INTRODUCTION

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Skeletal muscle differentiation is characterized by terminal withdrawal from the cell cycle, the coordinated activation of muscle-specific gene expression, and the fusion of myoblasts into multinucleated myotubes. As in most cell types, proliferation and differentiation of skeletal myoblasts are mutually exclusive events. Established mouse myogenic cell lines have allowed the identification of muscle-specific transcription factors, belonging to the MyoD family, which determine the initiation and the maintenance of the myogenic program (reviewed in references 8, 18, 60, and 94). Muscle regulatory factors (MRFs) are basic helix-loop-helix transcription factors, which promote skeletal muscle differentiation by binding to a consensus sequence, termed E-box, present in the regulatory region of many muscle-specific genes (12, 95).

Besides regulating tissue-specific gene expression, the activity of MRFs is also involved in promoting cell cycle arrest (37, 42). Although the molecular mechanisms responsible for the coupling of cell cycle arrest with terminal differentiation of muscle cells have not been completely elucidated, several functional interactions between myogenic factors and cell cycle regulatory proteins have now been clarified. We have previously reported that MyoD induces transcription of the retinoblastoma growth suppressor gene (pRb) by a mechanism independent of direct binding of MyoD to the Rb gene promoter (46). It has also been shown that MyoD can mediate the transcriptional induction of the cell cycle inhibitor p21 (28).

A critical role for pRb activity in muscle cells was first suggested by the finding that the ability of DNA tumor virus oncoproteins, such as adenovirus E1A, simian virus 40 (SV40) large T antigen, and polyomavirus large T antigen, to inhibit myogenic differentiation is related to their ability to bind (and hence inactivate) the pRb family of proteins (10, 25, 43, 84). The importance of pRb in myogenesis is also indicated by the observation that muscle differentiation is associated with induced expression of pRb (19), which shows enhanced nuclear affinity and a hypophosphorylated, active state (25, 86). Further studies have more directly demonstrated that pRb function is required for myoblast differentiation; in fact, by using cells derived from mouse embryos specifically deficient for pRb, it has been demonstrated that pRb activity is required both for MyoD-mediated activation of muscle structural genes and irreversible cell cycle withdrawal (58, 78). Moreover, physical interaction between pRb and MyoD has been found both in vitro and in vivo (25), though the question of how such interaction regulates MyoD or pRb activity remains unanswered.

The function of pRb is known to be inactivated through phosphorylation by cyclin-dependent kinases (cdk's), which act in conjunction with their regulatory partners, the cyclins (reviewed in references 54, 57, 72, 80). As expected, the expression in muscle cells of most cyclins is down-regulated at the onset of terminal differentiation, as cells arrest in the G₀/G₁ phase of the cell cycle (33, 70, 91), with the notable exception of cyclin D3, whose expression is actually induced during terminal differentiation (36, 71). The activity of cdk's is negatively regulated by cdk inhibitors, which bind to either cdk or cyclin-cdk complexes, inhibiting their activity and blocking cell cycle progression (reviewed in references 30 and 81). In addition, the cdk inhibitor p21 can also function as a direct inhibitor of DNA polymerase by binding to the proliferating cell nuclear antigen (PCNA) subunit (89). Increased expression of the p21 and p18 cdk inhibitors has been associated with the process of terminal muscle differentiation (1, 27, 28, 52, 65, 66).

In addition to pRb, another important cellular protein, p300, targeted by viral oncoproteins and involved in cell cycle control, was first suggested as a regulator of myogenic differentiation based on E1A's requirement for p300 binding to inhibit differentiation (10, 56). Subsequently, p300 has been identified as a transcriptional adapter which assists the function of several transcriptional activators by mediating their communication with the basal transcription machinery (2, 16, 39). Recently, we and others have demonstrated that p300 enhances the transcriptional activity of MyoD and is involved in both the cell growth arrest and the myogenic activity of MyoD (17, 67, 77, 102).

