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Autocrine Growth Factor Signaling by Insulin-like Growth Factor-II Mediates MyoD-stimulated Myocyte Maturation

2003; Elsevier BV; Volume: 278; Issue: 42 Linguagem: Inglês

10.1074/jbc.c300299200

ISSN

1083-351X

Autores

Elizabeth M. Wilson, Marlene M. Hsieh, Peter Rotwein,

Tópico(s)

Genetics and Neurodevelopmental Disorders

Resumo

Skeletal muscle differentiation, maturation, and regeneration are regulated by interactions between intrinsic genetic programs controlled by myogenic transcription factors, including members of the MyoD and MEF2 families, and environmental cues mediated by hormones and growth factors. Insulin-like growth factors (IGFs) also play key roles in muscle development, and in the maintenance and repair of mature muscle, but their mechanisms of interaction with other muscle regulatory networks remain undefined. To evaluate the potential interplay between MyoD and IGF signaling pathways, we have studied muscle differentiation in C3H 10T1/2 fibroblasts acutely converted to myoblasts by quantitative infection with a recombinant adenovirus encoding mouse MyoD. In these cells, IGF-II gene and protein expression are induced as early events in differentiation, and the IGF-I receptor and downstream signaling molecules, including Akt, are rapidly activated. Interference with IGF-II production by a tetracycline-inhibited adenovirus expressing an IGF-II cDNA in the antisense orientation reversibly inhibited both production of muscle-specific structural proteins and myocyte fusion to form multinucleated myotubes. Similar results were achieved with a tetracycline-inhibited adenovirus expressing dominant-negative Akt. Our observations identify a robust autocrine amplification network in which MyoD enhances the later steps in muscle differentiation by induction of a locally acting growth factor. Skeletal muscle differentiation, maturation, and regeneration are regulated by interactions between intrinsic genetic programs controlled by myogenic transcription factors, including members of the MyoD and MEF2 families, and environmental cues mediated by hormones and growth factors. Insulin-like growth factors (IGFs) also play key roles in muscle development, and in the maintenance and repair of mature muscle, but their mechanisms of interaction with other muscle regulatory networks remain undefined. To evaluate the potential interplay between MyoD and IGF signaling pathways, we have studied muscle differentiation in C3H 10T1/2 fibroblasts acutely converted to myoblasts by quantitative infection with a recombinant adenovirus encoding mouse MyoD. In these cells, IGF-II gene and protein expression are induced as early events in differentiation, and the IGF-I receptor and downstream signaling molecules, including Akt, are rapidly activated. Interference with IGF-II production by a tetracycline-inhibited adenovirus expressing an IGF-II cDNA in the antisense orientation reversibly inhibited both production of muscle-specific structural proteins and myocyte fusion to form multinucleated myotubes. Similar results were achieved with a tetracycline-inhibited adenovirus expressing dominant-negative Akt. Our observations identify a robust autocrine amplification network in which MyoD enhances the later steps in muscle differentiation by induction of a locally acting growth factor. Skeletal muscle differentiation, maturation, maintenance, and repair require ongoing cooperation and coordination between an intrinsic regulatory program controlled by myogenic transcription factors and signaling pathways activated by hormones and growth factors (1Lassar A. Munsterberg A. Curr. Opin. Cell Biol. 1994; 6: 432-442Crossref PubMed Scopus (143) Google Scholar, 2Naya F.S. Olson E. Curr. Opin. Cell Biol. 1999; 11: 