke B.A. Poueymirou W.T. Panaro F.J. Na E. Dharmarajan K. Pan Z.Q. Valenzuela D.M. DeChiara T.M. Stitt T.N. Yancopoulos G.D. Glass D.J. Science. 2001; 294: 1704-1708Crossref PubMed Scopus (2633) Google Scholar). MAFbx levels increased after both 8 and 24 h of DEX treatment (Fig. 1C). Duplicate cultures were used for assessment of DEX-induced mRNA changes. Microarray Analysis Identifies Over 500 Genes Significantly Regulated in IGF-induced Hypertrophy or DEX-induced Atrophy Models—Microarray analysis was performed to identify genes regulated during DEX-induced atrophy and IGF-1-induced hypertrophy. In this first microarray experiment, only genes whose expression changed