- Email: [email protected]
Increased nuclear proteins in muscle satellite cells in aged animals as compared to young growing animals
Experimental Gerontology 39 (2004) 1521–1525 www.elsevier.com/locate/expgero
Increased nuclear proteins in muscle satellite cells in aged animals as compared to young growing animals Shuichi Machidaa, Frank W. Bootha,b,* a
Department of Biomedical Sciences, University of Missouri-Columbia, E102 Veterinary Medical Building, 1600 East Rollins Road, Columbia, MO 65211, USA b Department of Medical Pharmacology and Physiology, Dalton Cardiovascular Center, University of Missouri-Columbia, Columbia, MO 65211, USA Received 14 May 2004; received in revised form 9 July 2004; accepted 20 August 2004 Available online 11 September 2004
Abstract Evidence implies that satellite cells could play some limiting role in aged muscle undergoing repair or maintenance of mass, which is of potential clinical concern as this could contribute to sarcopenia. Further, insufficient information is available concerning the cellular mechanisms responsible for the lower rat satellite cell proliferation in old animals. Thus, it was hypothesized that the following proteins would be increased in nuclei of satellite cells from old rat skeletal muscle: the cyclin-dependent kinase (CDK) inhibitors p21WAF1/CIP1 and p27Kip1 as well as the transcription factors p53 and Forkhead box, subgroup O1 (FOXO1). In addition, the NADC-dependent histone deacetylase SIRT1, the mammalian ortholog of the yeast SIR2 (silence information regulator 2) and a member of the Sirtuin family, was hypothesized to decrease in satellite cell nuclei of old rats. Old satellite cells (30-months old) exhibited a lesser number of BrdU-positive cells as compared to satellite cells (3-months old) from young growing animals. Western blot analysis demonstrated that nuclei of old satellite cells accumulated the cell cycle inhibitors p21WAF1/CIP1 and p27Kip1. In addition, nuclear p53 and FOXO1 proteins were also higher in old satellite cells than in cells from young growing animals. These data indicated both p53/p21WAF1/CIP1- and FOXO1/p27Kip1-dependent pathways might contribute to the age-associated decrease in satellite cell proliferation. Cytoplasmic manganese superoxide dismutase (MnSOD), a gene driven by FOXO1, was higher in old satellite cells. Unexpectedly, nuclear SIRT1 was also increased in old satellite cells compared with satellite cells from young growing animals. The physiological significance of enhanced nuclear SIRT1 expression in old satellite cells remains elusive at this time. In summary, satellite cells in old rats have nuclear accumulation of proteins inhibiting the cell cycle as compared to young, growing animals. q 2004 Elsevier Inc. All rights reserved. Keywords: Forkhead; p21; p27; p53; Sir2
1. Introduction Aged skeletal muscle has impaired capacities of regeneration (Brooks and Faulkner, 1990; Grounds, 1998) and regrowth (Chakravarthy et al., 2000b). Conboy et al. (2003) attribute the dramatic age-related decline in myoblast generation in response to injury an impairment
* Corresponding author. Address: Department of Biomedical Sciences, University of Missouri-Columbia, E102 Veterinary Medical Building, 1600 East Rollins Road, Columbia, MO 65211, USA. Tel.: C1-573-882-6652; fax: C1-573-884-6890. E-mail address: [email protected] (F.W. Booth). 0531-5565/$ - see front matter q 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.exger.2004.08.009
of activation of satellite cells. Satellite cells are the myogenic cells responsible for post-natal growth, as well as regeneration and repair of injured muscle (Hawke and Garry, 2001). Satellite cell replication has been reported to be limiting in skeletal muscle growth. Elimination of proliferation by satellite cells with low-level g-irradiation in young animals limits muscle hypertrophy produced by mechanical overload (Rosenblatt and Parry, 1992; Adams et al., 2002). A second example is that myoblast transfer in gene therapy is limited by history of satellite cell proliferation. The replication of human satellite cells for an additional 15 divisions in culture resulted in a 6-fold