683-688Crossref PubMed Scopus (258) Google Scholar). The basic helix-loop-helix (bHLH) 1The abbreviations used are: bHLH, basic helix-loop-helix; IGF, insulin-like growth factor; IGF-IR, IGF-I receptor; DMEM, Dulbecco's modified Eagle's medium; MHC, myosin heavy chain; DM, differentiation medium; RT, reverse transcriptase. transcription factors of the MyoD family (MyoD, Myf-5, myogenin, and MRF4) are central regulators of myogenesis in vivo and in cell culture (3Weintraub H. Cell. 1993; 75: 1241-1244Abstract Full Text PDF PubMed Scopus (931) Google Scholar, 4Buckingham M. Curr. Opin. Genet. Dev. 2001; 11: 440-448Crossref PubMed Scopus (351) Google Scholar). These proteins bind as heterodimers with other widely expressed bHLH transcription factors to DNA control regions termed E boxes that are often found in the promoters of muscle-specific genes (4Buckingham M. Curr. Opin. Genet. Dev. 2001; 11: 440-448Crossref PubMed Scopus (351) Google Scholar). Subsequent interactions with transcriptional co-activators are necessary for gene activation (5Sartorelli V. Puri P.L. Hamamori Y. Ogryzko V. Chung G. Nakatani Y. Wang J.Y. Kedes L. Mol. Cell. 1999; 4: 725-734Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar, 6Polesskaya A. Naguibneva I. Fritsch L. Duquet A. Ait-Si-Ali S. Robin P. Vervisch A. Pritchard L.L. Cole P. Harel-Bellan A. EMBO J. 2001; 20: 6816-6825Crossref PubMed Scopus (100) Google Scholar). As established by gene knock-out studies in mice, MyoD and Myf-5 act redundantly at an early step in myoblast specification. Mice expressing either factor alone form muscle but do not if both proteins are absent (7Rudnicki M.A. Schnegelsberg P.N. Stead R.H. Braun T. Arnold H.H. Jaenisch R. Cell. 1993; 75: 1351-1359Abstract Full Text PDF PubMed Scopus (1327) Google Scholar). MyoD also is required for normal muscle regeneration in the adult (8Sabourin L.A. Girgis-Gabardo A. Seale P. Asakura A. Rudnicki M.A. J. Cell. Biol. 1999; 144: 631-643Crossref PubMed Scopus (273) Google Scholar). Myogenin acts downstream of MyoD/Myf-5 to promote differentiation (9Hasty P. Bradley A. Morris J.H. Edmondson D.G. Venuti J.M. Olson E.N. Klein W.H. Nature. 1993; 364: 501-506Crossref PubMed Scopus (1028) Google Scholar, 10Nabeshima Y. Hanaoka K. Hayasaka M. Esumi E. Li S. Nonaka I. Nabeshima Y. Nature. 1993; 364: 532-535Crossref PubMed Scopus (729) Google Scholar), while MRF4 plays a more limited role in muscle formation in vivo (reviewed in Ref. 11Olson E.N. Arnold H.H. Rigby P.W. Wold B.J. Cell. 1996; 85: 1-4Abstract Full Text Full Text PDF PubMed Scopus (437) Google Scholar). Insulin-like growth factors (IGF-I and -II) also play key roles in normal muscle development in the embryo (12Liu J.P. Baker J. Perkins A.S. Robertson E.J. Efstratiadis A. Cell. 1993; 75: 59-72Abstract Full Text PDF PubMed Scopus (2585) Google Scholar, 13Powell-Braxton L. Hollingshead P. Warburton C. Dowd M. Pitts-Meek S. Dalton D. Gillett N. Stewart T.A. Genes Dev. 1993; 7: 2609-2617Crossref PubMed Scopus (677) Google Scholar) and are important for coordinating muscle regeneration and re-innervation following injury (14Caroni P. Schneider C. Kiefer M.C. Zapf J. J. Cell. Biol. 1994; 125: 893-902Crossref PubMed Scopus (102) Google Scholar, 15Barton E.R. Morris L. Musaro A. Rosenthal N. Sweeney H.L. J. Cell. Biol. 2002; 157: 137-148Crossref PubMed Scopus (399) Google Scholar). IGF action additionally may be critical for sustaining muscle mass during aging (16Barton-Davis E.R. Shoturma D.I. Musaro A. Rosenthal N. Sweeney H.