We have examined the role of protein phosphorylation in the modulation of the key muscle-specific transcription factor MyoD. We show that MyoD is highly phosphorylated in growing myoblasts and undergoes substantial dephosphorylation during differentiation. MyoD can be efficiently phosphorylated in vitro by either purified cdk1-cyclin B or cdk1 and cdk2 immunoprecipitated from proliferative myoblasts. Comparative two-dimensional tryptic phosphopeptide mapping combined with site-directed mutagenesis revealed that cdk1 and cdk2 phosphorylate MyoD on serine 200 in proliferative myoblasts. In addition, when the seven proline-directed sites in MyoD were individually mutated, only substitution of serine 200 to a nonphosphorylatable alanine (MyoD-Ala200) abolished the slower-migrating hyperphosphorylated form of MyoD, seen either in vitro after phosphorylation by cdk1-cyclin B or in vivo following overexpression in 10T1/2 cells. The MyoD-Ala200 mutant displayed activity threefold higher than that of wild-type MyoD in transactivation of an E-box-dependent reporter gene and promoted markedly enhanced myogenic conversion and fusion of 10T1/2 fibroblasts into muscle cells. In addition, the half-life of MyoD-Ala200 protein was longer than that of wild-type MyoD, substantiating a role of Ser200 phosphorylation in regulating MyoD turnover in proliferative myoblasts. Taken together, our data show that direct phosphorylation of MyoD Ser200 by cdk1 and cdk2 plays an integral role in compromising MyoD activity during myoblast proliferation.
Previous works have established a unique function of MyoD in the control of muscle gene expression during DNA damage response in myoblasts. Phosphorylation by DNA damage-activated ABL tyrosine kinase transiently inhibits MyoD-dependent activation of transcription in response to genotoxic stress. We show here that ABL-MyoD signaling is also an essential component of the DNA repair machinery in myoblasts exposed to genotoxic stress. DNA damage promoted the recruitment of MyoD to phosphorylated Nbs1 (pNbs1)-containing repair foci, and this effect was abrogated by either ABL knockdown or the ABL kinase inhibitor imatinib. Upon DNA damage, MyoD and pNbs1 were detected on the chromatin to MyoD target genes without activating transcription. DNA damage-mediated tyrosine phosphorylation was required for MyoD recruitment to target genes, as the ABL phosphorylation-resistant MyoD mutant (MyoD Y30F) failed to bind the chromatin following DNA damage, while retaining the ability to activate transcription in response to differentiation signals. Moreover, MyoD Y30F exhibited an impaired ability to promote repair in a heterologous system, as compared with MyoD wild type (WT). Consistently, MyoD-null satellite cells (SCs) displayed impaired DNA repair that was rescued by reintroduction of MyoD WT but not by MyoD Y30F. In addition, inhibition of ABL kinase prevented MyoD WT-mediated rescue of DNA repair in MyoD-null SCs. These results identify an unprecedented contribution of MyoD to DNA repair and suggest that ABL-MyoD signaling coordinates DNA repair and transcription in myoblasts.
MyoD; ABL; DNA damage; chromatin; DNA repair
MyoD is a basic helix-loop-helix transcription factor involved in the activation of genes encoding skeletal muscle-specific proteins. Independent of its ability to transactivate muscle-specific genes, MyoD can also act as a cell cycle inhibitor. MyoD activity is regulated by transcriptional and posttranscriptional mechanisms. While MyoD can be found phosphorylated, the functional significance of this posttranslation modification has not been established. MyoD contains several consensus cyclin-dependent kinase (CDK) phosphorylation sites. In these studies, we examined whether a link could be established between MyoD activity and phosphorylation at putative CDK sites. Site-directed mutagenesis of potential CDK phosphorylation sites in MyoD revealed that S200 is required for MyoD hyperphosphorylation as well as the normally short half-life of the MyoD protein. Additionally, we determined that turnover of the MyoD protein requires the proteasome and Cdc34 ubiquitin-conjugating enzyme activity. Results of these studies demonstrate that hyperphosphorylated MyoD is targeted for rapid degradation by the ubiquitin pathway. The targeted degradation of MyoD following CDK phosphorylation identifies a mechanism through which MyoD activity can be regulated coordinately with the cell cycle machinery (CDK2 and CDK4) and/or coordinately with the cellular transcriptional machinery (CDK7, CDK8, and CDK9).
The transcription factors MyoD and Myf-5 control myoblast identity and differentiation. MyoD and Myf-5 manifest opposite cell cycle-specific expression patterns. Here, we provide evidence that MyoD plays a pivotal role at the G2/M transition by controlling the expression of p21Waf1/Cip1 (p21), which is believed to regulate cyclin B-Cdc2 kinase activity in G2. In growing myoblasts, MyoD reaccumulates during G2 concomitantly with p21 before entry into mitosis; MyoD is phosphorylated on Ser5 and Ser200 by cyclin B-Cdc2, resulting in a decrease of its stability and down-regulation of both MyoD and p21. Inducible expression of a nonphosphorylable MyoD A5/A200 enhances the MyoD interaction with the coactivator P/CAF, thereby stimulating the transcriptional activation of a luciferase reporter gene placed under the control of the p21 promoter. MyoD A5/A200 causes sustained p21 expression, which inhibits cyclin B-Cdc2 kinase activity in G2 and delays M-phase entry. This G2 arrest is not observed in p21−/− cells. These results show that in cycling cells MyoD functions as a transcriptional activator of p21 and that MyoD phosphorylation is required for G2/M transition.