1522
S. Machida, F.W. Booth / Experimental Gerontology 39 (2004) 1521–1525
reduction in the number of myoblasts that were incorporated into muscle fibers (Cooper et al., 2003). Cooper et al. (2003) explained that their result substantiates the notion that the approach to replicative senescence for satellite cells, and therefore, the loss of replicative potential is important for successful host cell muscle regeneration after myoblast transplantation. However, insufficient cellular information is available to explain the decreased satellite cell proliferation in old animals/humans. The hypothesis of the current study was that proteins involved in cell cycle arrest would be elevated in nuclei of satellite cells isolated from old rats as compared to those from young, growing rats since the proliferation of old satellite cells is less. An experimental strategy was to follow-up previously published experiments on young, growing and old rats. Skeletal muscles in old rats showed no regrowth from limb immobilization whereas young muscles completely regrew (Pattison et al., 2003). The notion was then that proteins that inhibit the cell cycle would be less in young, growing than in old satellite cells. We therefore selected for study proteins with inhibitory actions on the cell cycle. p21WAF1/CIP1 and p27Kip1 induce cell cycle arrest (Coqueret, 2003), so a sub-hypothesis was made that nuclear level of p21WAF1/CIP1 and p27Kip1 proteins would be higher in satellite cells from old as compared to young, growing muscles, and thus be associated with decreased satellite cell proliferation. In response to various types of stress, including increased oxidative stress, p53 accumulates in the nucleus, where it transcriptionally activates genes that are involved in cell cycle arrest, such as p21WAF1/CIP1 (el-Deiry et al., 1993), and thus p53 induces cell cycle arrest (Donehower, 2002; Vogelstein et al., 2000). Therefore, an additional sub-hypothesis was generated that p53 protein would be more in nuclei of satellite cells from old than young, growing animals. The rationale was that oxidative stress is higher in old skeletal muscle (Ji, 2001), so p53 could be lower in the nuclei of young, growing satellite cells. The Forkhead box, subgroup O1, (FOXO1) transcription factor is of interest to aging studies because it binds to promoters of specific genes, stimulating cell cycle arrest by upregulating p27Kip1 (Dijkers et al., 2000b; Medema et al., 2000) and stimulates manganese superoxide dismutase (MnSOD) expression. Increased MnSOD activity leads to decreased cell growth due to prolonged cell cycle transition times in G1 and S phases without significant changes in G2/M phase (Kim et al., 2004), so an increase in MnSOD in old satellite cells could contribute to their decreased proliferation, as well as to minimize oxidative stress. Consequently, another sub-hypothesis was that FOXO1 and MnSOD proteins would be higher in nuclei from satellite cells of old than young, growing animals. NADC-dependent histone deacetylase Sir2 (silence information regulator 2) (designated as SIRT1 (sirtuin 1) in the current paper) overexpression in yeast and C. elegans extends lifespan and has been linked to longevity regulation
(Lin et al., 2000; Tissenbaum and Guarente, 2001). Previously we have shown that satellite cells isolated from old rats have decreased longevity in culture (Chakravarthy et al., 2000b); therefore, our final sub-hypothesis is that SIRT1 would be less in old satellite cell nuclei.
