Inhibition of Myogenesis by Multiple Cyclin-Cdk Complexes

During skeletal myogenesis, cell cycle withdrawal accompanies the expression of the contractile phenotype. Here we show that ectopic expression of each D-type cyclin is sufficient to inhibit the transcriptional activation of the muscle-specific creatine kinase (MCK) gene. In contrast, ectopic expression of cyclin A or cyclin E inhibits MCK expression only when they are co-expressed with their catalytic partner cyclin-dependent kinase 2 (Cdk2). For each of these conditions, myogenic transcriptional inhibition is reversed by the ectopic co-expression of the general Cdk inhibitor p21. Inhibition of MCK expression by cyclins or cyclin-Cdk combinations correlates with E2F activation, suggesting that the inhibition is mediated by the overall Rb-kinase activities of the Cdk complexes. In support of this hypothesis, a hyperactive mutant of Rb was found to partially reverse the inhibition of MCK expression by cyclin D1 and by the combination of cyclin A and Cdk2. These data demonstrate that the inhibition of myogenic transcriptional activity is a general feature of overall Cdk activity which is mediated, at least in part, by an pocket protein/E2F-dependent pathway. MCK promoter activity is also inhibited by ectopic E2F1 expression, but this inhibition is not reversed by the co-expression of p21. Analyses of a series of E2F1 mutants revealed that the transcriptional activation, leucine zipper, basic, and cyclin A/Cdk2-binding domains are dispensable, but the helix-loop-helix region is essential for myogenic inhibition. These data demonstrate that myocyte proliferation and differentiation are coordinated at the level of E2F and that these opposing activities are regulated by different E2F domains. During skeletal myogenesis, cell cycle withdrawal accompanies the expression of the contractile phenotype. Here we show that ectopic expression of each D-type cyclin is sufficient to inhibit the transcriptional activation of the muscle-specific creatine kinase (MCK) gene. In contrast, ectopic expression of cyclin A or cyclin E inhibits MCK expression only when they are co-expressed with their catalytic partner cyclin-dependent kinase 2 (Cdk2). For each of these conditions, myogenic transcriptional inhibition is reversed by the ectopic co-expression of the general Cdk inhibitor p21. Inhibition of MCK expression by cyclins or cyclin-Cdk combinations correlates with E2F activation, suggesting that the inhibition is mediated by the overall Rb-kinase activities of the Cdk complexes. In support of this hypothesis, a hyperactive mutant of Rb was found to partially reverse the inhibition of MCK expression by cyclin D1 and by the combination of cyclin A and Cdk2. These data demonstrate that the inhibition of myogenic transcriptional activity is a general feature of overall Cdk activity which is mediated, at least in part, by an pocket protein/E2F-dependent pathway. MCK promoter activity is also inhibited by ectopic E2F1 expression, but this inhibition is not reversed by the co-expression of p21. Analyses of a series of E2F1 mutants revealed that the transcriptional activation, leucine zipper, basic, and cyclin A/Cdk2-binding domains are dispensable, but the helix-loop-helix region is essential for myogenic inhibition. These data demonstrate that myocyte proliferation and differentiation are coordinated at the level of E2F and that these opposing activities are regulated by different E2F domains. INTRODUCTIONIrreversible cell cycle withdrawal is a key component of myogenic differentiation, but little is known about the interplay between the myogenic transcription factors and the cell cycle regulatory proteins. Previous studies have shown that the retinoblastoma susceptibility gene product (Rb) 1The abbreviations used are: Rbretinoblastoma susceptibility gene productMCKmuscle-specific creatine kinaseCdkcyclin-dependent kinaseLucluciferaseDMEMDulbecco's modified Eagle's mediumCMVcytomegalovirusPAGEpolyacrylamide gel electrophoresisPBSphosphate-buffered saline. has an essential role in maintaining the postmitotic state of differentiated myotubes (1Gu W. Schneider J.W. Condorelli G. Nadal-Ginard B. Cell. 1993; 72: 309-324Google Scholar, 2Schneider J.W. Gu W. Zhu L. Mahdavi V. Nadal-Ginard B. Science. 