The activities of myogenic basic helix-loop-helix (bHLH) factors are regulated by a number of different positive and negative signals. Extensive information has been published about the molecular mechanisms that interfere with the process of myogenic differentiation, but little is known about the positive signals. We previously showed that overexpression of rat Mos in C2C12 myoblasts increased the expression of myogenic markers whereas repression of Mos products by antisense RNAs inhibited myogenic differentiation. In the present work, our results show that the rat mos proto-oncogene activates transcriptional activity of MyoD protein. In transient transfection assays, Mos promotes transcriptional transactivation by MyoD of the muscle creatine kinase enhancer and/or a reporter gene linked to MyoD-DNA binding sites. Physical interaction between Mos and MyoD, but not with E12, is demonstrated in vivo by using the two-hybrid approach with C3H10T1/2 cells and in vitro by using the glutathione S-transferase (GST) pull-down assays. Unphosphorylated MyoD from myogenic cell lysates and/or bacterially expressed MyoD physically interacts with Mos. This interaction occurs via the helix 2 region of MyoD and a highly conserved region in Mos proteins with 40% similarity to the helix 2 domain of the E-protein class of bHLH factors. Phosphorylation of MyoD by activated GST-Mos protein inhibits the DNA-binding activity of MyoD homodimers and promotes MyoD-E12 heterodimer formation. These data support a novel function for Mos as a mediator (coregulator) of muscle-specific gene(s) expression.
Background: Hedgehog (Hh) signaling regulates skeletal myogenesis; however, the molecular mechanisms involved are not fully understood.
Results: Gli2, a transactivator of Hh signaling, associates with MyoD gene elements, regulating MyoD expression, and binds to MyoD protein, regulating its ability to induce myogenesis.
Conclusion: Hh signaling is linked to MyoD gene expression and MyoD protein function.
Significance: Novel mechanistic insight is gained into the Hh-regulated myogenesis.
The inhibition of MyoD expression is important for obtaining muscle progenitors that can replenish the satellite cell niche during muscle repair. Progenitors could be derived from either embryonic stem cells or satellite cells. Hedgehog (Hh) signaling is important for MyoD expression during embryogenesis and adult muscle regeneration. To date, the mechanistic understanding of MyoD regulation by Hh signaling is unclear. Here, we demonstrate that the Hh effector, Gli2, regulates MyoD expression and associates with MyoD gene elements. Gain- and loss-of-function experiments in pluripotent P19 cells show that Gli2 activity is sufficient and required for efficient MyoD expression during skeletal myogenesis. Inhibition of Hh signaling reduces MyoD expression during satellite cell activation in vitro. In addition to regulating MyoD expression, Hh signaling regulates MyoD transcriptional activity, and MyoD activates Hh signaling in myogenic conversion assays. Finally, Gli2, MyoD, and MEF2C form a protein complex, which enhances MyoD activity on skeletal muscle-related promoters. We therefore link Hh signaling to the function and expression of MyoD protein during myogenesis in stem cells.
Development; Embryonic Stem Cell; Gene Expression; Myogenesis; Skeletal Muscle; Transcription; Gli; Hedgehog Signaling; MEF2; MyoD
MyoD is a transcription factor implicated in the regulation of adult muscle gene expression. Distinguishing the expression of MyoD in satellite myoblasts and muscle fibres has proved difficult in vivo leading to controversy over the significance of MyoD expression within adult innervated muscle fibres. Here we employ the MD6.0-lacZ transgenic mouse, in which the 6 kb proximal enhancer/promoter (DRR/PRR) of MyoD drives lacZ, to show that MyoD is present and transcriptionally active in many adult muscle fibres.
In culture, MD6.0-lacZ expresses in myotubes but not myogenic cells, unlike endogenous MyoD. Reporter expression in vivo is in muscle fibre nuclei and is reduced in MyoD null mice. The MD6.0-lacZ reporter is down-regulated both in adult muscle fibres by denervation or muscle disuse and in cultured myotubes by inhibition of activity. Activity induces and represses MyoD through the DRR and PRR, respectively. During the postnatal period, accumulation of β-galactosidase correlates with maturation of innervation. Strikingly, endogenous MyoD expression is up-regulated in fibres by complete denervation, arguing for a separate activity-dependent suppression of MyoD requiring regulatory elements outside the DRR/PRR.
The data show that MyoD regulation is more complex than previously supposed. Two factors, MyoD protein itself and fibre activity are required for essentially all expression of the 6 kb proximal enhancer/promoter (DRR/PRR) of MyoD in adult fibres. We propose that modulation of MyoD positive feedback by electrical activity determines the set point of MyoD expression in innervated fibres through the DRR/PRR element.
