Glycogen Synthase Kinase 3β Suppresses Myogenic Differentiation through Negative Regulation of NFATc3

Skeletal muscle atrophy is a prominent and disabling feature in many chronic diseases. Prevention or reversal of muscle atrophy by stimulation of skeletal muscle growth could be an important therapeutic strategy. Glycogen synthase kinase 3β (GSK-3β) has been implicated in the negative regulation of skeletal muscle growth. Since myogenic differentiation is an essential part of muscle growth, we investigated if inhibition of GSK-3β is sufficient to stimulate myogenic differentiation and whether this depended on regulation of the transcription factor nuclear factor of activated T-cells (NFAT). In both myogenically converted mouse embryonic fibroblasts and C2C12 myoblasts, deficiency of GSK-3β protein (activity) resulted in enhanced myotube formation and muscle-specific gene expression during differentiation, which was reversed by reintroduction of wild type but not kinase-inactive (K85R) GSK-3β. In addition, GSK-3β inhibition restored myogenic differentiation following calcineurin blockade, which suggested the involvement of NFAT. GSK-3β-deficient mouse embryonic fibroblasts or myoblasts displayed enhanced nuclear translocation of NFATc3 and elevated NFAT-sensitive promoter transactivation, which was reduced by reintroducing wild type, but not K85R GSK-3β. Overexpression of NFATc3 increased muscle gene promoter transactivation, which was abolished by co-expression of wild type GSK-3β. Finally, stimulation of muscle gene expression observed following GSK-3β inhibition was strongly attenuated in NFATc3-deficient myoblasts, indicating that this response requires NFATc3. Collectively, our data demonstrate negative regulation of myogenic differentiation by GSK-3β through a transcriptional mechanism that depends on NFATc3. Inhibition of GSK-3β may be a potential strategy in prevention or treatment of muscle atrophy. Skeletal muscle atrophy is a prominent and disabling feature in many chronic diseases. Prevention or reversal of muscle atrophy by stimulation of skeletal muscle growth could be an important therapeutic strategy. Glycogen synthase kinase 3β (GSK-3β) has been implicated in the negative regulation of skeletal muscle growth. Since myogenic differentiation is an essential part of muscle growth, we investigated if inhibition of GSK-3β is sufficient to stimulate myogenic differentiation and whether this depended on regulation of the transcription factor nuclear factor of activated T-cells (NFAT). In both myogenically converted mouse embryonic fibroblasts and C2C12 myoblasts, deficiency of GSK-3β protein (activity) resulted in enhanced myotube formation and muscle-specific gene expression during differentiation, which was reversed by reintroduction of wild type but not kinase-inactive (K85R) GSK-3β. In addition, GSK-3β inhibition restored myogenic differentiation following calcineurin blockade, which suggested the involvement of NFAT. GSK-3β-deficient mouse embryonic fibroblasts or myoblasts displayed enhanced nuclear translocation of NFATc3 and elevated NFAT-sensitive promoter transactivation, which was reduced by reintroducing wild type, but not K85R GSK-3β. Overexpression of NFATc3 increased muscle gene promoter transactivation, which was abolished by co-expression of wild type GSK-3β. Finally, stimulation of muscle gene expression observed following GSK-3β inhibition was strongly attenuated in NFATc3-deficient myoblasts, indicating that this response requires NFATc3. Collectively, our data demonstrate negative regulation of myogenic differentiation by GSK-3β through a transcriptional mechanism that depends on NFATc3. Inhibition of GSK-3β may be a potential strategy in prevention or treatment of muscle atrophy. Maintenance of muscle mass is critical for health, and loss of skeletal muscle mass compromises human physical condition and survival