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15603-15607Crossref PubMed Scopus (603) Google Scholar, 17Bodine 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 (1921) Google Scholar, 18Musaro 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 (890) Google Scholar, 19Paul A.C. Rosenthal N. J. Cell. Biol. 2002; 156: 751-760Crossref PubMed Scopus (101) Google Scholar). In muscle and in other cell types the actions of both IGFs are mediated by the IGF-I receptor (IGF-IR), a ligand-stimulated membrane-spanning tyrosine protein kinase that activates several intracellular signaling pathways through a series of adaptor molecules (20Nakae J. Kido Y. Accili D. Endocr. Rev. 2001; 22: 818-835Crossref PubMed Scopus (357) Google Scholar). IGF action also is modified through high affinity interactions with a series of extracellular IGF-binding proteins, IGFBP-1 through -6 that limit access to the IGF-IR, but also enhance growth factor half-life (21Clemmons D.R. Cytokine Growth Factor Rev. 1997; 8: 45-62Crossref PubMed Scopus (435) Google Scholar). In contrast to differences observed among bHLH proteins in vivo, tissue culture studies have demonstrated similar functions for these transcription factors. In culture, each protein has been found to direct a range of cell types toward the myoblast lineage, to promote cell cycle arrest, and to stimulate differentiation (1Lassar A. Munsterberg A. Curr. Opin. Cell Biol. 1994; 6: 432-442Crossref PubMed Scopus (143) Google Scholar, 3Weintraub H. Cell. 1993; 75: 1241-1244Abstract Full Text PDF PubMed Scopus (931) Google Scholar), in part through cooperation with members of the MEF2 family of transcriptional factors, which act as accessory regulators of muscle gene expression and differentiation (2Naya F.S. Olson E. Curr. Opin. Cell Biol. 1999; 11: 683-688Crossref PubMed Scopus (258) Google Scholar). Here using C3H 10T1/2 fetal fibroblasts as a model, we identify another mechanism by which MyoD enhances its myogenic actions, through induction of IGF-II gene and protein expression early in differentiation. Secreted IGF-II in turn activates the IGF-IR and downstream signaling molecules, including Akt. As inhibition of IGF-II expression or impairment of Akt prevents production of muscle structural proteins and blocks formation of multinucleated myofibers, these results define an autocrine amplification pathway by which myogenic bHLH proteins stimulate the later events of muscle differentiation through production of a locally acting growth factor. Materials—Fetal calf serum, newborn calf serum, horse serum, and trypsin were purchased from Invitrogen Inc. Dulbecco's modified Eagle's medium (DMEM) and phosphate-buffered saline were from Mediatech-Cellgrow (Herndon, VA). R3IGF-I was from Gro-Pep (Adelaide, Australia). Doxycycline was from Clontech (Palo Alto, CA) and was dissolved in distilled water at a concentration of 500 μg/ml and stored at –20 °C until use. Protease inhibitor tablets were purchased from Roche Applied Sciences, okadaic acid from Alexis Biochemicals (San Diego, CA), and sodium orthovanadate from Sigma. Effectene was from Qiagen Inc. (Valencia, CA), and TransIT-LT-1 was from Mirus Corp. (Madison, WI). The BCA protein assay kit was from Pierce, and nitrocellulose was from Osmonics (Westborough, MA). Restriction enzymes, buffers, ligases, and polymerases were purchased from Roche Applied Sciences, BD Biosciences (Clontech), and Fermentas (Hanover, MD). Reagents for enhanced chemifluorescence were from Amersham Biosciences. Several monoclonal antibodies were purchased from the Developmental Studies Hybridoma Bank (Iowa City, IA), including F5D (anti-myogenin, W. E. Wright), MF20 (anti-myosin heavy chain (MHC), D. A. Fischman), and CT3 (anti-troponin T, J. J.