1994; 264: 1467-1471Google Scholar). Rb is hypophosphorylated upon terminal differentiation and this state of dephosphorylation is maintained when differentiated myotubes are re-exposed to high mitogen media. Hypophosphorylated Rb inactivates the E2F transcription factor which is essential for the expression of genes required for DNA synthesis (3Nevins J.R. Science. 1992; 258: 424-429Google Scholar). Myocytes from Rb-deficient mice can differentiate into myotubes, but these myotubes can resynthesize DNA upon mitogen stimulation (2Schneider J.W. Gu W. Zhu L. Mahdavi V. Nadal-Ginard B. Science. 1994; 264: 1467-1471Google Scholar). The phosphorylation status of Rb is regulated, at least in part, by the activity of cyclin-dependent kinases including Cdk2 and Cdk4 (4Sherr C.J. Cell. 1994; 79: 551-555Google Scholar). Recently, the general Cdk inhibitor p21 was shown to be dramatically induced during skeletal muscle differentiation, and a high level of p21 expression is sustained when myotubes are re-exposed to high mitogen media (5Parker S.B. Eichele G. Zhang P. Rawls A. Sands A.T. Bradley A. Olson E.N. Harper J.W. Elledge S.J. Science. 1995; 267: 1024-1027Google Scholar, 6Halevy O. Novitch B.G. Spicer D.B. Skapek S.X. Rhee J. Hannon G.J. Beach D. Lassar A.B. Science. 1995; 267: 1018-1021Google Scholar, 7Guo K. Wang J. Andrés V. Smith R.C. Walsh K. Mol. Cell. Biol. 1995; 15: 3823-3829Google Scholar). p21 is induced early during the differentiation program (8Andrés V. Walsh K. J. Cell Biol. 1996; 132: 657-666Google Scholar), and its expression is critical for myocyte viability (9Wang J. Walsh K. Science. 1996; 273: 359-361Google Scholar). In myotubes, p21 is the predominant inhibitory subunit of the Cdk complex (7Guo K. Wang J. Andrés V. Smith R.C. Walsh K. Mol. Cell. Biol. 1995; 15: 3823-3829Google Scholar, 10Wang J. Walsh K. Cell Growth Differ. 1996; 7: 1471-1478Google Scholar). Thus p21 is likely to function to inhibit the phosphorylation of Rb or other Rb family members during myocyte differentiation.Surprisingly, it has been reported that overexpression of cyclin D1, but not cyclins A, B, D2, D3, or E, can block myogenesis as detected by the transcriptional activation of an MCK-reporter construct in transiently transfected cultures of differentiating C2C12 cells (11Rao S.S. Chu C. Kohtz D.S. Mol. Cell. Biol. 1994; 14: 5259-5267Google Scholar). More recently it was reported that the cyclin D1-mediated inhibition of myogenic transcriptional activation could be reversed by the co-expression of the Cdk inhibitors p21 or p16 (12Skapek S.X. Rhee J. Spicer D.B. Lassar A.B. Science. 1995; 267: 1022-1024Google Scholar). These data have led to the proposal that cyclin D1 down-regulation may be a nodal point in the coordination of myocyte differentiation and cell cycle activity. Further, it was proposed that myogenic inhibition by cyclin D1 occurs through an Rb-independent pathway because the overexpression of cyclin A or cyclin E, the regulatory subunits of the Cdk2 Rb-kinase, do not inhibit myogenesis (12Skapek S.X. Rhee J. Spicer D.B. Lassar A.B. Science. 1995; 267: 1022-1024Google Scholar).To test the hypothesis that cyclin D1 functions uniquely to coordinate cell cycle with myogenic transcription through an Rb-independent mechanism, we analyzed the effects of additional cell cycle regulatory molecules on the same MCK transcriptional assay that was employed in these previous studies (11Rao S.S. Chu C. Kohtz D.S. Mol. Cell. Biol. 1994; 14: 5259-5267Google Scholar, 12Skapek S.X. Rhee J. Spicer D.B. Lassar A.B. Science. 1995; 267: 1022-1024Google Scholar). Here we report that in addition to cyclin D1, the overexpression of other D-type cyclins (D2 and D3) or the combinations of Cdk2 and cyclin A or cyclin E also inhibit MCK transcriptional activation, and that this inhibition is reversed by the co-expression of p21. MCK transcription is inversely correlated with expression from an E2F-reporter gene construct, and a hyperactive Rb mutant partially reverses the cyclin-Cdk-mediated inhibition of MCK transcription. Collectively these data indicate that the inhibition of myogenic transcription by cell cycle components can be mediated by a pocket protein/E2F