in chronic diseases, such as chronic obstructive pulmonary disease (1Schols A.M. Broekhuizen R. Weling-Scheepers C.A. Wouters E.F. Am. J. Clin. Nutr. 2005; 82: 53-59Crossref PubMed Scopus (598) Google Scholar, 2Vestbo J. Prescott E. Almdal T. Dahl M. Nordestgaard B.G. Andersen T. Sorensen T.I. Lange P. Am. J. Respir. Crit. Care Med. 2006; 173: 79-83Crossref PubMed Google Scholar). Restoring lost muscle mass is important for improving quality of life and ultimately disease prognosis (3Schols A.M. Slangen J. Volovics L. Wouters E.F. Am. J. Respir. Crit. Care Med. 1998; 157: 1791-1797Crossref PubMed Scopus (887) Google Scholar). To restore muscle mass and improve muscle function in various diseases conditions, a better understanding of the molecular mechanisms of skeletal muscle (re)growth is required. Skeletal muscle differentiation is a critical element of certain types of postnatal growth of the skeletal musculature and is mainly dependent on satellite cells (quiescent myoblasts), which upon activation proliferate, differentiate, and fuse with existing muscle fibers or with each other to form new myofibers (4Allen D.L. Monke S.R. Talmadge R.J. Roy R.R. Edgerton V.R. J. Appl. Physiol. 1995; 78: 1969-1976Crossref PubMed Scopus (204) Google Scholar). Myoblast fusion allows additional muscle growth by myonuclear accretion, beyond the limitations imposed by the myonuclear domain (i.e. the maximal cytoplasm/nucleus ratio) (5Allen D.L. Roy R.R. Edgerton V.R. Muscle Nerve. 1999; 22: 1350-1360Crossref PubMed Scopus (370) Google Scholar). The protein kinase GSK-3 2The abbreviations used are:GSK-3glycogen synthase kinase 3βNFATnuclear factor of activated T-cellsGMgrowth mediumDMEMDulbecco's modified Eagle's mediumFBSfetal bovine serumDMdifferentiation mediumMEFmouse embryo fibroblastTnItroponin IshRNAshort hairpin RNAshGSKshort hairpin glycogen synthase kinaseWTwild typecaconstitutively activatedIGFinsulin-like growth factorpRSpRetro-Super is ubiquitously expressed, and, although it was originally identified as a suppressor of glycogen synthase (6Embi N. Rylatt D.B. Cohen P. Eur. J. Biochem. 1980; 107: 519-527Crossref PubMed Scopus (812) Google Scholar), GSK-3 has been implicated in a myriad of metabolic and signaling pathways (7Frame S. Cohen P. Biochem. J. 2001; 359: 1-16Crossref PubMed Scopus (1280) Google Scholar). Recent studies have identified GSK-3β as a negative regulator of both cardiac and skeletal muscle hypertrophy (8Haq S. Choukroun G. Kang Z.B. Ranu H. Matsui T. Rosenzweig A. Molkentin J.D. Alessandrini A. Woodgett J. Hajjar R. Michael A. Force T. J. Cell Biol. 2000; 151: 117-130Crossref PubMed Scopus (335) Google Scholar, 9Rommel 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 (1213) Google Scholar) as well as muscle differentiation (10van der Velden J.L. Langen R.C. Kelders M.C. Wouters E.F. Janssen-Heininger Y.M. Schols A.M. Am. J. Physiol. 2006; 290: C453-C462Crossref PubMed Scopus (86) Google Scholar). glycogen synthase kinase 3β nuclear factor of activated T-cells growth medium Dulbecco's modified Eagle's medium fetal bovine serum differentiation medium mouse embryo fibroblast troponin I short hairpin RNA short hairpin glycogen synthase kinase wild type constitutively activated insulin-like growth factor pRetro-Super Regulation of the transcriptional regulator nuclear factor of activated T-cells (NFAT) depends on the balance between inhibitory phosphorylation by GSK-3β and stimulatory dephosphorylation by the calcium-dependent serine/threonine phosphatase calcineurin. Activated calcineurin exposes the nuclear localization signal of NFAT, resulting in its nuclear translocation (11Chin E.R. Olson E.N. Richardson J.A. Yang Q. Humphries C. Shelton J.M. Wu H. Zhu W. Bassel-Duby R. Williams R.S. Genes Dev. 1998; 12: 2499-2509Crossref PubMed Scopus (836) Google Scholar). Conversely, GSK-3β-dependent phosphorylation masks the nuclear localization signal, resulting in nuclear export of NFAT and termination of calcineurin-induced gene transcription (12Beals C.R. Sheridan C.M. Turck C.W. Gardner P. Crabtree G.R. Science. 