-C. Lin). A monoclonal antibody to MyoD was from BD Biosciences (Pharmingen, San Diego, CA). Polyclonal antibodies to Akt and phospho-Akt (Ser473) were from Cell Signaling Technology (Beverly, MA), and the polyclonal antibody to IGF-II was purchased from Abcam, Ltd. (Cambridge, UK). A monclonal antibody to phosphotyrosine was from Santa Cruz Biotechnology (Santa Cruz, CA), as was a polyclonal antibody to the β subunit of the IGF-I receptor. Antibody conjugates were purchased from Molecular Probes (Eugene, OR): goat anti-mouse IgG1-Alexa 488, goat anti-mouse IgG2b-Alexa 594, anti-rabbit IgG-alkaline phosphatase, and anti-mouse IgG-alkaline phosphatase. The AdEasy adenoviral recombinant kit was from Q-BIO Gene (Carlsbad, CA). All other chemicals were reagent grade and were purchased from commercial suppliers. Cell Culture—C3H 10T1/2 mouse embryonic fibroblasts (ATCC catalog number CCL226) were incubated on gelatin-coated tissue culture dishes in growth media (DMEM with 10% heat-inactivated fetal bovine serum and 10% newborn calf serum) at 37 °C in humidified air with 5% CO2, until they reached 50% of confluent density for acute infection with recombinant adenoviruses. Differentiation was initiated 1 day later after cells reached ∼95% of confluent density after washing with phosphate-buffered saline by addition of differentiation medium (DM), consisting of DMEM plus 2% horse serum (22Tureckova J. Wilson E.M. Cappalonga J.L. Rotwein P. J. Biol. Chem. 2001; 276: 39264-39270Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). In some experiments DM consisted of DMEM plus 0.5% horse serum. Construction and Use of Recombinant Adenovirus—A FLAG epitope tag followed by a stop codon and XbaI site were added to the 3′ end of the coding region of mouse MyoD by polymerase chain reaction. The modified cDNA was sequenced, digested with SalI and XbaI restriction endonucleases, and ligated into the pShuttle:CMV vector. A dominant-negative Akt (AktDN) was prepared by modifying a human Akt-1 cDNA with a T7 epitope tag at its 3′ end (a gift from Dr. Richard Roth, Stanford University School of Medicine) by mutating codons for lysine 179, threonine 308, and serine 473 to alanines using the QuikChange mutagenesis kit (Stratagene, La Jolla, CA). Each mutation was verified by restriction enzyme mapping and by DNA sequencing. The AktDN cDNA was then subcloned via SalI and XbaI restriction endonuclease sites into a modified pShuttle plasmid containing a tetracycline-regulated promoter (22Tureckova J. Wilson E.M. Cappalonga J.L. Rotwein P. J. Biol. Chem. 2001; 276: 39264-39270Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar), as was the coding region of mouse IGF-II in the antisense orientation (23Stewart C.E. Rotwein P. J. Biol. Chem. 1996; 271: 11330-11338Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar). All recombinant adenoviruses were generated and isolated via a protocol supplied by Q-BIO Gene. The adenovirus encoding a tetracyline-inhibited transactivator (Ad-tTA) has been described (22Tureckova J. Wilson E.M. Cappalonga J.L. Rotwein P. J. Biol. Chem. 2001; 276: 39264-39270Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). A recombinant adenovirus encoding β-galactosidase (Ad-β-Gal) was a gift from Dr. J. Molkentin, University of Cincinnati School of Medicine. All viruses were purified on discontinuous cesium chloride gradients and titered by optical density. For infections, recombinant adenoviruses (Ad-tTA at a multiplicity of infection of 125, others at 250) were diluted in DMEM plus 2% fetal bovine serum, filtered through a Gelman syringe filter (0.45 μm), and added to cells at 37 °C for 120 min. After addition of an equal volume of DMEM with 20% each of fetal bovine serum and newborn calf serum, cells were incubated for a further 24 h and then were placed in DM. Under these conditions, ∼90% of cells were infected. Immunoblotting—Whole cell protein lysates were prepared after washing cells with phosphate-buffered saline and incubating on ice for 15 min in RIPA Buffer (50 mm Tris-Cl, pH 7.5, 150 mm NaCl, 0.1% SDS, 0.5% sodium deoxycholate, and 1% IGEPAL CA-630). Lysates were