pathway. Myogenic transcription is also inhibited by the ectopic expression of the E2F1 transcription factor. Surprisingly, mutations in the activation, leucine zipper, basic, and cyclin A-Cdk2 binding domains of E2F1 have no effect on its ability to inhibit myogenic transcription, but this activity was abolished by mutations in the E2F1 helix-loop-helix region. Taken together, these data demonstrate that the inhibition of myogenesis is a general feature of cyclin-Cdk activity and that myogenic differentiation is coordinated with cell cycle activity at the level of E2F.DISCUSSIONHere we investigated the molecular links between cell cycle activity and myogenic differentiation. These analyses extend the studies of Rao et al. (11Rao S.S. Chu C. Kohtz D.S. Mol. Cell. Biol. 1994; 14: 5259-5267Google Scholar), which showed that the overexpression of cyclin D1, but not cyclins A, B, or E, represses MCK transcriptional activation, and the studies of Skapek et al. (12Skapek S.X. Rhee J. Spicer D.B. Lassar A.B. Science. 1995; 267: 1022-1024Google Scholar), which showed that the cyclin D1-mediated repression is overcome by the ectopic expression of Cdk inhibitors. Based upon these prior observations it was proposed that cyclin D1 functions uniquely to coordinate cell cycle withdrawal and myogenic differentiation through an Rb-independent mechanism (12Skapek S.X. Rhee J. Spicer D.B. Lassar A.B. Science. 1995; 267: 1022-1024Google Scholar). However, this hypothesis is not supported by the data from this current study. Here it is shown that the inhibition of myogenic transcription is a function shared by many cyclins or cyclin-Cdk combinations. Further, these data indicate that the inhibitory effects of the cyclin-Cdk complexes are largely mediated by Rb, or a related pocket proteins, and that the E2F transcription factor plays a central role in coordinating myogenic differentiation and cell cycle activity.Here it is shown that the overexpression of each D-type cyclin is sufficient to inhibit MCK promoter activity. However, the forced expression of cyclins A or E have little if any effect on MCK transcriptional activation (Fig. 2) (11Rao S.S. Chu C. Kohtz D.S. Mol. Cell. Biol. 1994; 14: 5259-5267Google Scholar, 12Skapek S.X. Rhee J. Spicer D.B. Lassar A.B. Science. 1995; 267: 1022-1024Google Scholar). To investigate the differential effects of these cyclins on myogenic transcriptional activation, the expression patterns of their cognate catalytic subunits were compared. During myogenesis the expression of the Cdk2 protein is markedly down-regulated, but Cdk4 levels do not detectably change (Fig. 4A) (also see 7Guo K. Wang J. Andrés V. Smith R.C. Walsh K. Mol. Cell. Biol. 1995; 15: 3823-3829Google Scholar, 11Rao S.S. Chu C. Kohtz D.S. Mol. Cell. Biol. 1994; 14: 5259-5267Google Scholar, and 12Skapek S.X. Rhee J. Spicer D.B. Lassar A.B. Science. 1995; 267: 1022-1024Google Scholar). Thus, we reasoned that cyclin A or cyclin E overexpression might inhibit myogenesis only when they are co-expressed with their catalytic partner Cdk2. On the other hand, overexpression of the D-type cyclins might be sufficient to block MCK transcription because their catalytic partner, Cdk4, is not down-regulated during myogenesis. Consistent with this hypothesis are the observations that MCK promoter activity is inhibited, albeit with different efficiencies, by the transfection of cyclin D1, D2, or D3 expression vectors (Fig. 3). MCK promoter activity was also inhibited by combinations of expression vectors for cyclin A and Cdk2 or cyclin E and Cdk2, but not by the transfection of these individual plasmids (Fig. 2, Fig. 3, Fig. 4B). In all cases, the inhibition of MCK transcription was overcome by the co-expression of p21. Collectively, these data indicate MCK transcriptional inhibition is dependent on the overall cell cycle activity, rather than the action of an individual cell cycle component (i.e. cyclin D1). This hypothesis is consistent with the observation of an inverse correlation between MCK promoter expression and E2F transcriptional activity in cells transfected with different combinations of these cell cycle factors (Fig. 5).Vectors expressing hyperactive and inactive mutants of Rb were utilized to test the potential