MyoD is a tissue-specific transcriptional activator that acts as a master switch for skeletal muscle differentiation. Its activity is induced during the transition from proliferating, nondifferentiated myoblasts to resting, well-differentiated myotubes. Like many other transcriptional regulators, it is a short-lived protein; however, the targeting proteolytic pathway and the underlying regulatory mechanisms involved in the process have remained obscure. It has recently been shown that many short-lived regulatory proteins are degraded by the ubiquitin system. Degradation of a protein by the ubiquitin system proceeds via two distinct and successive steps, conjugation of multiple molecules of ubiquitin to the target protein and degradation of the tagged substrate by the 26S proteasome. Here we show that MyoD is degraded by the ubiquitin system both in vivo and in vitro. In intact cells, the degradation is inhibited by lactacystin, a specific inhibitor of the 26S proteasome. Inhibition is accompanied by accumulation of high-molecular-mass MyoD-ubiquitin conjugates. In a cell-free system, the proteolytic process requires both ATP and ubiquitin and, like the in vivo process, is preceded by formation of ubiquitin conjugates of the transcription factor. Interestingly, the process is inhibited by the specific DNA sequence to which MyoD binds: conjugation and degradation of a MyoD mutant protein which lacks the DNA-binding domain are not inhibited. The inhibitory effect of the DNA requires the formation of a complex between the DNA and the MyoD protein. Id1, which inhibits the binding of MyoD complexes to DNA, abrogates the effect of DNA on stabilization of the protein.
Myogenesis is a multistep process, in which myoblasts withdraw from the cell cycle, cease to divide, elongate and fuse to form multinucleated myotubes. Cell cycle transition is controlled by a family of cyclin-dependent protein kinases (CDKs) regulated by association with cyclins, negative regulatory subunits and phosphorylation. Muscle differentiation is orchestrated by myogenic regulatory factors (MRFs), such as MyoD and Myf-5. DNA methylation is crucial in transcriptional control of genes involved in myogenesis. Previous work has indicated that treatment of fibroblasts with the DNA-demethylating agent 5-azacytidine (AZA) promotes MyoD expression. We studied the effects of AZA on cell cycle regulation and MRFs synthesis during myoblast proliferation and early myogenesis phases in C2C12 cells. During the proliferation phase, cells were incubated in growth medium with 5µM AZA (GMAZA) or without AZA (GM) for 24 hours. At 70% confluence, cells were kept in growth medium in order to spontaneously achieve differentiation or transferred to differentiation medium with 5μM AZA (DMAZA) or without AZA (DM) for 12 and 24 hours. Cells used as control were unstimulated.
In the proliferation phase, AZA-treated cells seemed to lose their characteristic circular shape and become elongated. The presence of AZA resulted in significant increases in the protein contents of Cyclin-D (FC:1.23 GMAZA vs GM p≤0.05), p21 (FC: 1.23 GMAZA vs GM p≤0.05), Myf-5 (FC: 1.21 GMAZA vs GM p≤0.05) and MyoD (FC: 1.20 GMAZA vs GM p≤0.05). These results propose that AZA could inhibit cell proliferation.
During 12 hours of differentiation, AZA decreased the downregulation of genes involved in cell cycle arrest and in restriction point (G1 and G1/S phase) and the expression of several cyclins, E2F Transcription Factors, cyclin-dependent kinase inhibitors, specific genes responsible of cell cycle negative regulation. During 24 hours of differentiation, AZA induced an increment in the protein expression of Myf-5 (FC: 1.57 GMAZA vs GM p≤0.05), MyoD (FC: 1.14 DM vs GM p≤0.05; FC: 1.47 DMAZA vs GM p≤0.05), p21 (FC: 1.36 GMAZA vs GM p≤0.01; FC: 1.49 DM vs GM p≤0.05; FC: 1.82 DMAZA vs GM p≤0.01) and MyHC (FC: 1.40 GMAZA vs GM p≤0.01; FC: 2.39 DM vs GM p≤0.05; FC: 3.51 DMAZA vs GM p≤0.01). Our results suggest that AZA-induced DNA demethylation can modulate cell cycle progression and enhance myogenesis. The effects of AZA may open novel clinical uses in the field of muscle injury research and treatment.