passed through a 22-gauge needle and centrifuged at 15,000 rpm at 4 °C to remove insoluble material, and protein concentrations were determined using the BCA protein assay kit. Protein samples (30 μg each) were separated by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and incubated with antibodies as described previously (22Tureckova J. Wilson E.M. Cappalonga J.L. Rotwein P. J. Biol. Chem. 2001; 276: 39264-39270Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). Antibodies were used at the following dilutions: anti-MHC (1:500), anti-myogenin (1:500), anti-troponin T (1:1000), anti-Akt (1:2000), anti-phospho-Akt (Ser473) (1:1000), anti-IGF-II (1:1000), anti-IGF-I receptor β subunit (1:500), anti-phospho-tyrosine (1:1000). IGF-II was detected by immunoblotting after extraction from conditioned DM using Sep-Pak Vac 1cc C18 columns (Waters Corp., Milford, MA), as described previously (24Florini J.R. Magri K.A. Ewton D.Z. James P.L. Grindstaff K. Rotwein P.S. J. Biol. Chem. 1991; 266: 15917-15923Abstract Full Text PDF PubMed Google Scholar). The detection limit was 20 ng (∼3nm) of purified IGF-II. Akt enzymatic activity was assessed by immunoblotting using GSK-3β as substrate and anti-phospho-GSK-3β for detection after immunoprecipitation of 200 μg of cell lysates with anti-Akt conjugated to agarose beads, according to a protocol supplied by the manufacturer (Cell Signaling Technology). Tyrosine phosphorylation of the β subunit of the IGF-I receptor was evaluated by two methods, with comparable results. Cell lysates (500 μg) were immunoprecipitated with an antibody to the β subunit of the IGF-I receptor (1:150 dilution), followed by immunoblotting with anti-phosphotyrosine; alternatively, lysates (100 μg) were immunoprecipitated with anti-phosphotyrosine (1:40 dilution), followed by immunoblotting with anti-IGF-I receptor β subunit. Immunocytochemistry—Cells were fixed in 4% paraformaldehyde for 15 min at 20 °C and permeabilized with a 50:50 mixture of methanol and acetone for 2 min before blocking in 0.25% normal goat serum for >1hat20 °C. Primary antibodies diluted in blocking buffer were added for 16 h at 4 °C (anti-MHC, 1:250 dilution, anti-myogenin, 1:250 dilution). After a washing step, cells were incubated for 2 h at 20 °C in goat anti-mouse IgG2b-Alexa 594 (red) and goat anti-mouse IgG1-Alexa 488 (green), each diluted to 1:1000 in blocking buffer. Images were captured with a Roper Scientific Cool Snap FX CCD camera attached to a Nikon Eclipse T300 fluorescent microscope using IP Labs 3.5 software. RNA Isolation and Analysis—Whole cell RNA was isolated as described (23Stewart C.E. Rotwein P. J. Biol. Chem. 1996; 271: 11330-11338Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar). RNA concentration was determined spectrophotometrically at 260 nm, and its quality assessed by agarose gel electrophoresis. RNA (2.5 μg) was reverse-transcribed in a final volume of 20 μl using a RT-PCR kit (Invitrogen) with oligo(dT) primers. Each PCR reaction contained 1.0 μl of cDNA. Primer sequences were as follows: for IGF-II, 5′-TCAAGCCGTGCCAACCGTCGC-3′ (sense strand), 5′-CTCCGAAGAGGCTCCCCCGTG-3′ (antisense); for IGF-I, 5′-CTCTTCAGTTCGTGTGTGGACC-3′(sense), 5′-CCACACTGACATGCCCAAGAC-3′ (antisense); for myogenin, 5′-ATGGAGCTGTATGAGACATCC-3′ (sense), 5′-GACGAAACCATGCCCAACTGA-3′ (antisense); for S17, 5′-ATCCCCAGCAAGAAGCTTCGGAACA-3′ (sense), 5′-TATGGCATAACAGATTAAACAGCTC-3′ (antisense). The linear range of product amplification was established in pilot studies for each primer pair, and the cycle number representing the approximate midpoint was used in final experiments. This varied from 20 to 25 cycles. Results were quantified by densitometry after electrophoresis through 1.2% agarose gels. Akt Activity Is Required for MyoD-mediated Myoblast Differentiation—Recently