role of Rb (or a related pocket protein) as a mediator of myogenic transcriptional inhibition. Ectopic expression of the hyperactive (p56), but not an inactive (p56/H209), Rb mutant partially reversed the MCK transcriptional inhibition that resulted from the overexpression cyclin D1 or the combination of Cdk2 and cyclin A (Fig. 7C). These data further indicate that MCK transcription is inhibited by overall cell cycle activity and that this inhibition is mediated by the Rb/E2F pathway. This notion is further supported by the finding that E2F1 overexpression inhibits MCK transcription (Fig. 6) and overall myogenic differentiation (27Wang J. Helin K. Jin P. Nadal-Ginard B. Cell Growth Differ. 1995; 6: 1299-1306Google Scholar). MCK promoter inhibition by E2F1 is reversed by the co-expression of the hyperactive Rb mutant, but not by p21 (Fig. 6, Fig. 7); presumably because E2F1 acts at a step that is downstream from p21 (28DeGregori J. Leone G. Ohtani K. Miron A. Nevins J.R. Genes Dev. 1995; 9: 2873-2887Google Scholar).The data from this study are consistent with the model depicted in Fig. 11. Various cyclins, through interactions with their catalytic subunits, inhibit the transcription of muscle-specific genes via the phosphorylation of Rb or other pocket proteins. This phosphorylation leads to the activation of the E2F family of transcription factors, which induce the expression of essential S-phase genes and also inhibit the expression of muscle-specific genes through an unknown mechanism. The induction of p21 during myogenesis, a broad-specificity Cdk inhibitor, enhances differentiation by blocking the action of multiple cyclin-Cdk complexes. Finally, E2F also activates the expression of cyclin A and cyclin E (29DeGregori J. Kowalik T. Nevins J.R. Mol. Cell Biol. 1995; 15: 4215-4224Google Scholar), but this pathway appears to have a minimal effect on MCK transcription because the inhibition of MCK promoter activity by forced E2F1 expression is not reversed by p21.The data presented here suggest that E2F functions to coordinate the opposing cellular fates of proliferation and differentiation during myogenesis. Support for the hypothesis comes from the finding that different domains within E2F1 function to promote cell proliferation and inhibit myogenic differentiation (Fig. 8). The transcriptional activation, leucine zipper and helix-loop-helix domains are required for the expression of S phase genes and cell cycle progression. However, only the helix-loop-helix region of E2F1 is essential for the myogenic inhibitory activity, while other regions of the protein are dispensable (Fig. 10, Fig. 11). The inhibition of myogenic transcription by E2F1 overexpression may result from its ability to inactivate E proteins that are essential for myogenic differentiation. Further analyses on the mechanism of E2F-mediated inhibition of myogenic differentiation will be of interest. INTRODUCTIONIrreversible cell cycle withdrawal is a key component of myogenic differentiation, but little is known about the interplay between the myogenic transcription factors and the cell cycle regulatory proteins. Previous studies have shown that the retinoblastoma susceptibility gene product (Rb) 1The abbreviations used are: Rbretinoblastoma susceptibility gene productMCKmuscle-specific creatine kinaseCdkcyclin-dependent kinaseLucluciferaseDMEMDulbecco's modified Eagle's mediumCMVcytomegalovirusPAGEpolyacrylamide gel electrophoresisPBSphosphate-buffered saline. has an essential role in maintaining the postmitotic state of differentiated myotubes (1Gu W. Schneider J.W. Condorelli G. Nadal-Ginard B. Cell. 1993; 72: 309-324Google Scholar, 2Schneider J.W. Gu W. Zhu L. Mahdavi V. Nadal-Ginard B. Science. 1994; 264: 1467-1471Google Scholar). Rb is hypophosphorylated upon terminal differentiation and this state of dephosphorylation is maintained when differentiated myotubes are re-exposed to high mitogen media. Hypophosphorylated Rb inactivates the E2F transcription factor which is essential for the expression of genes required for DNA synthesis (3Nevins J.R. Science. 1992; 258: 424-429Google Scholar). Myocytes from Rb-deficient mice can differentiate into myotubes, but these myotubes can resynthesize DNA upon mitogen stimulation (2Schneider J.W. Gu W. Zhu L. Mahdavi V. Nadal-Ginard B. Science. 