1997; 275: 1930-1934Crossref PubMed Scopus (635) Google Scholar, 13Neal J.W. Clipstone N.A. J. Biol. Chem. 2001; 276: 3666-3673Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). Although NFAT was identified in T-cells, several studies have postulated a role for NFAT in skeletal muscle gene expression (14Corin S.J. Levitt L.K. O'Mahoney J.V. Joya J.E. Hardeman E.C. Wade R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6185-6189Crossref PubMed Scopus (47) Google Scholar, 15Musaro A. McCullagh K.J. Naya F.J. Olson E.N. Rosenthal N. Nature. 1999; 400: 581-585Crossref PubMed Scopus (557) Google Scholar). Five NFAT isoforms have been identified (16Olson E.N. Williams R.S. BioEssays. 2000; 22: 510-519Crossref PubMed Scopus (221) Google Scholar), and four isoforms (NFATc1, -c2, -c3, and -5) are expressed in skeletal muscle (17Abbott K.L. Friday B.B. Thaloor D. Murphy T.J. Pavlath G.K. Mol. Biol. Cell. 1998; 9: 2905-2916Crossref PubMed Scopus (196) Google Scholar, 18O'Connor R.S. Mills S.T. Jones K.A. Ho S.N. Pavlath G.K. J. Cell Sci. 2007; 120: 149-159Crossref PubMed Scopus (97) Google Scholar). NFATc1, -c2, -c3, and -5 have overlapping but also nonredundant functions in skeletal muscle, including control of myogenic differentiation, myoblast migration and fusion, and fiber type specification (18O'Connor R.S. Mills S.T. Jones K.A. Ho S.N. Pavlath G.K. J. Cell Sci. 2007; 120: 149-159Crossref PubMed Scopus (97) Google Scholar, 19Horsley V. Friday B.B. Matteson S. Kegley K.M. Gephart J. Pavlath G.K. J. Cell Biol. 2001; 153: 329-338Crossref PubMed Scopus (203) Google Scholar, 20Kegley K.M. Gephart J. Warren G.L. Pavlath G.K. Dev. Biol. 2001; 232: 115-126Crossref PubMed Scopus (102) Google Scholar, 21Schulz R.A. Yutzey K.E. Dev. Biol. 2004; 266: 1-16Crossref PubMed Scopus (237) Google Scholar). Calcineurin has been implicated in the regulation of skeletal muscle growth (22Dunn S.E. Burns J.L. Michel R.N. J. Biol. Chem. 1999; 274: 21908-21912Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar), regrowth (23Miyazaki M. Hitomi Y. Kizaki T. Ohno H. Katsumura T. Haga S. Takemasa T. Med. Sci. Sports Exerc. 2006; 38: 1065-1072Crossref PubMed Scopus (22) Google Scholar), and myogenic differentiation (24Delling U. Tureckova J. Lim H.W. P. Molkentin J.D. Mol. Cell. Biol. 2000; PubMed Scopus Google Scholar). NFATc1, -c2, and -c3, but not are to calcineurin C. J. A. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar), and stimulation of myogenic differentiation activation of calcineurin NFATc3 (17Abbott K.L. Friday B.B. Thaloor D. Murphy T.J. Pavlath G.K. Mol. Biol. Cell. 1998; 9: 2905-2916Crossref PubMed Scopus (196) Google Scholar, U. Tureckova J. Lim H.W. P. Molkentin J.D. Mol. Cell. Biol. 2000; PubMed Scopus Google Scholar). In this we that inhibition of GSK-3β myogenic differentiation by NFATc3 nuclear localization and transcriptional myogenic to the of of GSK-3β on skeletal muscle differentiation and NFAT The negative regulation of myogenic differentiation by GSK-3β through of NFATc3 nuclear localization and inhibition of muscle gene Cell skeletal muscle C2C12 the was in growth medium of Dulbecco's modified Eagle's medium and and fetal bovine serum or differentiation medium which with and type and mouse embryo fibroblast J. Woodgett Nature. 