cell cycle; DNA methylation; myogenic transcription factors; myogenic phenotype; myogenesis
The murine p204 protein level is highest in heart and skeletal muscle. During the fusion of cultured myoblasts to myotubes, the p204 level increases due to transcription dependent on the muscle-specific MyoD protein, and p204 is phosphorylated and translocated from the nucleus to the cytoplasm. p204 overexpression accelerates myoblast fusion in differentiation medium and triggers this process even in growth medium. Here we report that p204 is required for the differentiation of C2C12 myoblasts. We propose that it enables the differentiation, at least in part, by overcoming the inhibition of the activities of the MyoD and E47 proteins by the Id proteins: Id1, Id2, and Id3. These are known to inhibit skeletal muscle differentiation by binding and blocking the activity of MyoD, E12/E47, and other myogenic basic helix-loop-helix (bHLH) proteins. Our hypothesis is based on the following findings. (i) A decrease in the p204 level in C2C12 myoblasts by antisense RNA (a) increased the level of the Id2; (b) inhibited the MyoD-, E12/E47-, and other bHLH protein-dependent accumulation of the muscle-specific myosin heavy-chain protein; and (c) inhibited the fusion of myoblasts to myotubes in differentiation medium. (ii) p204 bound to the Id proteins in vitro and in vivo. (iii) In the binding of p204 to Id2, the b segment of p204 and the HLH segment of Id2 were involved. (iv) Addition of p204 overcame the inhibition by the Id proteins of the binding of MyoD and E47 to DNA in vitro. (v) Overexpression of p204 in myoblasts (a) decreased the level of the Id proteins, even in a culture in growth medium, and (b) overcame the inhibition by the Id proteins of MyoD- and E47 dependent transcription and also overcame the inhibition by Id2 of the fusion of myoblasts to myotubes.
Mice lacking MyoD exhibit delayed skeletal muscle regeneration and markedly enhanced numbers of satellite cells. Myoblasts isolated from MyoD-/- myoblasts proliferate more rapidly than wild type myoblasts, display a dramatic delay in differentiation, and continue to incorporate BrdU after serum withdrawal.
Primary myoblasts isolated from wild type and MyoD-/- mutant mice were examined by microarray analysis and further characterized by cell and molecular experiments in cell culture.
We found that NF-κB, a key regulator of cell-cycle withdrawal and differentiation, aberrantly maintains nuclear localization and transcriptional activity in MyoD-/- myoblasts. As a result, expression of cyclin D is maintained during serum withdrawal, inhibiting expression of muscle-specific genes and progression through the differentiation program. Sustained nuclear localization of cyclin E, and a concomitant increase in cdk2 activity maintains S-phase entry in MyoD-/- myoblasts even in the absence of mitogens. Importantly, this deficit was rescued by forced expression of IκBαSR, a non-degradable mutant of IκBα, indicating that inhibition of NF-κB is sufficient to induce terminal myogenic differentiation in the absence of MyoD.
MyoD-induced cytoplasmic relocalization of NF-κB is an essential step in linking cell-cycle withdrawal to the terminal differentiation of skeletal myoblasts. These results provide important insight into the unique functions of MyoD in regulating the switch from progenitor proliferation to terminal differentiation.
Skeletal muscle; Myoblasts; MyoD; NF-κB; IKK; IκB; Differentiation; Myogenesis
The effects of transforming growth factor beta (TGF-beta) were studied in closely related human mammary epithelial cells (HMEC), both finite-life-span 184 cells and immortal derivatives, 184A1S, and 184A1L5R, which differ in their cell cycle responses to TGF-beta but express type I and type II TGF-beta receptors and retain TGF-beta induction of extracellular matrix. The arrest-resistant phenotype was not due to loss of cyclin-dependent kinase (cdk) inhibitors. TGF-beta was shown to regulate p15INK4B expression at at least two levels: mRNA accumulation and protein stability. In TGF-beta-arrested HMEC, there was not only an increase in p15 mRNA but also a major increase in p5INK4B protein stability. As cdk4- and cdk6-associated p15INK4B increased during TGF-beta arrest of sensitive cells, there was a loss of cyclin D1, p21Cip1, and p27Kip1 from these kinase complexes, and cyclin E-cdk2-associated p27Kip1 increased. In HMEC, p15INK4B complexes did not contain detectable cyclin. p15INK4B from both sensitive and resistant cells could displace in vitro cyclin D1, p21Cip1, and p27Kip1 from cdk4 isolated from sensitive cells. Cyclin D1 could not be displaced from cdk4 in the resistant 184A1L5R cell lysates. Thus, in TGF-beta arrest, p15INK4B may displace already associated cyclin D1 from cdks and prevent new cyclin D1-cdk complexes from forming. Furthermore, p27Kip1 binding shifts from cdk4 to cyclin E-cdk2 during TGF-beta-mediated arrest. The importance of posttranslational regulation of p15INK4B by TGF-beta is underlined by the observation that in TGF-beta-resistant 184A1L5R, although the p15 transcript increased, p15INK4B protein was not stabilized and did not accumulate, and cyclin D1-cdk association and kinase activation were not inhibited.
