Departments of 1Internal Medicine and 2Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-8573
Submitted 7 February 2003 ; accepted in final form 21 June 2003
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ABSTRACT |
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myoblasts; stem cells; apoptosis; differentiation
Cell cycle progression is primarily controlled by two regulatory complexes: the cyclin/cyclin dependent kinases (Cdks), which promote cell cycle progression, and the Cdk inhibitors, which repress cell cycle progression. In skeletal muscle, particular interest has been focused on the Cdk inhibitor p21CIP1/WAF1 (hereafter referred to as p21), for its regulatory role in muscle growth and differentiation. Early during MPC differentiation, p21 is induced by myogenic factors such as MyoD (9). The induction of p21 blocks progression through the cell cycle by binding to proliferating cell nuclear antigen (PCNA) and the G1 phase cyclin/Cdk complexes to prevent their enzymatic activity (for review, see Ref. 21). Mice deficient in p21 (p21-/-) develop into phenotypically normal adults with no increase in spontaneous malignancies (6) and no defects in migration-associated differentiation within small intestine cell lineages (4). These results suggest that functionally redundant proteins are capable of compensating for the absence of p21, and this suggestion is supported by reports of mice lacking both p21 and p57 (25). These double-mutant mice display severely arrested muscle development, suggesting that p57 is the redundant Cdk inhibitor compensating for the absence of p21 during skeletal muscle development. Collectively, these studies demonstrate that functionally redundant proteins are capable of compensating for the lack of p21 during development.
We hypothesized that adult p21-/- mice would demonstrate enhanced skeletal muscle regeneration on the basis our findings of increased MPC number and proliferative capacity in p21-/- skeletal muscle. After cardiotoxin-induced muscle injury, p21-/- skeletal muscle displayed attenuated regeneration coincident with an increase in proliferating and apoptotic nuclei throughout the regenerative period. Serum withdrawal in p21-/- MPC cultures resulted in a marked impairment in differentiation with an increase in differentiation-induced apoptosis compared with wild-type MPCs. The protein expression of other Cdk inhibitors, p27 and p57, was not upregulated in differentiating p21-/- MPCs, suggesting that other redundant pathways may be responsible for the regeneration that ultimately occurs in adult p21-/- skeletal muscle after injury.
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MATERIALS AND METHODS |
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Primary MPC cultures. Asynchronously dividing primary MPC cultures were harvested from hindlimb skeletal muscle of neonatal (2-5 day old) mice (16). Cells were preplated twice for 30 min and grown on collagen coated plates in F-10 growth medium (20% fetal bovine serum, 0.5% penicillin/streptomycin, 25 ng/ml basic fibroblast growth factor). In selected experiments, MPCs were induced to undergo differentiation with the use of a DMEM differentiation medium containing 2% normal horse serum, 0.5% penicillin/streptomyocin, HEPES buffer (50 mM), insulin (10 µg/ml), and transferrin (10 µg/ml).
Cell cycle and cell size profiling. Proliferating primary MPC cultures from wild-type (n = 9) and p21-/- (n = 5) skeletal muscle were fixed in 4% paraformaldehyde, permeabilized with 0.3% Triton X-100, and incubated with a propidium iodide staining solution (1.8 mg/ml RNAse A, 50 µg/ml propidium iodide) for >3 h to label DNA. Using a FACScan (Becton Dickinson, Franklin Lakes, NJ) flow cytometer, we sorted >10,000 cells on the basis of DNA content. The data were processed, and the percentage of cells in each phase of the cell cycle was quantified using Cellquest software (BD Biosciences). Cell size/diameter differences between wild-type and p21 null MPCs (n = 4 for each group) were analyzed using FSC-H (3).
Western analysis. Differentiating wild-type and p21-/- primary MPCs were harvested for Western analysis at 0, 2, 3, and 5 days after exposure to differentiation medium (n = 3-4 at each time point). Whole cell protein extracts were prepared from differentiating cells as described previously (1). Western membranes were probed with anti-myoglobin (DAKO; rabbit polyclonal; 1:1,000), anti-p27, anti-Rb, anti-p53 (BD Biosciences; mouse monoclonal; 1:1,000), anti-Fas, or anti-p57 (Santa Cruz Biotechnologies; rabbit polyclonal: 1:1,000). Signals were visualized using Supersignal chemiluminescent reagent (Pierce Biotechnology) according to the supplier's instructions. Equal loading control was performed using -tubulin (Sigma Chemical; mouse monoclonal; 1:2,000) and Ponceau stain (Sigma Chemical; not shown).