2. Materials and methods 2.1. Materials Cell culture medium and reagents were purchased from Life Technologies, Inc. (Rockville, MD). Antibodies against anti- p21WAF1/CIP1 (Catalog # 05-345), p27Kip1 (#06-445), MnSOD (#06-984) and Sir2 (#07-131) were obtained from Upstate Biotechnology (Lake Placid, NY). Mouse monoclonal antibody to p53 (#2524) and polyclonal rabbit anti-FKHR (FOXO1) (#9462) were purchased from Cell Signaling (Beverly, MA). Anti-paxillin (#P13520) was purchased from Transduction Laboratories (Lexington, KY). D3 mouse monoclonal anti-desmin was acquired from the Developmental Studies Hybridoma Bank (Iowa City, IA). MoAb5.8A mouse monoclonal anti-MyoD antibody was purchased from Pharmingen (San Diego, CA). 5-bromo-2 0 -deoxyuridine-5 0 -monophosphate (BrdU) was obtained from Sigma (St Louis, MO) and mouse monoclonal antibody to BrdU from Roche Diagnostics (Indianapolis, IN). 2.2. Animals Pathogen-free, F1 generation of Fisher 344! Brown Norway male rats (3- and 30-months old) were obtained from Harlan Labs (National Institute of Aging). Since old rats exhibit defects in muscle regrowth from atrophy (Chakravarthy et al., 2000b), age comparisons were selected between young, growing rats and old rats experiencing muscle loss in order to determine differences in inhibitory cell cycle proteins in nuclei of satellite cells. All the animals were housed at 21 8C in a 12-h light/12-h dark cycle. Rat chow and water were provided ad libitum. The animals were allowed to acclimatize to their new surrounding for 2 weeks before the cell isolation procedures were performed. All animal experimental protocols were approved by the University of Missouri Institutional Animal Care and Use Committee. 2.3. Isolation of satellite cells Animals were anesthetized with a single intraperitoneal injection of a cocktail (2.0 ml/kg body weight) consisting of 75 mg/ml ketamine, 3 mg/ml xylazine, and 5 mg/ml acepromazine. Satellite cells were isolated from the gastrocnemius, plantaris, tibialis anterior, extensor digitorum longus, and quadriceps muscles of individual rats according to the previously described methods used in our
S. Machida, F.W. Booth / Experimental Gerontology 39 (2004) 1521–1525
lab (Machida et al., 2004). No-passage, isolated cells from a single rat for every observation were seeded separately for immunocytochemistry, for BrdU, and for Westerns. To determine the percentage of myogenic cells present, the isolated cells were stained with the MyoD antibody (dilution; 1:100) and desmin antibody (dilution; 1:3) by immunocytochemical analysis performed as previously described by us (Machida et al., 2004) with the following modifications. Cells were seeded at 5000 cells per well. For the visualization of the primary antibody binding, the diaminobenzidine (DAB) substrate kit (Vector Laboratories, California) for peroxidase was used. At least a total of 200 cells were counted. The isolated cells of each age group contained more than 90% both MyoD- and desmin-positive cells (data not shown). All cultures were maintained at 37 8C in a humid air atmosphere containing 6% CO2.
1523
Fig. 1. Nuclear proteins isolated from satellite cells of young (Y) and old (O) rats do not contain detectable paxillin in Western blots. Cytoplasmic proteins from the young satellite cells was loaded as a positive control. All lanes were loaded with equal protein (30 mg). MW stands for the lane containing the molecular weight markers (Right-hand arrows indicate position of markers).