Two clonal rat rhabdomyosarcoma cell lines BA-Han-1B and BA-Han-1C with different capacities for myogenic differentiation have been examined for the expression of muscle regulatory basic helix-loop-helix (bHLH) proteins of the MyoD family. Whereas cells of the BA-Han-1C subpopulation constitutively expressed MyoD1 and could be induced to differentiate with retinoic acid (RA), BA-Han-1B cells did not express any of the myogenic control factors and appeared to be largely differentiation-defective. Upon induction with RA, BA-Han-1C cells expressed also myogenin, in contrast to BA-Han-1B cells which never activated any of the genes encoding muscle bHLH factors. The onset of myogenin transcription in BA-Han-1C cells required de novo protein synthesis and DNA replication suggesting that RA probably did not act directly on the myogenin gene. Although MyoD1 was expressed in proliferating BA-Han-1C myoblasts, muscle-specific reporter genes were not activated indicating that MyoD was biologically inactive. However, transfections with plasmid expressing additional MyoD1 protein resulted in the transactivation of muscle genes even in the absence of RA. mRNA encoding the negative regulatory HLH protein Id was expressed in proliferating BA-Han-1C cells and disappeared later after RA induction which suggested that it may be involved in the regulation of MyoD1 activity. The myogenic differentiation of malignant rhabdomyosarcoma cells strictly correlated with the activation of the myogenin gene. In fact, stable transfections of BA-Han-1C cells with myogenin expressing plasmids resulted in spontaneous differentiation. Together, our results suggest that the transformed and undifferentiated phenotype of BA-Han- 1C rhabdomyosarcoma cells is associated with the inactivation of the myogenic factor MyoD1 as well as lack of myogenin expression. RA alleviates the inhibition of myogenic differentiation, probably by activating MyoD protein and myogenin gene transcription. BA-Han-1B cells did not respond to RA and the differentiated phenotype could not be restored by overexpression of MyoD1 or myogenin.
Skeletal muscle differentiation, maturation, and regeneration are regulated by interactions between signaling pathways activated by hormones and growth factors, and intrinsic genetic programs controlled by myogenic transcription factors, including members of the MyoD and myocyte enhancer factor 2 (MEF2) families. Insulin-like growth factors (IGFs) play key roles in muscle development in the embryo, and in the maintenance and hypertrophy of mature muscle in the adult, but the precise signaling pathways responsible for these effects remain incompletely defined. To study mechanisms of IGF action in muscle, we have developed a mouse myoblast cell line termed C2BP5 that is dependent on activation of the IGF-I receptor and the phosphatidyl inositol 3-kinase (PI3-kinase)-Akt pathway for initiation of differentiation. Here, we show that differentiation of C2BP5 myoblasts could be induced in the absence of IGF action by recombinant adenoviruses expressing MyoD or myogenin, but it was reversibly impaired by the PI3-kinase inhibitor LY294002. Similar results were observed using a dominant-negative version of Akt, a key downstream component of PI3-kinase signaling, and also were seen in C3H 10T1/2 fibroblasts. Inhibition of PI3-kinase did not prevent accumulation of muscle differentiation-specific proteins (myogenin, troponin T, or myosin heavy chain), did not block transcriptional activation of E-box containing muscle reporter genes by MyoD or myogenin, and did not inhibit the expression or function of endogenous MEF2C or MEF2D. An adenovirus encoding active Akt could partially restore terminal differentiation of MyoD-expressing and LY294002-treated myoblasts, but the resultant myofibers contained fewer nuclei and were smaller and thinner than normal, indicating that another PI3-kinase-stimulated pathway in addition to Akt is required for full myocyte maturation. Our results support the idea that an IGF-regulated PI3-kinase pathway functions downstream of or in parallel with MyoD, myogenin, and MEF2 in muscle development to govern the late steps of differentiation that lead to multinucleated myotubes.
To investigate the mode of action of the p16INK4a tumor suppressor protein, we have established U2-OS cells in which the expression of p16INK4a can be regulated by addition or removal of isopropyl-β-d-thiogalactopyranoside. As expected, induction of p16INK4a results in a G1 cell cycle arrest by inhibiting phosphorylation of the retinoblastoma protein (pRb) by the cyclin-dependent kinases CDK4 and CDK6. However, induction of p16INK4a also causes marked inhibition of CDK2 activity. In the case of cyclin E-CDK2, this is brought about by reassortment of cyclin, CDK, and CDK-inhibitor complexes, particularly those involving p27KIP1. Size fractionation of the cellular lysates reveals that a substantial proportion of CDK4 participates in active kinase complexes of around 200 kDa. Upon induction of p16INK4a, this complex is partly dissociated, and the majority of CDK4 is found in lower-molecular-weight fractions consistent with the formation of a binary complex with p16INK4a. Sequestration of CDK4 by p16INK4a allows cyclin D1 to associate increasingly with CDK2, without affecting its interactions with the CIP/KIP inhibitors. Thus, upon the induction of p16INK4a, p27KIP1 appears to switch its allegiance from CDK4 to CDK2, and the accompanying reassortment of components leads to the inhibition of cyclin E-CDK2 by p27KIP1 and p21CIP1. Significantly, p16INK4a itself does not appear to form higher-order complexes, and the overwhelming majority remains either free or forms binary associations with CDK4 and CDK6.