BrdU incorporation and TUNEL assays of proliferating and differentiating MPCs. To assess cellular proliferation, we incubated primary MPCs grown on collagen-coated cover-slips for 7 h with 10 µM 5'-bromo-2'-deoxyuridine (BrdU; Roche Molecular Biochemicals). After incubation, cells were fixed for 5 min with 4% paraformaldehyde and stained with anti-BrdU monoclonal serum (1:25 dilution; Roche Molecular Biochemicals), followed by a FITC-conjugated goat anti-mouse secondary antibody (1:25 dilution). Cells were costained with propidium iodide (50 ng/ml) to label all nuclei. Cellular proliferation was quantified as a percentage of BrdU-positive nuclei (proliferating cells) to propidium iodide-positive nuclei (total cells). A total of >1,500 propidium iodide-positive nuclei were counted for each genotype. The time for wild-type and p21-/- MPCs to complete the cell cycle was calculated on the basis of the percentage of cells that were BrdU positive after 7 h of BrdU incubation.
To assess apoptotic cell death in differentiating cells, we grew primary MPCs in chamber slides to 80% confluency and then exposed them to differentiation medium. After 60 h in differentiation medium, cells were washed twice with ice-cold PBS and fixed for 10 min with ice-cold 4% paraformaldehyde. After fixation, apoptosis was assessed using a fluorometric TdT-mediated dUTP nick end labeling (TUNEL) assay kit (Promega) according to the supplier's instructions.
Electron microscopy for MPC identification and quantitation. Tibialis anterior (TA) muscles from three wild-type and p21-/- mice were harvested after perfusion fixation with 3% glutaraldehyde. Samples were postfixed with buffered 1% osmium tetroxide, dehydrated with ethanol, embedded in Spurr resin, and polymerized overnight at 60°C. Sections (80 nm) were suspended on 200-mesh copper grids, stained with uranyl acetate and lead citrate, and examined using a JEOL 1200EXII transmission electron microscope. MPCs and myonuclei were identified and quantified according to criteria previously described (18), with a total of 500 myonuclei counted for p21-/- and wild-type skeletal muscle.
Cardiotoxin injury of skeletal muscle. TA muscles from wild-type and p21-/- mice (n = 3 for each time point) were injected with cardiotoxin (100 µl/muscle of 10 µM cardiotoxin, Naja nigricollis; Calbiochem) (7). TA muscles were harvested at 0 (uninjured), 5, 10, 14, and 21 days post-cardiotoxin injection and fixed overnight with 4% paraformaldehyde. Fixed tissues were processed and paraffin-embedded for further analyses.
Staining and immunohistochemical analyses of regenerating skeletal muscle. Paraffin-embedded sections were stained with Masson's trichrome to assess skeletal muscle fiber architecture. Immunohistochemical detection for desmin was undertaken with sections incubated overnight at 40°C with the polyclonal rabbit anti-desmin serum (Biogenex; 1:40). All sections were blocked with appropriate normal sera, and endogenous peroxidase activity was quenched by incubating sections in 0.6% hydrogen peroxide/methanol. Biotinylated goat anti-rabbit (Vector Laboratories; 1:200) secondary antibody was visualized with FITC-conjugated streptavidin (Vector Laboratories; 1:50). All negative control sections (PBS substituted for primary antibody) had an absence of signal.
The degree of apoptosis in regenerating wild-type and p21-/- skeletal muscle was assessed using a fluorometric TUNEL assay kit (previously described). Proliferating cells in regenerating wild-type and p21-/- skeletal muscles were evaluated using anti-PCNA rabbit polyclonal serum (Santa Cruz Biotechnologies; 1:100), followed by FITC-labeled goat anti-rabbit secondary serum (1:500 dilution). All sections were costained with propidium iodide (50 ng/ml) to label all nuclei. An average of >1,000 propidium iodide nuclei were counted from four to five different fields for each time period assessed. The percentage of PCNA-positive or TUNEL-positive nuclei was calculated as the total number of PCNA- or TUNEL-positive nuclei divided by the number of propidium iodide-positive nuclei (n = 4, >1,000 cells counted). All negative control sections (PBS substituted for primary antibody) had an absence of signal.
Imaging. Images were viewed on a Leica Laborlux S photomicroscope or an Olympus IMT2 inverted photomicroscope (cell culture images) equipped with epifluorescence optics and were collected using an Optronics VI 470 charge-coupled device camera and Scion Image 1.62 imaging software.
Data analysis. Student's t-tests were performed to identify significant differences (P < 0.05) in data obtained from wild-type and p21-/- mice. Data are presented as means ± SE.