2000; PubMed Scopus Google Scholar) by J. R. Woodgett and in or which with and types on in as R.C. Velden J.L. Schols A.M. Kelders M.C. Wouters E.F. Janssen-Heininger Y.M. J. 2004; PubMed Scopus Google Scholar). and in for or of or was of differentiation and the cells with and or to I by of was used as a for the of muscle-specific transcription NFAT-sensitive J. N. Olson E.N. Bassel-Duby R. Williams R.S. 2000; PubMed Google Scholar) used as a for NFAT transcriptional and a constitutively activated form of NFATc3 E. Molkentin J.D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), by The used for myogenic of was a of GSK-3β and GSK-3β K85R by Proc. Natl. Acad. Sci. U. S. A. 1995; 92: PubMed Scopus Google Scholar). was to for in To and cells in and and to the RNA and hairpin by of the the pRetro-Super R. R. Science. PubMed Scopus Google Scholar, R. R. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar), by R. by of cells G. in and and with the GSK-3β or the control by the medium was through a and the was used for of C2C12 cells the of for and to for with cells with for and was by for GSK-3β or and as a the the the the RNA for the in NFATc3 or NFATc3 protein was by cells in and by the of and on for by nuclear and cells in and the was by the of and a on for the was and the the nuclear was with and in and for on a and for of the was for protein and and was by of the for and protein was by the protein to the and of protein was and on a or by to a The was for in in by with a for GSK-3β Cell or NFATc3 or a of the with a and by to the of differentiation of C2C12 cells was of muscle kinase and by of the myogenic cells and in for in in and of the with a for and the was in for of protein or in the of bovine serum was a G. W. E. Clin. Chem. 22: PubMed Scopus Google Scholar) was for which was by the Biochem. PubMed Scopus Google Scholar). To myoblast fusion, the myogenic as the of in cells or by the of was in on and in differentiation cells in in and in to the The myogenic was by a and the of was RNA and of RNA C2C12 cells was the to the RNA was in and of RNA was to the with and by and was the following for I and in by of for each and the the The expression of the of was to in an or control myoblasts on and and or of in for NFATc3 a and a The with a a to a both of in the of of fibroblasts by of the muscle factor is a to activation of muscle-specific gene transcription H. S.J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). To the of GSK-3β on by we myogenic of and by of the GSK-3β displayed increased promoter with GSK-3β expression by with a GSK-3β promoter transactivation, expression of a (K85R) not that GSK-3β kinase muscle-specific gene a negative role for GSK-3β in myogenic GSK-3β of Skeletal Muscle of GSK-3β in in embryonic J. Woodgett Nature. 2000; PubMed Scopus Google Scholar), we investigated the of loss of GSK-3β upon myogenic differentiation, RNA of GSK-3β in C2C12 In myoblasts with or an of GSK-3β protein was observed in with control C2C12 myoblasts differentiation for cells displayed enhanced myotube formation with control cells which was by a in the myogenic GSK-3β resulted in increased with C2C12 cells expression was also strongly in with control myoblasts during myogenic differentiation a transcriptional of GSK-3β loss on expression the that loss of GSK-3β signaling myogenic of GSK-3β of myogenic differentiation by calcineurin in in the or of for of and and was expressed as myoblasts in the of and or was are of Inhibition of GSK-3β of by Calcineurin of the transcriptional regulator NFAT is dependent on the balance between inhibitory phosphorylation by GSK-3β and stimulatory dephosphorylation by calcineurin (12Beals C.R. Sheridan C.M. Turck C.W. Gardner P. Crabtree G.R. Science. 1997; 275: 1930-1934Crossref PubMed Scopus (635) Google Scholar). have that calcineurin is for muscle differentiation (22Dunn S.E. Burns J.L. Michel R.N. J. Biol. Chem. 1999; 274: 21908-21912Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar). To whether inhibition of calcineurin resulted in of myogenic differentiation in our C2C12 myoblasts in for with or the calcineurin and was was in a in response to In inhibition of GSK-3β by resulted in increased and (10van der Velden J.L. Langen R.C. Kelders M.C. Wouters E.F. Janssen-Heininger Y.M. Schols A.M. Am. J. Physiol. 