ROR/RZR is an orphan nuclear receptor that has no known ligand in the 'classical sense'. In the present study we demonstrate that RORalpha is constitutively expressed during the differentiation of proliferating myoblasts to post-mitotic multinucleated myotubes, that have acquired a contractile phenotype. Exogenous expression of dominant negative RORalpha1DeltaE mRNA in myogenic cells significantly reduces the endogenous expression of RORalpha1 mRNA, represses the accumu-lation and delays the activation of mRNAs encoding MyoD and myogenin [the muscle-specific basic helix-loop-helix (bHLH) proteins] and p21(Waf-1/Cip-1) (a cdk inhibitor). Immunohistochemistry demonstrates that morpho-logical differentiation is delayed in cells expressing the RORDeltaE transcript. Furthermore, the size and development of mutlinucleated myotubes is impaired. The E region of RORalpha1 interacts with p300, a cofactor that functions as a coactivator in nuclear receptor and MyoD-mediated transactivation. Consistent with the functional role of RORalpha1 in myogenesis, we observed that RORalpha1 directly interacts with the bHLH protein MyoD. This interaction was mediated by the N-terminal activation domain of the bHLH protein, MyoD, and the RORalpha1 DNA binding domain/C region. Furthermore, we demonstrated that p300, RORalpha1 and MyoD interact in a non-competitive manner. In conclusion, this study provides evidence for a biological role and positive influence of RORalpha1 in the cascade of events involved in the activation of myogenic-specific markers and cell cycle regulators and suggests that crosstalk between theretinoid-relatedorphan (ROR) nuclear receptors and the myogenic bHLH proteins has functional consequences for differentiation.
When mouse myoblasts or satellite cells differentiate in culture, the expression of myogenic regulatory factor, MyoD, is downregulated in a subset of cells that do not differentiate. The mechanism involved in the repression of MyoD expression remains largely unknown. Here we report that a stress-response pathway repressing MyoD transcription is transiently activated in mouse-derived C2C12 myoblasts growing under differentiation-promoting conditions. We show that phosphorylation of the α subunit of the translation initiation factor 2 (eIF2α) is followed by expression of C/EBP homology protein (CHOP) in some myoblasts. ShRNA-driven knockdown of CHOP expression caused earlier and more robust differentiation, whereas its constitutive expression delayed differentiation relative to wild type myoblasts. Cells expressing CHOP did not express the myogenic regulatory factors MyoD and myogenin. These results indicated that CHOP directly repressed the transcription of the MyoD gene. In support of this view, CHOP associated with upstream regulatory region of the MyoD gene and its activity reduced histone acetylation at the enhancer region of MyoD. CHOP interacted with histone deacetylase 1 (HDAC1) in cells. This protein complex may reduce histone acetylation when bound to MyoD regulatory regions. Overall, our results suggest that the activation of a stress pathway in myoblasts transiently downregulate the myogenic program.
Peptide growth factors control diverse cellular functions by regulating distinct signal transduction pathways. In cultured myoblasts, insulin-like growth factors (IGFs) stimulate differentiation and promote hypertrophy. IGFs also maintain muscle cell viability. We previously described C2 skeletal muscle lines lacking expression of IGF-II. These cells did not differentiate, but underwent progressive apoptotic death when incubated in differentiation medium. Viability could be sustained and differentiation enabled by IGF analogues that activated the IGF-I receptor; survival was dependent on stimulation of phosphatidylinositol 3-kinase (PI3-kinase). We now find that IGF action promotes myoblast survival through two distinguishable PI3-kinase–regulated pathways that culminate in expression of the cyclin-dependent kinase inhibitor, p21. Incubation with IGF-I or transfection with active PI3-kinase led to rapid induction of MyoD and p21, and forced expression of either protein maintained viability in the absence of growth factors. Ectopic expression of MyoD induced p21, and inhibition of p21 blocked MyoD-mediated survival, thus defining one PI3-kinase–dependent pathway as leading first to MyoD, and then to p21 and survival. Unexpectedly, loss of MyoD expression did not impede IGF-mediated survival, revealing a second pathway involving activation by PI3-kinase of Akt, and subsequent induction of p21. Since inhibition of p21 caused death even in the presence of IGF-I, these results establish a central role for p21 as a survival factor for muscle cells. Our observations also define a MyoD-independent pathway for regulating p21 in muscle, and demonstrate that distinct mechanisms help ensure appropriate expression of this key protein during differentiation.
insulin-like growth factors; p21; MyoD; phosphatidyl inositol 3-kinase; Akt
The human papillomavirus (HPV) E7 protein promotes S-phase reentry in a fraction of postmitotic, differentiated keratinocytes. Here we report that these cells contain an inherent mechanism that opposes E7-induced DNA replication. In organotypic raft cultures of primary human keratinocytes, neither cyclin E nor p21cip1 is detectable in situ. However, E7-transduced differentiated cells not in S phase accumulate abundant cyclin E and p21cip1. We show that normally p21cip1 protein is rapidly degraded by proteasomes. In the presence of E7 or E6/E7, p21cip1, cyclin E, and cyclin E2 proteins were all up-regulated. The accumulation of p21cip1 protein is a posttranscriptional event, and ectopic cyclin E expression was sufficient to trigger it. In constract, cdk2 and p27kip1 were abundant in normal differentiated cells and were not significantly affected by E7. Cyclin E, cdk2, and p21cip1 or p27kip1 formed complexes, and relatively little kinase activity was found associated with cyclin E or cdk2. In patient papillomas and E7 raft cultures, all p27kip1-positive cells were negative for bromodeoxyuridine (BrdU) incorporation, but only some also contained cyclin E and p21cip1. In contrast, all cyclin E-positive cells also contained p27kip1. When the expression of p21cip1 was reduced by rottlerin, a PKC δ inhibitor, p27kip1- and BrdU-positive cells remained unchanged. These observations show that high levels of endogenous p27kip1 can prevent E7-induced S-phase reentry. This inhibition then leads to the stabilization of cyclin E and p21cip1. Since efficient initiation of viral DNA replication requires cyclin E and cdk2, its inhibition accounts for heterogeneous viral activities in productively infected lesions.