2.4. Western blots No-passage, isolated cells from a single rat were seeded in a 100-mm collagen-coated dish at a density of 1.5–2!105 cells/dish and cultured 3–4 days in the growth medium until cells for both young- and old-derived cells were 60–70% confluent. Five rats composed each age group. Cells were lysed and scraped in ice-cold hypotonic buffer (HB) (25 mM Tris (pH 7.5), 1 mM MgCl2, 5 mM KCl, 0.05% NP-40, 10% glycerol, 1 mM Na3VO4, 1 mM benzamidine, 1 mM DTT, 10 mg/ml leupeptin, 10 mg/ml aprotinin, 10 mg/ml pepstatin, 10 mg/ml tosyl-L-phenylalanine chloromethyl ketone, 10 mg/ml Na-tosyl-L-lysine chloromethyl ketone, and 2 mM Pefabloc SC Plus). The scraped cells were lysed while slowly rotated for 10 min at 4 8C. Nuclei were pelleted by centrifugation at 500 g for 5 min, washed two times in HB, and lysed in extract buffer (50 mM HEPES (pH 7.5), 300 mM NaCl, 5 mM EDTA, 0.065% NP-40, 10% glycerol, plus inhibitors described above). The nuclei were lysed while slowly rotated for 30 min at 4 8C. Nuclear protein was pelleted by centrifugation at 15,000 g for 10 min. The separation of the nuclear fraction was confirmed by the absence of paxillin in Western blotting (Fig. 1). Paxillin is a cytoplasmic protein that localizes to focal adhesions at the sarcolemma (Schaller, 2001). Protein concentrations were determined by Bradford assay (Biorad). Equal amounts of total protein from young and old (nZ5/age group) were resolved on the same SDS-PAGE gel and subsequently transferred to a nitrocellulose membrane. All blots were then incubated with Ponceau S (Sigma) and verified equal loading in all lanes (data not shown). The membranes were then blocked with 5% nonfat dry milk in Tris-buffer saline with 0.1% Tween (TBST) for p53, FOXO1 and Sir2 antibodies, 3% milk in PBS for p27Kip1, p21WAF1/CIP1 and MnSOD, and 2.5% milk and 1% bovine serum albumin (BSA) in TBST for paxillin. All membranes were then probed overnight with the appropriate antibody. Antibodies were used at the following
concentrations: p53 (1:2000 in 5% BSA-TBST), Sir2 (1:10,000 in 5% BSA-TBST), FOXO1 (1:1000 in 5% BSA-TBST), p27Kip1 (1:5000 in 3% milk-PBS), p21WAF1/CIP1(1:2000 in 3% milk-PBS), MnSOD (1:2000 in 3% milk-PBS), and paxillin (1:10,000 in 2.5% milk and 1% BSA-TBST). The membranes, except for p53 protein, were washed and then incubated for 1 h with horseradish peroxidase-conjugated rabbit or mouse secondary antibody (1:2000 or 1:3000 rabbit, 1:1000 or 1:2000 mouse, Amersham Pharmacia Biotech, Piscataway, NJ. For p53 protein detection, the membrane was incubated with biotinylated anti-mouse IgG (1:200; Vector Laboratories, Burlingame, CA) in the blocking buffer for 30 min. The blot was then washed and incubated with the VECTASTAIN elite ABC reagent (1:50; Vector Laboratories) in the blocking buffer for an additional 30 min. Immunocomplexes were visualized using the enhanced chemiluminescence reagent (NEN Life Science Products, Boston, MA). The membrane was exposed to Hyperfilme ECL (Amersham Pharmacia Biotech, Piscataway, NJ) with the exposure time adjusted to keep the integrated optical densities (IOD) within a linear and non-saturated range. The signal bands were scanned utilizing the Personal Densitometer SI (Molecular Dynamics, Sunnyvale, CA) and quantified using ImageQuant software (Molecular Dynamics). Linear responses to loaded protein concentrations of 50 mg have been shown for p21WAF1/CIP1, p27Kip1, and SIRT1 (unpublished observations). 2.5. Pulse labeling proliferation index To evaluate proliferation, BrdU incorporation was examined Briefly, the isolated cells were seeded at 10,000 cells per well on the collagen-coated glass chamber slides (Lab-Tek II, Nunc) in 600 (L of F-10 with high serum condition. After a 48-h-incubation, cultures were pulse labeled for 1 h with 10 mM bromodeoxyuridine
1524
S. Machida, F.W. Booth / Experimental Gerontology 39 (2004) 1521–1525
(BrdU), followed by immunocytochemistry for detection of BrdU according to the manufacturer’s directions (Roche Diagnostics, Indianapolis, IN). For the visualization of the BrdU antibody binding, the diaminobenzidine (DAB) substrate kit (Vector Laboratories, California) for peroxidase was used. At least a total of 200 cells were counted. The percentage of BrdU labeled cells was used as an indicator of proliferating cells. 2.6. Statistics The data are shown as mean G SE and were analyzed by Student’s t-test. P!0.05 was considered statistically significant.