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RESULTS |
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MPC number in 2- to 4-mo-old uninjured wild-type and p21-/- TA skeletal muscle was quantitated using electron microscopy (n = 3 for each group). Skeletal muscle lacking p21 displayed a small increase in MPC number compared with wild-type skeletal muscle (3.58 ± 0.37 vs. 2.86 ± 0.05%, respectively; P = 0.06).
Attenuated regeneration after myotrauma in p21 null skeletal muscle. After cardiotoxin-induced injury, p21-/- skeletal muscle regeneration was significantly impaired compared with wild-type skeletal muscle (Fig. 2). At 5 days postinjury, no visible difference between regenerating wild-type and p21-/- muscle was apparent. By 10 days postinjury, extensive regeneration had occurred in wild-type skeletal muscle with numerous newly regenerated myofibers containing centrally located nuclei. In contrast, widespread damage was still observable in the regenerating p21-/- skeletal muscle, which continued up to 21 days postinjury.
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Desmin is an intermediate filament protein that is expressed early during muscle differentiation, and its expression continues into the mature adult myofiber. Using immunohistochemical techniques, we used desmin expression to further evaluate the impaired regeneration of p21-/- skeletal muscle. Ten days after injury, desmin expression was visibly reduced in p21-/- skeletal muscle compared with wild-type muscle (Fig. 3). Reduced expression of desmin was still observable at 21 days of regeneration in the p21-/- skeletal muscle. Whereas wild-type desmin expression was homogeneous throughout the regenerated muscle at 21 days postinjury, expression in regenerating p21 null skeletal muscle was heterogeneous with visibly smaller myofibers. The smaller myofiber size in the regenerating p21-/- muscle, compared with wild-type skeletal muscle, implies that the regenerating p21-/- myofibers are less mature.
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Cellular proliferation in regenerating muscle, as assessed by PCNA expression, was increased in p21 null skeletal muscle compared with wild-type skeletal muscle up to 3 wk after injury (Fig. 4, A and D). Many of the PCNA-positive nuclei observed at 14 days of regeneration in wild-type skeletal muscle surrounded the regenerated myofibers (Fig. 4B), suggesting that these were MPC nuclei taking on a sublaminar position to replenish the quiescent MPC population. In contrast, the PCNA-positive nuclei in the p21-/- skeletal muscle were associated with mononuclear cells located interstitially, indicative of MPC nuclei in the active proliferation phase (as shown in 5-day regenerating wild-type skeletal muscle; Fig. 4, A and D). Interestingly, the increased proliferative response observed in p21-/- skeletal muscle was coincident with an increase in the total number of apoptotic nuclei compared with regenerating wild-type muscle. The increased apoptosis in p21-/- skeletal muscle was significant at 5 days of regeneration and remained significantly elevated until 21 days postinjury (Fig. 4, C and D).
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Impaired differentiation of p21-/- MPCs. We were interested to determine whether the defect in MPC differentiation was the result of impaired differentiation cues/signals in p21-/- regenerating muscle rather than a cell autonomous defect in p21-/- MPC differentiation. To evaluate this, we differentiated wild-type and p21-/- primary MPCs into myotubes through exposure to a low-serum differentiation medium. At day 1 in differentiation medium, p21-/- MPCs were indistinguishable from wild-type MPC cultures (Fig. 5A). However, MPCs lacking p21 appeared vacuolated and were nonviable or apoptotic after 3 days in differentiation medium. By day 4, significantly fewer myotubes were observed in the p21-/- cultures compared with wild-type cultures (Fig. 5B). Protein expression of myoglobin (a marker of differentiated myofibers) was reduced in p21-/- MPCs undergoing differentiation compared with wild-type MPCs, corroborating the impairment in p21-/- MPC differentiation (Fig. 5C). Notably, differentiating p21-/- MPCs were more prone to undergo apoptosis than wild-type MPCs (Fig. 5D), consistent with the increase in apoptotic nuclei observed during regeneration in p21-/- skeletal muscle. The increase in apoptotic nuclei observed in differentiating p21-/- MPCs using a TUNEL assay was corroborated by Western analysis of Fas (Fig. 5C). Fas, a member of the TNF- family, is a cell surface apoptosis-signaling molecule, and its increased expression is consistent with an increase in apoptosis. These results together support the conclusion that the impaired differentiation capacity of p21-/- MPCs is a cell autonomous defect rather than an impairment of differentiation signals within the regenerating p21-/- skeletal muscle.