To further clarify the functional links between myogenic factors and inhibitors of cell proliferation, in this study we have addressed two questions. The first question is whether the mechanism by which MyoD induces Rb, p21, and cyclin D3 during muscle differentiation is common to these three genes, and in particular, whether they are direct targets of MyoD transactivation or whether a new synthesis of factors is needed. Furthermore, we wanted to determine whether p300 and/or pRb function is implicated in this induction by MyoD. The second question concerns the functional role of a normally proliferative factor such as cyclin D3 in terminally differentiated myotubes. To investigate these problems, we used cells expressing a hormone-inducible MyoD protein and cells in which myogenic differentiation is blocked at different stages by adenovirus E1A mutants.

	MATERIALS AND METHODS

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Plasmids and probes. Many of the probes used in this study have been described previously (46). The mouse p21 probe was prepared by EcoRI plus HindIII digestion from the pGDSV7S-mwaf1 plasmid, obtained from C. Schneider (Trieste, Italy). The mouse cyclin D3 probe was obtained by EcoRI digestion from the pcN2.cyl3 plasmid (50).

Cells and DNA transfections and luciferase assays. Clone 7 of the C2 line of mouse myoblasts (101) was obtained from M. Buckingham (Institut Pasteur, Paris, France). C2 myoblasts were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 20% fetal bovine serum (HyClone). Myoblasts were carefully passaged before cell-cell contact, to avoid selection. To induce differentiation, 2 × 10⁵ cells, seeded in 100-cm-diameter dishes and allowed to grow for 3 days (until they reached 80 to 90% confluence), were exposed for 2 days to DMEM containing 2% fetal bovine serum. E1A-expressing C2 cell lines, generated by stable transfection of wt and E1A mutant derivatives, have been previously described and characterized (10).

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Microinjection experiments and immunofluorescence. Microinjection experiments were performed as previously described (24, 67). However, 3-day-old C2 myotubes, cultured in 2% fetal calf serum (FCS)-enriched medium, were injected into the nuclei with 0.2 µg of the E1A N-terminal mutant (12S E1A RG2) and either 0.6 µg of the cyclin D3 expression vector Rc/CMV-CycD3 or 0.6 µg of the Rc/CMV empty vector; all the DNA preparations were in Tris-EDTA (TE; pH 7.6). Twelve hours before the injection, cells were shifted to 0.1% FCS, and 4 h after the injection, bromodeoxyuridine (BrdU) was added for an additional 18 h. Cells were then washed in phosphate-buffered saline (PBS), fixed, and processed for immunofluorescence. To detect E1A expression, cells were fixed in a 1:2 methanol-acetone solution, dried, preincubated with 5% bovine serum albumin (BSA) in PBS, and incubated for 30 min at 37°C with a 1:5 dilution of hybridoma-conditioned medium containing the M73 mouse monoclonal anti-E1A antibody (29). Specifically bound antibody was visualized by incubation with rhodamine-conjugated second-step anti-mouse immunoglobulin (Ig) antibody (Cappel). Immunofluorescence for detection of BrdU, as DNA synthesis indicator, was performed with an anti-BrdU antibody directly conjugated with fluorescein (Boehringer), according to the manufacturer's instructions. After immunofluorescence treatment, nuclei were stained by a 3-min incubation in a 1-µg/ml solution of 4',6-diamidino-2-phenylindole (DAPI) in PBS.

Northern blot analysis. Total cellular RNA was isolated by using the method of Chomczynski and Sacchi (11). Ten micrograms of each RNA sample was size fractionated on 1.2% agarose-30% formaldehyde gels and transferred to Qiabrane nylon filters (Qiagen), as described by Thomas (85). The integrity and the amount of RNA were checked by ethidium bromide staining of ribosomal RNA. DNA fragments, purified by low-melting agarose gels, were ³²P labelled by random priming and used to probe the filters. Hybridization was carried out for 24 h at 42°C in 50% formamide, 5× SSPE (1× SSPE is 0.15 M NaCl, 0.01 M sodium phosphate, 0.001 M EDTA [pH 7.7]), 5× Denhardt's solution (1× Denhardt's is 0.02% Ficoll, 0.02% polyvinyl-pyrrolidone, 0.02% BSA), 0.5% sodium dodecyl sulfate (SDS), 20 µg of salmon sperm DNA, and 2 × 10⁶ cpm of ³²P-labelled probes per ml. Filters were washed three times in 0.2× SSC (1× SSC is 0.15 M NaCl plus 0.015 M Na-citrate), 0.2% SDS, for 30 min at 58°C, followed by exposition to phosphorstorage screens (Molecular Dynamics).