3. Results 3.1. Pulse labeling proliferation index of cultured satellite cells, protein concentrations in satellite cell nuclei, and cytoplasmic MnSOD protein Immunohistochemical detection of BrdU-positive cells demonstrated that the percentage of labeled satellite cells was decreased by 61% in old as compared to young, growing muscles (Table 1). Nuclear p21WAF1/CIP1 and p27Kip1 proteins were 114 and 782%, respectively, higher in isolated satellite cells from old than young, growing rats (Table 1, Fig. 2). Nuclear p53 protein was 68% higher in primary satellite cells from old than young, growing rats (Table 1, Fig. 2). The nuclear expression of FOXO1 was increased by 131% in old satellite cells compared with young, growing satellite cells (Table 1, and Fig. 2). Nuclear SIRT1 protein was 75% higher in old satellite cells than young, growing satellite cells (Table 1, and Fig. 2). Nuclear levels of GSK-3b protein did not differ (pZ0.39) between young and old rats (data not shown), suggesting that the aforementioned increases in proteins in the nucleus were not a generalized response. Cytoplasmic MnSOD protein was
Table 1 Pulse labeling proliferation index and protein concentrations Measurement
Age a
BrDU incorporation Nuclear p21WAF1/CIP1b Nuclear p27Kip1b Nuclear p53b Nuclear FOXO1b Cytoplasmic MnSODb Nuclear SIRT1b †
3-months old
30-months old
31.3G2.32 320G38.4 0.0365G0.008 132G8.01 152G44.4 938G128 663G109
12.2G0.97‡ 684G98.2‡ 0.322G0.09‡ 222G25.5‡ 351G30.2‡ 1594G225† 1157G176†
p!0.05; ‡p!0.01. Values are the means G SEM. nZ5 per group. a % Positive cells. b Arbitrary densitometry units.
Fig. 2. Representative blots of nuclear p21WAF1/CIP1, p27Kip1, p53, FOXO1 and SIRT1 proteins and of cytoplasmic MnSOD protein in satellite cells isolated from young (Y) and old (O) rats. The isolated satellite cells were seeded in 100-mm collagen-coated dishes at a density 2!105 cells/dish. Nuclear and cytoplasmic extracts were isolated at 60–70% confluence without passage. All young and old samples were assayed on the same blot. See Table 1 for summary of results.
70% higher in satellite cells from old than young, growing rats (Table 1, Fig. 2).
4. Discussion In order to provide further insight into why satellite cell proliferation is lower in older animals Schultz and Lipton (1982) selected for comparison 6- and 30-months old rats, a similar evaluation strategy as employed in our microarray study (Pattison et al., 2003). The notion was that to enhance the detection of those factors responsible for loss of cell growth in old age, the comparison would be to muscles in growing rats. Changes in four of these molecules (p53, p21WAF1/CIP1, FOXO1, and p27Kip1) are in the direction known to inhibit the cell cycle. p21WAF1/CIP1and p27Kip1, which bind and inhibit cyclindependent kinase complexes, block cell cycle progression (Coqueret, 2003). p21WAF1/CIP1-null satellite cells display increased cell number and enhanced cell cycle progression compared with wild-type satellite cells (Hawke et al., 2003). Ectoptic expression of p27Kip1 decreases satellite cell proliferation (Chakravarthy et al., 2000a). Therefore, the higher levels of p21WAF1/CIP1and p27Kip1 protein in satellite cells isolated from old as compared to young, growing animals suggests that their increases could be playing some role in the lower satellite cell proliferation seen in old skeletal muscle. p53, perhaps the single most important human tumor suppressor, is commonly mutated in human cancers