2006; 290: C453-C462Crossref PubMed Scopus (86) Google Scholar). inhibition of GSK-3β and calcineurin the of calcineurin on myogenic differentiation on of and data demonstrate that calcineurin and GSK-3β have on myoblast differentiation, which may be through NFAT, which has been implicated in myogenic differentiation (24Delling U. Tureckova J. Lim H.W. P. Molkentin J.D. Mol. Cell. Biol. 2000; PubMed Scopus Google Scholar). GSK-3β NFATc3 in Skeletal Muscle NFATc3 nuclear localization was in myoblasts by during with C2C12 cells a in NFATc3 nuclear localization with control cells in which NFATc3 mainly in the NFATc3 nuclear in GSK-3β with control cells was by of nuclear NFATc3 in was not between the cells not that increased myogenic differentiation of myoblasts in of GSK-3β is with increased NFATc3 nuclear GSK-3β NFATc3 the of NFATc3 nuclear by NFATc3 was in and with a NFAT-sensitive promoter Overexpression of NFATc3 NFAT transcriptional activation to in GSK-3β-deficient with inhibition of GSK-3β by enhanced NFAT transcriptional in but not in To whether NFAT transcriptional is also by GSK-3β in skeletal muscle C2C12 cells with the NFAT and NFATc3. Overexpression of NFATc3 a of NFAT transcriptional which was increased by inhibition of GSK-3β of NFATc3 with GSK-3β but not kinase-inactive (K85R) GSK-3β NFAT transcriptional transcriptional of a of NFATc3 was not by GSK-3β data NFAT of GSK-3β signaling and a role of GSK-3β in transcriptional in C2C12 by whether NFATc3 myogenic differentiation, C2C12 cells with the promoter to muscle-specific gene expression during of NFATc3 increased promoter and following of differentiation promoter was enhanced by GSK-3β inhibition NFATc3 also increased promoter following myogenic of but not K85R promoter In increased promoter in response to NFAT was to GSK-3β promoter was also increased in C2C12 myoblasts NFATc3 was which was in by but not K85R GSK-3β Overexpression of NFATc3 also muscle-specific gene expression but was to GSK-3β that muscle gene expression during myogenic differentiation is by which in is by GSK-3β. of Muscle during by GSK-3β Inhibition we investigated whether stimulation of myogenic differentiation by inhibition of GSK-3β is dependent on NFATc3 the RNA as for GSK-3β C2C12 a NFATc3 with control NFATc3 protein was reduced in myoblasts and control myoblasts with the promoter to muscle-specific gene expression during promoter was strongly attenuated in myoblasts with reduced NFATc3 with control myoblasts of inhibition of GSK-3β in control myoblasts in with myoblasts in data demonstrate that stimulation of muscle gene expression during differentiation by inhibition of GSK-3β is dependent on NFATc3. Skeletal muscle growth including a in protein as well as differentiation, and fusion of satellite cells and expression of muscle-specific (5Allen D.L. Roy R.R. Edgerton V.R. Muscle Nerve. 1999; 22: 1350-1360Crossref PubMed Scopus (370) Google Scholar, Physiol. 2004; PubMed Scopus Google Scholar). and are by insulin-like growth of and signaling pathways 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 (1213) Google Scholar, S.C. Stitt T.N. M. G.L. R. E. A. Glass D.J. Yancopoulos G.D. Cell Biol. 2001; 3: PubMed Scopus Google Scholar). studies have that GSK-3β is a negative regulator of both cardiac and skeletal muscle growth 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 (1213) Google Scholar, S.E. J. PubMed Scopus Google Scholar, Am. J. Physiol. PubMed Scopus Google Scholar). GSK-3β was with myonuclear and myogenic differentiation in skeletal muscle atrophy (10van der Velden J.L. Langen R.C. Kelders M.C. Wouters E.F. Janssen-Heininger Y.M. Schols A.M. Am. J. Physiol. 2006; 290: C453-C462Crossref PubMed Scopus (86) Google Scholar). In addition, in studies have suggested that GSK-3β inhibition of GSK-3β was to stimulate myogenic differentiation (10van der Velden J.L. Langen R.C. Kelders M.C. Wouters E.F. Janssen-Heininger Y.M. Schols A.M. Am. J. Physiol. 