Immunoprecipitation and Western blot analyses. For immunoprecipitation experiments, exponentially growing or differentiated C2 cells were rinsed two times with PBS, and cell lysates were prepared by the addition of ice-cold Nonidet P-40 (NP-40) lysis buffer (Tris-HCl [pH 7.4], 50 mM; NaCl, 250 mM; NP-40, 0.5%; ATP, 5 mM; MgCl₂, 5 mM) or Triton lysis buffer (Tris-HCl [pH 7.4], 50 mM; NaCl, 250 mM; Triton, 1%; ATP, 5 mM; MgCl₂, 5 mM), both supplemented with protease and phosphatase inhibitors (leupeptin, 10 µg/ml; aprotinin, 10 µg/ml; phenylmethylsulfonyl fluoride [PMSF], 1 mM; Na₃VO₄, 0.1 mM; NaF, 50 mM). After clearing was performed by centrifugation at 14,000 rpm for 15 min, extracts were assayed for protein concentration by the Bradford assay (6); 500-µg aliquots were then precleared with either a rabbit preimmune serum or the total mouse IgG fraction (Sigma, St. Louis, Mo.) and protein A-agarose for 2 h at 4°C. After centrifugation at 12,000 rpm, the supernatants were incubated with protein A-agarose or protein G-agarose beads (Pierce) and with the appropriate antibodies for at least 2 to 4 h at 4°C. The immunoprecipitates were washed four times with ice-cold lysis buffer and then resuspended in 2× Laemmli buffer (1× Laemmli buffer is Tris-HCl [pH 6.8], 62.5 mM; SDS, 2%; glycerol, 10%; -mercaptoethanol, 5%), heat denatured, and run on SDS-polyacrylamide gels.

Immune complex kinase assay. Cells were suspended in lysis buffer containing 50 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) (pH 7.5), 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1 mM DTT, 0.1% Tween 20, 10% glycerol, 1 mM PMSF, 10 µg of leupeptin per ml, 5 µg of aprotinin per ml, 10 mM -glycerophosphate, 1 mM sodium orthovanadate, and 50 mM NaF (all protease inhibitors were obtained from Sigma Chemicals), followed by a 10-s sonication and clearing by centrifugation at 14,000 rpm for 15 min. Supernatants were assayed for protein concentration as described above; protein samples of 4 mg each were then immunoprecipitated for at least 2 to 4 h at 4°C with protein A-agarose beads precoated with saturating amounts of the appropriate antibody (5 µg). Immunoprecipitated proteins on beads were washed three times with 1 ml of lysis buffer and twice with kinase buffer (50 mM HEPES [pH 7.5], 1 mM DTT, 10 mM MgCl₂, plus protease inhibitors, as described above). The beads were resuspended in 50 µl of kinase buffer containing 2 µg of glutathione S-transferase (GST)-pRb (769-921) fusion protein (Santa Cruz Biotechnology, Inc.), 2.5 mM EGTA, 10 mM -glycerophosphate, 1 mM Na₃VO₄, 20 µM ATP, and 10 µCi of [-³²P]ATP (6,000 Ci/mmol; NEN Dupont, Boston, Mass.). After incubation for 30 min at 30°C, the samples were boiled in 2× Laemmli buffer and separated by SDS-PAGE. Phosphorylated proteins were visualized by exposure to phosphorstorage screens. The antibodies used were as follows: for cyclin D1, MAb clone DCS11, kindly provided by J. Bartek; for cyclin D3, MAb clone 18B6-10; and for cdk4 and cdk2, polyclonal rabbit antibodies obtained from Santa Cruz Biotechnology.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