published studies have demonstrated that Akt activity and protein expression are stimulated during differentiation of established muscle cell lines (17Bodine 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 (1921) Google Scholar, 22Tureckova J. Wilson E.M. Cappalonga J.L. Rotwein P. J. Biol. Chem. 2001; 276: 39264-39270Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 25Vandromme M. Rochat A. Meier R. Carnac G. Besser D. Hemmings B.A. Fernandez A. Lamb N.J. J. Biol. Chem. 2001; 276: 8173-8179Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 26Jiang B.H. Aoki M. Zheng J.Z. Li J. Vogt P.K. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2077-2081Crossref PubMed Scopus (227) Google Scholar, 27Rommel 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 (663) Google Scholar, 28Rommel 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 (1212) Google Scholar). Here we show that Akt enzymatic activity also is induced as an early event in differentiation in fibroblasts converted to myoblasts after acute infection with a recombinant adenovirus encoding mouse MyoD (Ad-MyoD) and that active Akt is needed for the later events in differentiation that culminate in myotube formation. Illustrated in Fig. 1 are results of time course experiments using 10T1/2 fibroblasts infected 1 day earlier with Ad-MyoD at an m.o.i. in which ∼90% of cells express MyoD (data not shown) and subsequently incubated in DM. As seen in Fig. 1A, Ad-MyoD-infected 10T1/2 cells underwent rapid and extensive muscle differentiation, with progressive expression of myogenin and MHC, and formation of large multinucleated myotubes within 2 days. Similarly robust muscle-specific protein expression was observed by immunoblotting (Fig. 1B). Myogenin and MHC were induced in Ad-MyoD-infected cells but were not detected in fibroblasts infected with an adenovirus encoding β-galactosidase (Ad-β-Gal). Also seen in Fig. 1B is evidence of activation of Akt beginning by 16 h after addition of DM, as indicated by a progressive increase in phosphorylation on serine 473. A similarly large induction of Akt enzymatic activity was observed, as shown in Fig. 1C. In Ad-MyoD infected cells, Akt kinase activity was stimulated by ∼20-fold by 1 day in DM, was increased by over 30-fold by 2 days, and was maintained at high levels for at least 3 days. Little Akt activity could be detected in fibroblasts acutely infected with Ad-β-Gal (data not shown). In contrast to these results, minimal activation of the MAP kinases, Erks 1 and 2 or p38, was seen in Ad-MyoD or Ad-β-Gal-infected cells during the same interval (data not shown). The significance of the rise in Akt kinase activity in MyoD-mediated muscle differentiation was tested by co-infecting 10T1/2 cells with recombinant adenoviruses expressing MyoD and a dominant-negative Akt under control of a tetracycline-regulated gene promoter (Ad-AktDN). In pilot studies, Ad-AktDN blocked IGF-induced Akt enzymatic activity (data not shown). As seen in Fig. 2, A and B, inhibition of endogenous Akt blocked MHC and troponin-T expression and myotube formation but had no effect on production of myogenin. In contrast, when expression of AktDN was impeded by the tetracycline analog, doxycycline, differentiation proceeded normally. Thus, Akt activity is required for MyoD-stimulated muscle differentiation. IGF-II mRNA and Protein Expression Are Induced, and the IGF-I Receptor Is Activated during MyoD-mediated Muscle Differentiation—We next investigated mechanisms responsible for activation of Akt during MyoD-stimulated myoblast differentiation. We first examined components of the IGF system, as IGF signaling has been found to stimulate Akt and to enhance differentiation of established muscle cell lines and in vivo (22Tureckova J. Wilson E.M. Cappalonga