During the terminal differentiation of skeletal myoblasts, the activities of myogenic factors regulate not only tissue-specific gene expressions but also the exit from the cell cycle. The induction of cell cycle inhibitors such as p21 and pRb has been shown to play a prominent role in the growth arrest of differentiating myoblasts. Here we report that, at the onset of differentiation, activation by MyoD of the Rb, p21, and cyclin D3 genes occurs in the absence of new protein synthesis and with the requirement of the p300 transcriptional coactivator. In differentiated myocytes, cyclin D3 also becomes stabilized and is found nearly totally complexed with unphosphorylated pRb. The detection of complexes containing cyclin D3, cdk4, p21, and PCNA suggests that cdk4, along with PCNA, may get sequestered into high-order structures held together by pRb and cyclin D3. Cyclin D3 up-regulation and stabilization is inhibited by adenovirus E1A, and this correlates with the ability of E1A to promote pRb phosphorylation; conversely, the overexpression of cyclin D3 in differentiated myotubes counteracts the E1A-mediated reactivation of DNA synthesis. These results indicate that cyclin D3 critically contributes to the irreversible exit of differentiating myoblasts from the cell cycle.
Estrogens induce proliferation of estrogen receptor (ER)-positive MCF-7 breast cancer cells by stimulating G1/S transition associated with increased cyclin D1 expression, activation of cyclin-dependent kinases (Cdks), and phosphorylation of the retinoblastoma protein (pRb). We have utilized blockade of cyclin D1-Cdk4 complex formation through adenovirus-mediated expression of p16INK4a to demonstrate that estrogen regulates Cdk inhibitor expression and expression of the Cdk-activating phosphatase Cdc25A independent of cyclin D1-Cdk4 function and cell cycle progression. Expression of p16INK4a inhibited G1/S transition induced in MCF-7 cells by 17-β-estradiol (E2) with associated inhibition of both Cdk4- and Cdk2-associated kinase activities. Inhibition of Cdk2 activity was associated with delayed removal of Cdk-inhibitory activity in early G1 and decreased cyclin A expression. Cdk-inhibitory activity and expression of both p21Cip1 and p27Kip1 was decreased, however, in both control and p16INK4a-expressing cells 20 h after estrogen treatment. Expression of Cdc25A mRNA and protein was induced by E2 in control and p16INK4a-expressing MCF-7 cells; however, functional activity of Cdc25A was inhibited in cells expressing p16INK4a. Inhibition of Cdc25A activity in p16INK4a-expressing cells was associated with depressed Cdk2 activity and was reversed in vivo and in vitro by active Cdk2. Transfection of MCF-7 cells with a dominant-negative Cdk2 construct inhibited the E2-dependent activation of ectopic Cdc25A. Supporting a role for Cdc25A in estrogen action, antisense CDC25A oligonucleotides inhibited estrogen-induced Cdk2 activation and DNA synthesis. In addition, inactive cyclin E-Cdk2 complexes from p16INK4a-expressing, estrogen-treated cells were activated in vitro by treatment with recombinant Cdc25A and in vivo in cells overexpressing Cdc25A. The results demonstrate that functional association of cyclin D1-Cdk4 complexes is required for Cdk2 activation in MCF-7 cells and that Cdk2 activity is, in turn, required for the in vivo activation of Cdc25A. These studies establish Cdc25A as a growth-promoting target of estrogen action and further indicate that estrogens independently regulate multiple components of the cell cycle machinery, including expression of p21Cip1 and p27Kip1.
The Cip/Kip family, namely, p21Cip1, p27Kip1, and p57Kip2, are stoichiometric cyclin-dependent kinase inhibitors (CKIs). Paradoxically, they have been proposed to also act as positive regulators of Cdk4/6-cyclin D by stabilizing these heterodimers. Loss of p21Cip1 and p27Kip1 reduces Cdk4/6-cyclin D complexes, although with limited phenotypic consequences compared to the embryonic lethality of Cdk4/6 or triple cyclin D deficiency. This milder phenotype was attributed to Cdk2 compensatory mechanisms. To address this controversy using a genetic approach, we generated Cdk2−/−
p27−/− mice. Triple-knockout mouse embryonic fibroblasts (MEFs) displayed minimal levels of D-type cyclins and Cdk4/6-cyclin D complexes. p57Kip2 downregulation in the absence of p21Cip1 and p27Kip1 aggravated this phenotype, yet MEFs lacking all Cip/Kip proteins exhibited increased retinoblastoma phosphorylation, together with enhanced proliferation and transformation capacity. In vivo, Cdk2 ablation induced partial perinatal lethality in p21−/−
p27−/− mice, suggesting partial Cdk2-dependent compensation. However, Cdk2−/−
p27−/− survivors displayed all phenotypes described for p27−/− mice, including organomegalia and pituitary tumors. Thus, Cip/Kip deficiency does not impair interphasic Cdk activity even in the absence of Cdk2, suggesting that their Cdk-cyclin assembly function is dispensable for homeostatic control in most cell types.