The expression of p21, Rb, and cyclin D3 is directly induced by MyoD and requires p300. Previous work has shown that terminal differentiation of muscle cells is accompanied by transcriptional induction of the retinoblastoma gene (Rb) and the accumulation of the hypophosphorylated (active) form of the Rb protein (pRb) (25, 46, 86). Although the cdk4 and cdk2 pRb kinases are constitutively expressed during myoblast differentiation, their regulatory subunits (cyclin D1, cyclin E, and cyclin A) are down-regulated (33, 71, 83, 91). In contrast, cyclin D3, which is known to function as a cdk4-activating subunit, is greatly induced in differentiating muscle cells (36, 71). The p21 cdk inhibitor is markedly induced upon skeletal muscle differentiation (27, 28, 65), contributing to the decrease of cdk activity (92). Because the increased expression of the Rb, p21, and cyclin D3 mRNAs is a typical feature of muscle cell differentiation, we wished to determine whether these three genes are direct targets of MyoD.

View larger version (58K): [in this window] [in a new window]   FIG. 1.   Direct activation of p21, Rb, and cyclin D3 by MyoD-ER. (A) Confluent cultures of C3H-ER-MyoD cells were exposed to differentiation medium (DM) for increasing periods of time in the presence or absence of estradiol as indicated. Total RNA was extracted from cells at each stage of differentiation and then subjected to Northern blot analysis. Identical filters were probed for myogenin, p21, cyclin D3, Rb, and myosin heavy chain (myosin HC). Ethidium bromide staining of rRNA, on one of the filters, was photographed with UV light. (B) C3H-ER-MyoD cells were induced to differentiate with estradiol (107 M) in the presence or absence of cycloheximide (CHX; 50 µg/ml) for 13 h. The effects of cycloheximide were determined also in C3H-ER-MyoD cells maintained in differentiation medium for 13 h in the absence of estradiol. Total RNA was extracted and then analyzed by Northern blotting, with probes for myogenin, p21, cyclin D3, and Rb. One of the filters was reprobed with a GAPDH probe to normalize the amounts of loaded RNA.

View larger version (57K): [in this window] [in a new window]   FIG. 2.   Levels of p21, cyclin D3, and Rb mRNAs in C2 cells expressing wt E1A and E1A mutant derivatives. Northern blotting was used to analyze the RNA isolated from C2 parental cells and C2-derived stable lines expressing either wt E1A or the E1A mutants described in Table 1. Identical filters were probed for myogenin, MyoD, p21, cyclin D3, Rb, and myosin heavy chain. Shown are results for growing cells (GM) and cells kept for 48 h in differentiation medium (DM). One of the filters was reprobed with a GAPDH probe to normalize the amounts of loaded RNA.

Cyclin D3 mediates the interaction of cdk4, p21, and PCNA with pRb in differentiated C2 cells. The increased levels of pRb, p21, and cyclin D3 during differentiation argued for a role of these proteins in terminally differentiated myotubes. It has been well established that pRb is essential for both cellular growth arrest and myogenic bHLH activity in skeletal muscle cells and that p21 contributes to the mechanism by which differentiating myocytes irreversibly exit the cell cycle (27, 28, 58, 78). By contrast, the significance of cyclin D3 induction still awaits elucidation.

View larger version (52K): [in this window] [in a new window]   FIG. 3.   Composition and activity of cdk4-cyclin D-p21-PCNA complexes in undifferentiated versus differentiated C2 cells. (A) Cell lysates from C2 myoblasts, either growing (GM) or exposed for 48 h to differentiation medium (DM), were immunoprecipitated by using antibodies against the proteins indicated at the top. The immunoprecipitated proteins were resolved by SDS-PAGE and then analyzed by immunoblotting with antibodies specific for cdk4, cyclin D3, cyclin D1, p21, and PCNA, as indicated on the left. (B) cdk4, cyclin D1, and cyclin D3 were immunoprecipitated from C2 cells either growing (GM) or exposed for 48 h to DM. The protein complexes, collected on protein A-Sepharose beads, were then incubated in the presence of [-32P]ATP with a bacterially produced GST-Rb fusion protein as a substrate (as detailed in Materials and Methods). Phosphorylated proteins were resolved on SDS-PAGE gels, and radioactivity was detected with a PhosphorImager (Molecular Dynamics). As a negative control, myoblast and myotube extracts were immunoprecipitated with total mouse IgGs; the Rb-kinase activity of these immunoprecipitates was measured as described above.