J.L. Rotwein P. J. Biol. Chem. 2001; 276: 39264-39270Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 28Rommel 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 (1212) Google Scholar). As shown in Fig. 3A, IGF-II gene expression, measured by semi-quantitative RT-PCR assay, was progressively induced in Ad-MyoD infected fibroblasts beginning by 8 h after incubation in DM. In contrast, little IGF-I mRNA could be detected over the same time frame, and transcripts for IGF-II and myogenin were not seen in fibroblasts infected with Ad-β-Gal and incubated in DM for the same time period. Transcripts for ribosomal protein S17 did not change in abundance. IGF-II protein expression also was induced in Ad-MyoD-infected fibroblasts. As assessed by immunoblotting of conditioned DM, IGF-II accumulation was observed in concentrated media from cells infected with Ad-MyoD after 1 day and showed a dramatic increase by 2 days (Fig. 3B). Little IGF-II could be seen by 12 h in DM (data not shown), and none was detected in concentrated conditioned media from fibroblasts infected with Ad-β-Gal. Thus, induction of IGF-II gene and protein expression are early events in MyoD-regulated myoblast differentiation. IGF-II activates intracellular signal transduction pathways by binding to the IGF-IR, a ligand-stimulated tyrosine protein kinase that undergoes autophosphorylation as an initial event in its activation (20Nakae J. Kido Y. Accili D. Endocr. Rev. 2001; 22: 818-835Crossref PubMed Scopus (357) Google Scholar). To assess phosphorylation of the IGF-IR in Ad-MyoD-infected 10T1/2 fibroblasts, lysates from cells incubated in DM were immunoprecipitated with an antibody to the β subunit of the receptor, followed by immunoblotting with an antibody to phosphotyrosine. As shown in Fig. 3C, progressively increasing tyrosine phosphorylation of the IGF-IR was detected beginning at 1 day after incubation of cells in DM. By 2 days, the extent of receptor tyrosine phosphorylation exceeded that induced in 10T1/2 cells after incubation with IGF-I for 15 min. By contrast, little receptor phosphorylation was seen after 12 h in DM in Ad-MyoD-infected fibroblasts (data not shown) or in Ad-β-Gal-infected 10T1/2 cells after up to 2 days in DM (Fig. 3C). IGF-II Action Is Required for MyoD-stimulated Muscle Differentiation—Experiments were performed next to assess the functional significance of production of IGF-II and activation of the IGF-IR for MyoD-mediated myoblast differentiation. Fibroblasts were co-infected with Ad-MyoD and a recombinant adenovirus encoding an antisense cDNA for mouse IGF-II under control of a tetracycline-inhibited promoter (Ad-IGF-IIAS). As shown in Fig. 4A, Ad-IGF-IIAS caused a marked decline in induction of IGF-II mRNA after a 1 day incubation of cells in DM, which was reversed by doxycycline. Secretion of IGF-II also was blocked in cells expressing IGF-IIAS mRNA, and tyrosine phosphorylation of the IGF-IR was impaired, being seen only at the 2-day time point (Fig. 4, B and C). This apparent discrepancy may be explained by the partial inhibition of IGF-II mRNA by Ad-IGF-IIAS and by the greater sensitivity of the assay for IGF-IR tyrosine phosphorylation than the assay for IGF-II protein (with a detection limit of ∼3 nm of IGF-II). Both were restored to normal with doxycycline (Fig. 4, B and C). Thus, Ad-IGF-IIAS impaired both IGF-II production and IGF-IR activation. Inhibition of IGF-II also diminished MyoD-mediated differentiation. As shown in Fig. 4, D and E, Ad-IGF-IIAS reduced the rise in myogenin accumulation, blocked MHC expression, prevented myotube formation, and delayed Akt phosphorylation. In contrast, when expression of IGF-IIAS mRNA was blocked by doxycycline, Akt phosphorylation was restored and differentiation proceeded normally. Thus, early production