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Despite the temporal impairment, p21-/- skeletal muscle ultimately regenerated to become indistinguishable from regenerated wild-type skeletal muscle. We attempted to identify possible compensatory proteins allowing for regeneration to occur in p21-/- skeletal muscle. Western analysis of p21-/- differentiating MPCs did not display an upregulation of the other CIP family Cdk inhibitors, p27 and p57 (Fig. 5C), consistent with the mRNA expression observed by others (6). There were no changes in p53 protein expression in differentiating p21-/- MPCs compared with wild-type MPCs (Fig. 5C). Analysis of retinoblastoma (Rb) expression demonstrated an increased phosphorylated state at 2 days of differentiation in p21-/- MPCs (Fig. 5C). These data are consistent with an increased number of p21-/- MPCs able to cycle even in the presence of differentiation medium. The phosphorylation of Rb was not different from that in wild-type MPCs at 3 or 5 days of differentiation medium (Fig. 5C).
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DISCUSSION |
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The increase in absolute number and cell cycle progression of p21-/- MPCs observed in the present study is consistent with the role of p21 as a cell cycle inhibitor and with previous reports demonstrating that the number and proliferative capacity of hematopoietic stem cells (HSCs) is elevated in p21-/- mice (5). An increased proliferative capacity is not a general phenotype of all p21-/- cells, however, because p21 null fibroblasts displayed no difference in proliferation compared with controls (4), suggesting that other Cdk inhibitors may be the primary regulators of cell cycle control in other cell types. The role for specific Cdk inhibitors within distinct cell populations is further supported by the finding that p57-/- mice display significant growth deficits attributed to impaired differentiation and increased apoptosis in chondrocytes, whereas no defects in proliferative capacity were observed within cardiac and skeletal muscle (24, 25).
The increased MPC number and cell cycle progression lead us to hypothesize that p21-/- skeletal muscle would display enhanced regeneration compared with wild type controls. Injection of cardiotoxin results in a highly reproducible myotrauma where >80% of the injected muscle undergoes a well-characterized pattern of degeneration/regeneration (7). We observed no difference between wild-type and p21-/- skeletal muscle during the early phases of regeneration (0-5 days), suggesting that it was not a defect associated with the acute phases of degeneration/regeneration that impaired regeneration in the absence of p21. However, in contrast to our initial hypothesis, a marked impairment in regeneration became apparent in p21-/- skeletal muscle during the later phases of regeneration, coincident with the period of MPC differentiation and maturation (10). Similar to the HSC response to myelotoxic stress (5), we observed a coincident increase in proliferating and apoptotic nuclei in regenerating p21-/- skeletal muscle compared with wild-type skeletal muscle. Though these cells were not specifically identified as MPCs, the increased proliferation and differentiation-induced apoptosis of MPCs observed in vitro strongly supports the hypothesis that the MPC population is undergoing coincident proliferation and apoptosis in regenerating p21-/- skeletal muscle. Therefore, it is conceivable that repeated injuries to p21 null skeletal muscle may result in premature exhaustion of the MPC pool and that future studies utilizing combinatorial matings of the p21-/- mice with myopathic mouse models (e.g., mdx mouse) would prove useful in addressing this issue
To examine the impaired myogenic differentiation capacity of p21-/- MPCs in greater detail, we utilized a cell culture strategy. Primary MPCs harvested from p21 null skeletal muscle demonstrated a significant impairment in their ability to differentiate upon serum withdrawal. Three days after exposure to differentiation medium, p21-/- MPCs were vacuolated and undergoing apoptosis, indicating that these cells were unable to coordinate cell cycle exit and myogenic differentiation, ultimately resulting in apoptotic cell death. This result is supported by the work of Brugarolas and colleagues (3), who reported that, unlike wild-type fibroblasts, p21 null fibroblasts were still capable of some proliferation when exposed to very low serum levels (<0.5%). Furthermore, induction of p21 coincides with an apoptotic-resistant phenotype in differentiating cells (22), supporting our findings of an increased apoptosis during MPC differentiation in the absence of p21.
The impaired differentiation of p21-/- MPCs observed in vitro provides strong support for a cell autonomous defect being primarily responsible for the attenuated regeneration in p21-/- skeletal muscle. However, it is possible that the lack of p21 in other cell lineages was involved in the attenuated regeneration. For example, if the absence of p21 in macrophages prolonged their presence in regenerating skeletal muscle, the increased cytokines and growth factors associated with macrophages (12) could account for the reduced ability of MPCs to differentiate appropriately. Future studies involving cell transplantation of p21-/- MPCs into wild-type mouse skeletal muscle would prove useful in addressing this issue.