View larger version (36K): [in this window] [in a new window]   FIG. 4.   (A) Analysis of protein complexes immunoprecipitated with anti-cdk4 or anti-pRb from C2 myotubes. Immunoprecipitation-coupled immunoblot analyses were performed by using extracts prepared from C2 cells harvested after 48 h in differentiation medium. Protein complexes, immunoprecipitated by using anti-cdk4 or anti-pRb antibodies, were resolved by SDS-PAGE and then subjected to immunoblot analyses with anti-cdk4, cyclin D3, p21, pRb, or PCNA antibodies (lanes 1 and 3). Following the first immunoprecipitation, cyclin D3, cdk4, p21, pRb, or PCNA were immunoprecipitated from the cdk4- and the pRb-depleted supernatants and detected by Western blotting by using the specific antibodies (lanes 2 and 4). (B) Time course of pRb protein induction in C2 differentiating cells. C2 myoblasts, cultured at low density (LD; approximately 40% confluent), were allowed to reach confluence (HD; high density) in growth medium (GM) and then transferred to differentiation medium (DM) for the times indicated. The cells, at each stage of differentiation, were extracted either in 0.2% SDS, 2% NP-40, 50 mM NaCl (mild extracting condition), or directly in SDS sample buffer (strong extracting condition). Proteins were separated on SDS-PAGE gels and subjected to immunoblot analysis with the MAb G 245 anti-pRb antibody, as detailed in Materials and Methods. Arrows indicate positions of the underphosphorylated (pRb) and hyperphosphorylated (ppRb) forms of the pRb protein. The staged cell extracts prepared in strong extracting conditions were also monitored for myogenin and myosin heavy chain (myosin HC) expression by using antibodies specific for these proteins.

pRb phosphorylation in E1A-expressing C2 cells. The sequence motif through which D-type cyclins interact with pRb is similar to the pRb-binding motif of some viral oncoproteins, and cyclin D-pRb complexes are disrupted by E1A and derived peptides (15, 20). Thus, we exploited E1A-expressing C2 cells in order to investigate the effect of the inhibition of the cyclin D3-pRb interaction on pRb phosphorylation.

View larger version (46K): [in this window] [in a new window]   FIG. 5.   pRb phosphorylation in E1A-expressing C2 cells. (A) Immunoblot analysis of pRb in C2 parental and C2-derived, E1A-expressing cell lines cultured either in growth (GM) or in differentiation (DM) medium for 48 h. For a description of the E1A mutants used to generate the E1A-expressing stable cell lines, see Table 1. Extracts were prepared by lysing cells in SDS sample buffer, proteins were then separated on SDS-PAGE gels and subjected to immunoblot analysis with the MAb G 245 anti-pRb antibody. The hypophosphorylated (pRb) and the hyperphosphorylated (ppRb) forms of the pRb proteins are indicated. (B) Levels of cyclins, cdk's, and cdk's in C2-derived stable lines expressing wt or mutant E1A. Cell extracts were prepared from C2 myotubes and from E1A-expressing C2 cells cultured in differentiation medium for 48 h. Levels of cyclin A, cyclin E, cyclin D3, cdk2, cdk4, p21, and p27 were monitored by immunoblot analysis by using antibodies specific for these proteins. (C) Determination of the kinase activity associated with cdk2 and cdk4 in E1A-expressing cells. cdk2 and cdk4 were immunoprecipitated from lysates of C2 myoblasts (growth medium; GM) or myotubes (differentiation medium; DM) or from C2-derived E1A-expressing cell lines cultured for 48 h in DM. Immunocomplexes, coupled to protein A-Sepharose, were incubated in the presence of [-32P]ATP with a bacterially expressed GST-Rb fusion protein or with commercial histone H1 as the substrate (see Materials and Methods). The 32P-labelled proteins were then separated on SDS-PAGE gels, and radioactivity was detected with a PhosphorImager (Molecular Dynamics). As a negative control, the C2 myotube extract was immunoprecipitated with total mouse IgGs; the pRb- and histone H1-kinase activities of this immunoprecipitate were measured as described above.