of IGF-II and activation of the IGF-IR are required for MyoD-stimulated muscle differentiation. The central role of MyoD and related bHLH transcription factors in muscle cell specification and differentiation has been known for over a decade (3Weintraub H. Cell. 1993; 75: 1241-1244Abstract Full Text PDF PubMed Scopus (931) Google Scholar), and numerous studies have demonstrated that MyoD can readily convert a range of cell types to myoblasts (3Weintraub H. Cell. 1993; 75: 1241-1244Abstract Full Text PDF PubMed Scopus (931) Google Scholar). We now find that an endogenously initiated signaling pathway, involving induction of IGF-II gene and protein expression, and stimulation of the IGF-IR and Akt, are additional key components of MyoD-mediated myoblast differentiation. IGF-II production, and IGF-IR and Akt activation, are relatively early events in the actions of MyoD in this model system, occurring soon after induction of myogenin, and this signaling pathway appears necessary for differentiation to proceed, as either inhibition of IGF-II or blockade of Akt impaired expression of MHC and troponin-T and formation of multinucleated myofibers. Thus, at least in the context of this model, our results indicate that IGF-II functions to initiate an essential autocrine amplification cascade for MyoD-mediated differentiation. Our observations also identify an approach that will be useful in defining the critical signaling pathways that act downstream of Akt in muscle cells. The biochemical mechanisms by which MyoD induces IGF-II gene expression are unknown. Previous studies have established that IGF-II gene transcription is stimulated during differentiation of established muscle cell lines (29Kou K. Rotwein P. Mol. Endocrinol. 1993; 7: 291-302PubMed Google Scholar) but have not identified key DNA response elements or defined critical transcription factors. The mouse IGF-II gene is complicated. It contains three tandem promoters, each with unique 5′ noncoding exons (30Rotwein P. Hall L.J. DNA Cell Biol. 1990; 9: 725-735Crossref PubMed Scopus (109) Google Scholar). IGF-II gene expression is also regulated by genomic imprinting, and the gene resides ∼70 kb 3′ to the H19 gene within a large imprinted locus on mouse chromosome 7 (31Issa J.P. Baylin S.B. Nat. Med. 1996; 2: 281-282Crossref PubMed Scopus (34) Google Scholar). The two genes are reciprocally imprinted with IGF-II being expressed from the paternally derived chromosome and H19 from the maternal chromosome (31Issa J.P. Baylin S.B. Nat. Med. 1996; 2: 281-282Crossref PubMed Scopus (34) Google Scholar). Investigation into the mechanisms of imprinting has led to identification of a key paternally methylated genomic region located just 5′ to H19 that functions as an insulator element and regulates reciprocal expression of the two genes (32Bell A.C. Felsenfeld G. Nature. 2000; 405: 482-485Crossref PubMed Scopus (1373) Google Scholar, 33Hark A.T. Schoenherr C.J. Katz D.J. Ingram R.S. Levorse J.M. Tilghman S.M. Nature. 2000; 405: 486-489Crossref PubMed Scopus (1230) Google Scholar). 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Genes Dev. 2000; 14: 1908-1919PubMed Google Scholar, 36Ainscough J.F. John R.M. Barton S.C. Surani M.A. Development (Camb.). 2000; 127: 3923-3930PubMed Google Scholar) are critical for MyoD-induced IGF-II gene activity in muscle. We thank Dr. Jay Nelson of the Oregon Health & Science University for Ad-tTA, Dr. Jeffrey Molkentin, University of Cincinnati, for Ad-β-Gal, Dr. Andrew Lassar, Harvard University Medical School, for mouse MyoD, and Dr. Richard Roth of Stanford University for wild-type human Akt-1 with a T7 epitope tag. We also appreciate helpful advice from other members of our laboratory during the course of this work.

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