Purpose. Rhabdomyosarcoma (RMS) is an embryonal tumor thought to arise from skeletal muscle cells that fail to
differentiate terminally. The majority of RMSs express MyoD, a protein essential to the differentiation of skeletal muscle.
It was recently shown that during myogenesis, MyoD activates the expression of the cyclin-dependent kinase inhibitor
(CDKi), p21, which itself plays a critical role in normal muscle development. To investigate the integrity of the MyoD/p21
pathway in RMS, we analyzed p21 and its relationship to MyoD expression in RMS.
Methods. A panel of RMS samples was assembled from primary biopsies and from cell lines. Integrity of p21 was analyzed
by single-strand conformation polymorphism (SSCP) and sequencing. Expression of p21 and MyoD was determined by
Northern blot analysis, and the ability of exogenous p21 to arrest the cell cycle of RMS cell line was determined by
Results. Our analysis indicates that although p21 is wild type in RMS, there is an inverse correlation between the levels
of p21 and MyoD in these tumors. Tumors that express significant amounts of MyoD fail to express p21. This does not
appear to be the result of mutations within the potential CACGTG sites present in the p21 promoter region or in the
coding region of p21. An additional group of RMSs express very high levels of p21 but express little, if any, MyoD.
Furthermore, RD, a RMS cell line which expresses high levels of endogenous p21, undergoes withdrawal from the cell
cycle following forced expression of p21, suggesting that the pathway which would lead to G1
arrest from endogenous p21 activity is defective.
Discussion. These data suggest that the interaction between p21 and MyoD is defective in RMS although the precise
nature of the defect remains to be elucidated.
The expression of retinoblastoma (pRb) and cyclin D3 proteins is highly induced during the process of skeletal myoblast differentiation. We have previously shown that cyclin D3 is nearly totally associated with hypophosphorylated pRb in differentiated myotubes, whereas Rb−/− myocytes fail to accumulate the cyclin D3 protein despite normal induction of cyclin D3 mRNA. Here we report that pRb promotes cyclin D3 protein accumulation in differentiating myoblasts by preventing cyclin D3 degradation. We show that cyclin D3 displays rapid turnover in proliferating myoblasts, which is positively regulated through glycogen synthase kinase 3β (GSK-3β)-mediated phosphorylation of cyclin D3 on Thr-283. We describe a novel interaction between pRb and cyclin D3 that maps to the C terminus of pRb and to a region of cyclin D3 proximal to the Thr-283 residue and provide evidence that the pRb-cyclin D3 complex formation in terminally differentiated myotubes hinders the access of GSK-3β to cyclin D3, thus inhibiting Thr-283 phosphorylation. Interestingly, we observed that the ectopic expression of a stabilized cyclin D3 mutant in C2 myoblasts enhances muscle-specific gene expression; conversely, cyclin D3-null embryonic fibroblasts display impaired MyoD-induced myogenic differentiation. These results indicate that the pRb-dependent accumulation of cyclin D3 is functionally relevant to the process of skeletal muscle cell differentiation.
To gain insight into the regeneration deficit of MyoD−/− muscle, we investigated the growth and differentiation of cultured MyoD−/− myogenic cells. Primary MyoD−/− myogenic cells exhibited a stellate morphology distinct from the compact morphology of wild-type myoblasts, and expressed c-met, a receptor tyrosine kinase expressed in satellite cells. However, MyoD−/− myogenic cells did not express desmin, an intermediate filament protein typically expressed in cultured myoblasts in vitro and myogenic precursor cells in vivo. Northern analysis indicated that proliferating MyoD−/− myogenic cells expressed fourfold higher levels of Myf-5 and sixfold higher levels of PEA3, an ETS-domain transcription factor expressed in newly activated satellite cells. Under conditions that normally induce differentiation, MyoD−/− cells continued to proliferate and with delayed kinetics yielded reduced numbers of predominantly mononuclear myocytes. Northern analysis revealed delayed induction of myogenin, MRF4, and other differentiation-specific markers although p21 was upregulated normally. Expression of M-cadherin mRNA was severely decreased whereas expression of IGF-1 was markedly increased in MyoD−/− myogenic cells. Mixing of lacZ-labeled MyoD−/− cells and wild-type myoblasts revealed a strict autonomy in differentiation potential. Transfection of a MyoD-expression cassette restored cytomorphology and rescued the differentiation deficit. We interpret these data to suggest that MyoD−/− myogenic cells represent an intermediate stage between a quiescent satellite cell and a myogenic precursor cell.
MyoD; myogenic regulatory factor; satellite cell; differentiation; proliferation