Cyclin D3 is involved in the permanent withdrawal of C2 myotubes from the cell cycle. It has been previously demonstrated that the expression of E1A in terminally differentiated myotubes, by either adenovirus infection or microinjection, triggers the reactivation of DNA synthesis and that this activity of E1A correlates with its ability to bind the pRb family members (67, 87). These observations and the results shown above indicate that the binding to pRb is required by E1A both to induce DNA synthesis and to suppress cyclin D3, suggesting that these two phenomena might be part of the same mechanism.

View larger version (36K): [in this window] [in a new window]   FIG. 6.   The overexpression of cyclin D3 can counteract the E1A-mediated reactivation of DNA synthesis in terminally differentiated myotubes. C2 myoblasts were allowed to differentiate for three days and then coinjected into the nuclei with 0.2 µg of the E1A N-terminal encoding plasmid in combination with 0.6 µg of either the Rc/CMV-cyclin D3 expression construct (d, e, and f), or 0.6 µg of the Rc/CMV empty vector (a, b, and c). Twelve hours before the injection, myotubes were shifted to 0.1% FCS, and 4 h after the injection BrdU was added for an additional 18 h. Cells were then fixed and stained for nuclear expression of E1A and BrdU incorporation. In panels a and d, E1A expressing nuclei were visualized by using an anti-E1A antibody (M73), followed by incubation with rhodamine-conjugated second-step antimouse antibody. In panels b and e, the nuclei were visualized by DAPI counterstaining. In panels c and f, the nuclei incorporating BrdU were visualized by staining with an anti-BrdU antibody directly conjugated with fluorescein. The injection of a single myotube nucleus resulted in the expression of the injected plasmids in all of the nuclei belonging to the same myotube; one representative field is shown. The results were reproduced in two independent experiments. In each of these experiments at least 50 nuclei positive for the expression of E1A were counted and scored for BrdU staining. When the E1A N-terminal expression construct was injected alone, almost 100% of the E1A-expressing nuclei were also positive for BrdU staining; in contrast, upon coinjection of the cyclin D3 expression vector, only 15% of the E1A-positive nuclei showed significant incorporation of BrdU.

View larger version (37K): [in this window] [in a new window]   FIG. 7.   Disruption of PCNA-cdk4 complexes in E1A-expressing C2 cells. Cell lysates prepared from C2 or from E1A-expressing C2 cells cultured in differentiation medium for 48 h were immunoprecipitated with the anti-cdk4 antibody. Immunoprecipitated proteins were then resolved by SDS-PAGE and analyzed by immunoblotting by using antibodies specific for p21, cyclin D3, or PCNA. The levels of PCNA in each cell line were detected by immunoblot analysis of whole-cell lysates (WCL).

The function of pRb is required for cyclin D3 stabilization in differentiating muscle cells. Two findings of the present study let us hypothesize that a pRb-dependent mechanism stabilizes cyclin D3 in differentiating C2 cells. The first finding was that nearly all the cyclin D3 of myotubes was complexed with pRb (Fig. 4A), and the second finding was that the inhibition of cyclin D3 accumulation in C2 cells expressing E1A mutants correlated with the ability of E1A to bind pRb (Fig. 5B). The simplest interpretation of these findings is that cyclin D3 was stabilized by its binding to pRb and that E1A disrupted such interaction and destabilized cyclin D3. If this was correct, the cyclin D3 protein would not be able to accumulate during the differentiation of muscle cells lacking pRb.