S. Machida, F.W. Booth / Experimental Gerontology 39 (2004) 1521–1525
(Grimberg, 2000). p21WAF1/CIP1 is one of a family of genes transactivated by p53 (el-Deiry, 1998) so its increase in satellite cells from old as compared young, growing muscles likely contributes to activation of the p21WAF1/CIP1 promoter. FOXO1 also is a transcription factor driving expression of a family of genes, including p27Kip1 and MnSOD (Birkenkamp and Coffer, 2003). The higher FOXO1 protein in the nuclei of satellite cells from old as compared to young, growing animals probably plays a role in the higher protein levels of p27Kip1 and MnSOD. It has been speculated that FOXO1’s upregulation of MnSOD counters some of the greater oxidative stress (Kops et al., 2002a). Finally, nuclear SIRT1 expression increased in old satellite cells. Interestingly while this article was under review, SIRT1’s deacetylation of FOXO1 was shown to enhance FOXO1’s transactivation of the MnSOD and p27Kip1 genes, which Daitoku et al. (2004) suggested is an event that could prolong lifespan. In summary, nuclei in satellite cells isolated from old as compared to young, growing rats exhibit changes in protein levels consistent with the diminished proliferation observed. Acknowledgements We would like to thank Tsghe Abraha for expert assistance and Dr Simon Lees for editorial suggestions. This work was supported by NIH grant AG18780 (FB). References Adams, G.R., Caiozzo, V.J., Haddad, F., Baldwin, K.M., 2002. Cellular and molecular responses to increased skeletal muscle loading after irradiation. Am. J. Physiol. Cell Physiol. 283, C1182–C1195. Birkenkamp, K.U., Coffer, P.J., 2003. Regulation of cell survival and proliferation by the FOXO (Forkhead box, class O) subfamily of Forkhead transcription factors. Biochem. Soc. Trans. 31 (Pt 1), 292–297. Brooks, S.V., Faulkner, J.A., 1990. Contraction-induced injury: recovery of skeletal muscles in young and old mice. Am. J. Physiol. 258 (3 Pt 1), C436–C442. Chakravarthy, M.V., Abraha, T.W., Schwartz, R.J., Fiorotto, M.L., Booth, F.W., 2000a. Insulin-like growth factor-I extends in vitro replicative life span of skeletal muscle satellite cells by enhancing G1/S cell cycle progression via the activation of phosphatidylinositol 3 0 kinase/Akt signaling pathway. J. Biol. Chem. 275, 35942–35952. Chakravarthy, M.V., Davis, B.S., Booth, F.W., 2000b. IGF-I restores satellite cell proliferative potential in immobilized old skeletal muscle. J. Appl. Physiol. 89, 1365–1379. Conboy, I.M., Conboy, M.J., Smythe, G.M., Rando, T.A., 2003. Notchmediated restoration of regenerative potential to aged muscle. Science 302, 1575–1577. Cooper, R.N., Thiesson, D., Furling, D., Di Santo, J.P., ButlerBrowne, G.S., Mouly, V., 2003. Extended amplification in vitro and replicative senescence: key factors implicated in the success of human myoblast transplantation. Hum. Gene Ther. 14, 1169–1179. Coqueret, O., 2003. New roles for p21 and p27 cell-cycle inhibitors: a function for each cell compartment? Trends Cell Biol. 13, 65–70.