1994; 264: 1467-1471Google Scholar). The phosphorylation status of Rb is regulated, at least in part, by the activity of cyclin-dependent kinases including Cdk2 and Cdk4 (4Sherr C.J. Cell. 1994; 79: 551-555Google Scholar). Recently, the general Cdk inhibitor p21 was shown to be dramatically induced during skeletal muscle differentiation, and a high level of p21 expression is sustained when myotubes are re-exposed to high mitogen media (5Parker S.B. Eichele G. Zhang P. Rawls A. Sands A.T. Bradley A. Olson E.N. Harper J.W. Elledge S.J. Science. 1995; 267: 1024-1027Google Scholar, 6Halevy O. Novitch B.G. Spicer D.B. Skapek S.X. Rhee J. Hannon G.J. Beach D. Lassar A.B. Science. 1995; 267: 1018-1021Google Scholar, 7Guo K. Wang J. Andrés V. Smith R.C. Walsh K. Mol. Cell. Biol. 1995; 15: 3823-3829Google Scholar). p21 is induced early during the differentiation program (8Andrés V. Walsh K. J. Cell Biol. 1996; 132: 657-666Google Scholar), and its expression is critical for myocyte viability (9Wang J. Walsh K. Science. 1996; 273: 359-361Google Scholar). In myotubes, p21 is the predominant inhibitory subunit of the Cdk complex (7Guo K. Wang J. Andrés V. Smith R.C. Walsh K. Mol. Cell. Biol. 1995; 15: 3823-3829Google Scholar, 10Wang J. Walsh K. Cell Growth Differ. 1996; 7: 1471-1478Google Scholar). Thus p21 is likely to function to inhibit the phosphorylation of Rb or other Rb family members during myocyte differentiation.Surprisingly, it has been reported that overexpression of cyclin D1, but not cyclins A, B, D2, D3, or E, can block myogenesis as detected by the transcriptional activation of an MCK-reporter construct in transiently transfected cultures of differentiating C2C12 cells (11Rao S.S. Chu C. Kohtz D.S. Mol. Cell. Biol. 1994; 14: 5259-5267Google Scholar). More recently it was reported that the cyclin D1-mediated inhibition of myogenic transcriptional activation could be reversed by the co-expression of the Cdk inhibitors p21 or p16 (12Skapek S.X. Rhee J. Spicer D.B. Lassar A.B. Science. 1995; 267: 1022-1024Google Scholar). These data have led to the proposal that cyclin D1 down-regulation may be a nodal point in the coordination of myocyte differentiation and cell cycle activity. Further, it was proposed that myogenic inhibition by cyclin D1 occurs through an Rb-independent pathway because the overexpression of cyclin A or cyclin E, the regulatory subunits of the Cdk2 Rb-kinase, do not inhibit myogenesis (12Skapek S.X. Rhee J. Spicer D.B. Lassar A.B. Science. 1995; 267: 1022-1024Google Scholar).To test the hypothesis that cyclin D1 functions uniquely to coordinate cell cycle with myogenic transcription through an Rb-independent mechanism, we analyzed the effects of additional cell cycle regulatory molecules on the same MCK transcriptional assay that was employed in these previous studies (11Rao S.S. Chu C. Kohtz D.S. Mol. Cell. Biol. 1994; 14: 5259-5267Google Scholar, 12Skapek S.X. Rhee J. Spicer D.B. Lassar A.B. Science. 1995; 267: 1022-1024Google Scholar). Here we report that in addition to cyclin D1, the overexpression of other D-type cyclins (D2 and D3) or the combinations of Cdk2 and cyclin A or cyclin E also inhibit MCK transcriptional activation, and that this inhibition is reversed by the co-expression of p21. MCK transcription is inversely correlated with expression from an E2F-reporter gene construct, and a hyperactive Rb mutant partially reverses the cyclin-Cdk-mediated inhibition of MCK transcription. Collectively these data indicate that the inhibition of myogenic transcription by cell cycle components can be mediated by a pocket protein/E2F pathway. Myogenic transcription is also inhibited by the ectopic expression of the E2F1 transcription factor. Surprisingly, mutations in the activation, leucine zipper, basic, and cyclin A-Cdk2 binding domains of E2F1 have no effect on its ability to inhibit myogenic transcription, but this activity was abolished by mutations in the E2F1 helix-loop-helix region. Taken together, these data demonstrate that the inhibition of myogenesis is a general feature of cyclin-Cdk activity and that myogenic differentiation is coordinated with cell cycle activity at the level of E2F.