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View larger version (34K): [in this window] [in a new window]   FIG. 8.   Cyclin D3 is a short-lived protein in Rb/ myocytes, and its degradation is mediated by the ubiquitin proteasome pathway. (A) Northern blot analysis of total RNA isolated from CC42 and C2 myogenic cells, either proliferating (GM) or maintained in differentiation medium for 48 h (DM). Identical filters were probed for Rb, myogenin, or cyclin D3. Ethidium bromide staining of rRNA on one of the filters was photographed with UV light. (B) Whole-cell extracts were prepared from C2 and CC42 cells, either proliferating (GM) or differentiating (48 h in differentiation medium [DM]). Equal amounts of proteins were separated on SDS-PAGE gels and subjected to immunoblot analysis with antibodies specific for pRb, myogenin, and cyclin D3. (C) Levels of cyclin D3 in differentiating CC42 cells treated with proteasome inhibitors. CC42 cells were exposed to differentiation medium for 24 h and then treated for 2 or 4 h with DMSO solvent alone or with MG132 (10 µM), LLnL (50 µM), or chloroquine (100 µM), as indicated. Whole-cell lysates, normalized for protein concentration, were immunoprecipitated with a cyclin D3 polyclonal antibody (sc182; Santa Cruz Biotechnology). Immunoprecipitated proteins were then resolved by SDS-PAGE and analyzed by immunoblotting by using an anti-cyclin D3 MAb (sc453; Santa Cruz Biotechnology).

Effect of ectopic expression of cyclin D3 in growing and differentiating myoblasts. The results reported thus far indicate that muscle differentiation accumulates high levels of cyclin D3 through both transcriptional and posttranscriptional mechanisms and that cyclin D3 contributes a function antagonistic to growth in terminally differentiated myotubes; this must be overcome by E1A to reactivate DNA synthesis in these postmitotic cells. To examine the role of cyclin D3 in differentiation with another, more direct approach, we determined whether the ectopic expression of cyclin D3 could activate the expression of muscle-specific genes in growing C2 myoblasts. We transfected growing C2 myoblasts with a luciferase reporter plasmid containing the MCK promoter-enhancer in the presence or absence of a cyclin D3-expression construct; a CMV--galactosidase expression construct was cotransfected to monitor transfection efficiencies. MCK transcriptional activity was then assessed in nonconfluent or mitogen-stimulated confluent cultures as well as in cultures under normal differentiating conditions. As shown in Fig. 9, the MCK promoter activity was slightly enhanced by ectopic cyclin D3 in nonconfluent and confluent cultures in growth medium.

View larger version (39K): [in this window] [in a new window]   FIG. 9.   Effect of ectopic expression of cyclin D3 on MCK expression in growing and differentiating C2 myoblasts. Proliferating C2 myoblasts were transfected with 0.5 µg of the MCK luc reporter plasmid in the presence of the indicated amount of the cyclin D3 expression construct (Rc/CMV-cycD3); the Rc/CMV expression vehicle without an insert was included to normalize DNA in all transfections. Eighteen hours after transfection, the cells were trypsinized; one half of the cells were plated onto 90-mm-diameter dishes, the other half were plated onto 60-mm-diameter dishes, and refed with growth medium. Parallel transfections were directly transferred to differentiation medium. After 72 h the cells in 90-mm-diameter dishes were subconfluent (A), those in 60-mm-diameter dishes were confluent (B), and those in differentiation medium were fully differentiated (C); at that time cells were collected and assayed for luciferase and -galactosidase activities. Equal amounts of proteins from each cell lysate were also assayed for cyclin D3 expression by Western blotting. The results shown are from one representative experiment. MCK-luc activity is expressed relative to the levels detected in the absence of cyclin D3. The experiments were done in duplicate and repeated three times. The mean values (expressed as fold induction relative to the baseline values) and the standard deviations (S.D.) of these experiments are shown at the bottom.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

The differentiation of skeletal myoblasts requires that these cells exit the cell cycle. Besides initiating muscle-specific gene expression, the myogenic bHLH factor MyoD has been implicated in promoting the cell cycle arrest, by inducing cell growth repressors, such as pRb and the kinase inhibitor p21. In this study, we show that cyclin D3, whose expression is strongly upregulated in differentiating muscle cells, also plays an important role in the irreversible cell cycle arrest of differentiated myocytes. The analysis of the mechanism of pRb, p21, and cyclin D3 induction during differentiation revealed that these genes are regulated similarly by MyoD.