2006; 290: C453-C462Crossref PubMed Scopus (86) Google Scholar, A. A. M. T. G. Mol. Biol. Cell. 2004; PubMed Scopus Google Scholar). The in the loss of GSK-3β was to stimulate muscle-specific gene expression and myotube formation during differentiation and regulation of NFATc3 nuclear translocation and transcriptional as the mechanism of of muscle gene expression by GSK-3β during In the of GSK-3β signaling increased muscle-specific promoter transactivation, muscle gene and myotube formation in of myogenic is in with the stimulatory of or inhibition of GSK-3β on myogenic differentiation by increased myotube formation and enhanced expression of muscle-specific such as and and (10van der Velden J.L. Langen R.C. Kelders M.C. Wouters E.F. Janssen-Heininger Y.M. Schols A.M. Am. J. Physiol. 2006; 290: C453-C462Crossref PubMed Scopus (86) Google Scholar). Conversely, reintroduction of but not kinase-inactive reduced muscle-specific gene indicating that GSK-3β kinase is for its on have that stimulation of myogenic differentiation be activation of which the phosphorylation of certain GSK-3β A. McCullagh K.J. Naya F.J. Olson E.N. Rosenthal N. Nature. 1999; 400: 581-585Crossref PubMed Scopus (557) Google Scholar, K.L. Friday B.B. Thaloor D. Murphy T.J. Pavlath G.K. Mol. Biol. Cell. 1998; 9: 2905-2916Crossref PubMed Scopus (196) Google Scholar, U. Tureckova J. Lim H.W. P. Molkentin J.D. Mol. Cell. Biol. 2000; PubMed Scopus Google Scholar). Conversely, inhibition of calcineurin by treatment and differentiation of skeletal muscle cells (17Abbott K.L. Friday B.B. Thaloor D. Murphy T.J. Pavlath G.K. Mol. Biol. Cell. 1998; 9: 2905-2916Crossref PubMed Scopus (196) Google Scholar). In with calcineurin inhibition not or myotube formation not and of and inhibition by or not of GSK-3β kinase not expression and myotube formation (10van der Velden J.L. Langen R.C. Kelders M.C. Wouters E.F. Janssen-Heininger Y.M. Schols A.M. Am. J. Physiol. 2006; 290: C453-C462Crossref PubMed Scopus (86) Google Scholar) but also restored the of calcineurin inhibition on myogenic differentiation, a role of GSK-3β calcineurin in muscle growth. In with calcineurin-induced cardiac hypertrophy is by cardiac activation of GSK-3β N. W. J. Shelton J.M. Richardson J.A. J.A. Olson E.N. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). of GSK-3β and calcineurin on muscle growth may be through NFAT (12Beals C.R. Sheridan C.M. Turck C.W. Gardner P. Crabtree G.R. Science. 1997; 275: 1930-1934Crossref PubMed Scopus (635) Google Scholar, 13Neal J.W. Clipstone N.A. J. Biol. Chem. 2001; 276: 3666-3673Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). NFAT is mainly by control of its nuclear which is to phosphorylation of the NFAT by including and dephosphorylation by calcineurin. In the C2C12 myoblasts with reduced GSK-3β increased nuclear localization and transcriptional activation of NFATc3 during differentiation, of negative regulation of NFATc3 by GSK-3β. is in with the of nuclear localization of NFAT GSK-3β was M. D. Crabtree G.R. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). our data that increased muscle gene expression and myotube formation in GSK-3β myoblasts or control myoblasts was to calcineurin that NFAT nuclear translocation by in the of both GSK-3β kinase and calcineurin phosphatase as was J. PubMed Scopus Google Scholar). of NFATc3 by GSK-3β phosphorylation of the NFAT NFAT transcriptional activation by an NFATc3 protein this was to the inhibitory of GSK-3β in fibroblasts or The not regulation of other NFAT isoforms by GSK-3β during differentiation, four NFAT to and are expressed in skeletal muscle (17Abbott K.L. Friday B.B. Thaloor D. Murphy T.J. Pavlath G.K. Mol. Biol. Cell. 1998; 9: 2905-2916Crossref PubMed Scopus (196) Google Scholar, 18O'Connor R.S. Mills S.T. Jones K.A. Ho S.N. Pavlath G.K. J. Cell