Previous studies have provided evidence that other cell cycle inhibitors, including p27, p57, and p53, are involved in skeletal muscle growth and development (19, 20, 25). We harvested whole cell lysates from differentiating wild-type and p21 null MPCs to determine whether an upregulation of other Cdk inhibitors was compensating for the absence of p21. p27, p53, and p57 protein expression were not upregulated in differentiating p21 null MPCs compared with wild-type MPCs, consistent with previous observations of p21-/- fibroblast gene expression (6). Zhang et al. (25) demonstrated that mice lacking p21 and p57 displayed profound developmental defects, including an increased proliferation and apoptosis in myoblasts and a failure to form myotubes. These results imply that during development, p57 may be a redundant Cdk inhibitor compensating for the absence of p21. Interestingly, these defects during development in double-mutant mice are similar to the present results showing that adult p21-/- MPCs displayed a dysregulated cell cycle progression, increased apoptosis, and impaired differentiation. It is interesting to speculate that during development, an array of environmental cues would promote an increased expression of p57, thus compensating for the absence of p21, whereas in injured adult skeletal muscle, these same cues are not present (or are of varied magnitude) to promote a similar compensation by p57.
In the present study, no change in p53 protein expression was observed in differentiating p21-/- MPCs compared with wild-type MPCs. Studies have demonstrated that p21-/- cancer cell lines were sensitized to apoptosis through a p53-mediated pathway (11), and a direct involvement of p53 (independent of p21) in myogenic cell differentiation has also been reported (19). Although we observed no increase in p53 in differentiating p21-/- MPCs, an increased p53 expression is not necessary to increase apoptosis, because it has been demonstrated that increased E2F1 (created by deletion of p21) can lead to increased p53 activity through a p14ARF-mediated pathway (2).
Changes in Rb protein, particularly the phosphorylation state, could compensate for the absence of p21 in differentiating MPCs. p21-/- MPCs displayed an increased phosphorylation state of Rb after 2 days in differentiation medium compared with wild-type MPCs. This observation is consistent with an increased number of p21-/- MPCs in a proliferative state at this time period. By 3 days in differentiation medium, the level of Rb phosphorylation in p21-/- MPCs was not significantly different from that in wild-type MPCs. The return to a wild-type level of Rb phosphorylation by 3 days in differentiation medium may be explained in two ways. First, the p21-/- MPC population is heterogeneous in nature such that cells that are unable to reduce Rb phosphorylation during differentiation will undergo apoptosis, whereas MPCs that are capable of reducing Rb phosphorylation will acquire an apoptosis-resistant phenotype and ultimately differentiate. Second, posttranslational modifications in other Cdk inhibitors or altered expression of other key cell cycle proteins may have compensated for the absence of p21.
Here we report that p21 is essential for the coordinated regulation of MPC cell cycle exit and myogenic differentiation during skeletal muscle regeneration. We hypothesize that the impaired regeneration observed in p21-/- skeletal muscle is the result of two factors: increased apoptosis and impaired differentiation within the MPC population. The increased apoptosis in the absence of p21 is consistent with the antiapoptotic effects of p21 during differentiation (23), an increased cell cycle rate reducing the potential for adequate DNA repair, and an increase in active E2F transcription factors resulting in p53-mediated apoptosis (2). Moreover, this hypothesis is consistent with the results of previously published gene disruption strategies including p57 and Rb. Mice lacking p57 display increased apoptosis within cardiac and skeletal muscles during development and impaired differentiation and increased apoptosis within chondrocytes, and Rb-/- myocytes display increased apoptosis and are unable to permanently exit the cell cycle despite forming multinucleated myotubes (22).
The impairment in muscle differentiation observed in the present study is in contrast to reports of a normal muscle phenotype during development in p21-/- mice (6). We propose that the environment associated with myotrauma in adult p21-/- skeletal muscle is dissimilar to that during embryonic development and that, as such, the signals/cues promoting the compensatory mechanisms are not occurring at the same rate and/or magnitude. Furthermore, because of the absence of p21, there is an increased phosphorylation of Rb by the cyclin/Cdks, resulting in Rb inactivation and increased E2F transcriptional activity. The increased activity of E2F promotes cell cycle progression through induction of cell cycle genes, and the phosphorylation of Rb reduces its ability to act as a coactivator of MyoD family members (8), further reducing signals necessary for myogenic differentiation.
In conclusion, the present study provides direct evidence that p21 is critical for normal cell cycle exit and myogenic differentiation within the adult MPC population. In the absence of p21, MPCs display an impaired differentiative capacity and an increased apoptosis, ultimately resulting in attenuated skeletal muscle regeneration following myotrauma.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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Present address of T. J. Hawke: Dept. of Pure and Applied Science, School of Kinesiology and Health Science, York University, 4700 Keele St., Toronto, ON, Canada M3J 1P3.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
* T. J. Hawke and A. P. Meeson contributed equally to this work.
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