The expression of p21, Rb, and cyclin D3 is directly regulated by MyoD and requires the function of p300. By using MyoD-ER cell lines, it has been previously shown that hormone-activated MyoD, in the absence of new protein synthesis, directly induces the expression of the endogenous MyoD gene and that of myogenin but does not induce several downstream muscle genes (32). This finding indicates that the initial activation of MyoD leads to the induction of early differentiation genes, whose function, in turn, is required to activate late muscle genes. We show here that p21, Rb, and cyclin D3 are induced by MyoD (like myogenin) without the requirement of newly synthesized factors, allowing these genes to be categorized as early differentiation markers. The MyoD-mediated induction of these genes is also dependent on p300 function.

View larger version (19K): [in this window] [in a new window]   FIG. 10.   (A) Schematic model of the multistep process of terminal differentiation. MyoD directly activates its own transcription and that of early differentiation genes through a mechanism that is cycloheximide (CHX) resistant and requires p300. Induction of late differentiation genes is CHX sensitive and requires pRb and myogenin. pRb, p21, and cyclin D3 contribute to cell cycle arrest of differentiating myoblasts. E1A, by targeting both p300 and pRb, interferes with the early and the late steps of muscle differentiation. (B) Schematic model of the E1A-mediated reinduction of DNA synthesis in terminally differentiated myotubes. pRb sequesters cyclin D3 along with inactive cdk's and PCNA; pRb binds E2F and recruits histone deacetylase (hDAC) to E2F. E1A, by binding the pRb pocket, inhibits the binding of E2F and cyclin D3 to pRb. Consequently, active E2F induces S-phase genes, and cyclin D3 is degraded; active cdk's and PCNA contribute to S-phase entry.

Regulation of cyclin D3 expression during muscle differentiation. It has been well established that D-type cyclins, whose expression is induced by serum growth factors (50), promote cellular proliferation by activating cdk4 and cdk6, both of which act as pRb kinases (48, 49, 51). Thus, as myoblasts become committed to terminal differentiation by serum growth factor withdrawal, the expression and/or activity of D-type cyclins would be expected to be down-regulated. Indeed, the activation of the terminal differentiation program in C2 myoblasts leads to the rapid disappearance of cyclin D1 mRNA and protein (33, 71, 91).

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Cyclin D3 complexes in differentiated C2 cells. The results discussed above indicate that the induction of cyclin D3 mRNA and the stabilization of the cyclin D3 protein are physiological features of muscle differentiation. Interestingly, it has been recently reported that cyclin D3 also accumulates to high levels in differentiating skeletal muscle in vivo during the late stages of mouse fetal development and the first weeks of postnatal life but not in fully differentiated adult skeletal muscle tissues, suggesting a role for cyclin D3 in the induction and/or establishment, rather than in the maintenance, of mammalian skeletal muscle differentiation (4).

The function of cyclin D3 is required for the irreversible cell cycle arrest of differentiated C2 cells. The cyclin D3-mediated associations discussed above delineate a self-sustaining mechanism able to ensure prompt pRb dephosphorylation and an irreversible cell cycle arrest during myogenic differentiation. In this mechanism, the critical role played by cyclin D3 is supported by the effects of E1A interference with the normal cell cycle regulation as follows: (i) the ability of E1A, when stably expressed in C2 cells, to induce pRb phosphorylation correlates with its ability to inhibit cyclin D3 expression, and (ii) E1A targets cyclin D3 to reactivate DNA synthesis in terminally differentiated myotubes.