Sci. 2007; 120: 149-159Crossref PubMed Scopus (97) Google Scholar). Although -c2, and have been a role in the regulation of myoblast differentiation, we on NFATc3 in this for a of is not to calcineurin our data and other studies V. Friday B.B. Matteson S. Kegley K.M. Gephart J. Pavlath G.K. J. Cell Biol. 2001; 153: 329-338Crossref PubMed Scopus (203) Google Scholar, U. Tureckova J. Lim H.W. P. Molkentin J.D. Mol. Cell. Biol. 2000; PubMed Scopus Google Scholar) that calcineurin signaling is for Conversely, stimulation of was also observed in studies by of calcineurin or calcineurin activation by studies have postulated a role for NFATc3 during stimulation of myoblast differentiation (17Abbott K.L. Friday B.B. Thaloor D. Murphy T.J. Pavlath G.K. Mol. Biol. Cell. 1998; 9: 2905-2916Crossref PubMed Scopus (196) Google Scholar, U. Tureckova J. Lim H.W. P. Molkentin J.D. Mol. Cell. Biol. 2000; PubMed Scopus Google Scholar). In with myoblasts with reduced NFATc3 protein displayed promoter during with our this a of NFATc3 regulation in which the balance between inhibitory phosphorylation by GSK-3β and stimulatory dephosphorylation by calcineurin is in of nuclear of NFATc3 during In calcineurin or GSK-3β in NFATc3 nuclear translocation and stimulation of myogenic In of this increased NFATc3 nuclear localization with of various muscle inhibition (10van der Velden J.L. Langen R.C. Kelders M.C. Wouters E.F. Janssen-Heininger Y.M. Schols A.M. Am. J. Physiol. 2006; 290: C453-C462Crossref PubMed Scopus (86) Google Scholar) or of GSK-3β. the of GSK-3β the and stimulation of muscle-specific gene transcription by NFATc3. of NFATc3 was sufficient to stimulate of the promoter during muscle NFATc3 was to negative regulation by GSK-3β kinase of GSK-3β the stimulatory of NFATc3 on muscle gene Finally, stimulation of muscle gene expression during myogenic differentiation by GSK-3β inhibition the of transcriptional activation of promoter in response to was strongly attenuated in myoblasts in which NFATc3 expression was of NFAT in muscle-specific gene expression has been and in with other transcription such as A. McCullagh K.J. Naya F.J. Olson E.N. Rosenthal N. Nature. 1999; 400: 581-585Crossref PubMed Scopus (557) Google Scholar), B.B. Pavlath G.K. J. Cell Sci. 2001; PubMed Google Scholar), and D.L. Dev. Biol. 2006; PubMed Scopus Google Scholar). In addition, NFATc3 was to the myogenic of (24Delling U. Tureckova J. Lim H.W. P. Molkentin J.D. Mol. Cell. Biol. 2000; PubMed Scopus Google Scholar). Since and not have this (24Delling U. Tureckova J. Lim H.W. P. Molkentin J.D. Mol. Cell. Biol. 2000; PubMed Scopus Google Scholar), this a role for NFATc3 in stimulation of muscle NFATc3 not enhanced differentiation following GSK-3β inhibition may have resulted the of NFATc3 protein additional signaling that are by GSK-3β may to increased differentiation in the of GSK-3β kinase factor is by GSK-3β S.R. J. Biochem. Cell Biol. 1999; PubMed Scopus Google Scholar), and of GSK-3β during muscle hypertrophy is with increased protein 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 (1213) Google Scholar), which also during myogenic differentiation (10van der Velden J.L. Langen R.C. Kelders M.C. Wouters E.F. Janssen-Heininger Y.M. Schols A.M. Am. J. Physiol. 2006; 290: C453-C462Crossref PubMed Scopus (86) Google Scholar). In addition, a in signaling is upon phosphorylation by GSK-3β H. A. J. A. R. J. 1997; PubMed Scopus Google Scholar). has been to several myogenic such as and H. S. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar), a role for in G. U. J. 1999; PubMed Scopus Google Scholar), which may be in the of GSK-3β. we that GSK-3β may as a of myogenic differentiation, such as and on which may control of myogenic In this GSK-3β as a potential for stimulation of myogenic differentiation NFATc3 to skeletal muscle growth or muscle atrophy. of for of the and the for the