1525
Daitoku, H., Hatta, M., Matsuzaki, H., Aratani, S., Ohshima, T., Miyagishi, M., Nakajima, T., Fukamizu, A., 2004. Silent information regulator 2 potentiates Foxo1-mediated transcription through its deacetylase activity. Proc. Natl Acad. Sci. USA 25. Epub ahead of print. Dijkers, P.F., Medema, R.H., Pals, C., Banerji, L., Thomas, N.S., Lam, E.W., Burgering, B.M., Raaijmakers, J.A., Lammers, J.W., Koenderman, L., Coffer, P.J., 2000. Forkhead transcription factor FKHR-L1 modulates cytokine-dependent transcriptional regulation of p27(KIP1). Mol. Cell Biol. 20, 9138–9148. Donehower, L.A., 2002. Does p53 affect organismal aging? J. Cell Physiol. 192, 23–33. el-Deiry, W.S., 1998. Regulation of p53 downstream genes. Semin. Cancer Biol. 8, 345–357. el-Deiry, W.S., Tokino, T., Velculescu, V.E., Levy, D.B., Parsons, R., Trent, J.M., Lin, D., Mercer, W.E., Kinzler, K.W., Vogelstein, B., 1993. WAF1, a potential mediator of p53 tumor suppression. Cell 75, 817– 825. Grimberg, A., 2000. P53 and IGFBP-3: apoptosis and cancer protection. Mol. Genet. Metab. 70, 85–98. Grounds, M.D., 1998. Age-associated changes in the response of skeletal muscle cells to exercise and regeneration. Ann. NY Acad. Sci. 854, 78– 91. Hawke, T.J., Garry, D.J., 2001. Myogenic satellite cells: physiology to molecular biology. J. Appl. Physiol. 91, 534–551. Hawke, T.J., Jiang, N., Garry, D.J., 2003. Absence of p21CIP rescues myogenic progenitor cell proliferative and regenerative capacity in Foxk1 null mice. J. Biol. Chem. 278, 4015–4020. Ji, L.L., 2001. Exercise at old age: does it increase or alleviate oxidative stress?. Ann. NY Acad. Sci. 928, 236–247. Kim, A., Zhong, W., Oberley, T.D., 2004. Reversible modulation of cell cycle kinetics in NIH/3T3 mouse fibroblasts by inducible overexpression of mitochondrial manganese superoxide dismutase. Antioxid. Redox. Signal 6, 489–500. Kops, G.J., Dansen, T.B., Polderman, P.E., Saarloos, I., Wirtz, K.W., Coffer, P.J., Huang, T.T., Bos, J.L., Medema, R.H., Burgering, B.M., 2002a. Forkhead transcription factor FOXO3a protects quiescent cells from oxidative stress. Nature 419, 316–321. Lin, S.J., Defossez, P.A., Guarente, L., 2000. Requirement of NAD and SIR2 for life-span extension by calorie restriction in Saccharomyces cerevisiae. Science 289, 2126–2128. Machida, S., Spangenburg, E.E., Booth, F.W., 2004. Primary rat muscle progenitor cells have decreased proliferation and myotube formation during passages. Cell Prolif. 37, 267–277. Medema, R.H., Kops, G.J., Bos, J.L., Burgering, B.M., 2000. AFX-like Forkhead transcription factors mediate cell-cycle regulation by Ras and PKB through p27Kip1. Nature 404, 782–787. Pattison, J.S., Folk, L.C., Madsen, R.W., Childs, T.E., Booth, F.W., 2003. Transcriptional profiling identifies extensive downregulation of extracellular matrix gene expression in sarcopenic rat soleus muscle. Physiol. Genomics 15, 34–43. Rosenblatt, J.D., Parry, D.J., 1992. Gamma irradiation prevents compensatory hypertrophy of overloaded mouse extensor digitorum longus muscle. J. Appl. Physiol. 73, 2538–2543. Schaller, M.D., 2001. Paxillin: a focal adhesion-associated adaptor protein. Oncogene 20, 6459–6472. Schultz, E., Lipton, B.H., 1982. Skeletal muscle satellite cells: changes in proliferation potential as a function of age. Mech. Ageing Dev. 20, 377– 383. Tissenbaum, H.A., Guarente, L., 2001. Increased dosage of a sir-2 gene extends lifespan in Caenorhabditis elegans. Nature 410, 227–230. Vogelstein, B., Lane, D., Levine, A.J., 2000. Surfing the p53 network. Nature 408, 307–310.
Increased nuclear proteins in muscle satellite cells in aged animals as compared to young growing animals Shuichi Machidaa, Frank W. Bootha,b,* a
Department of Biomedical Sciences, University of Missouri-Columbia, E102 Veterinary Medical Building, 1600 East Rollins Road, Columbia, MO 65211, USA b Department of Medical Pharmacology and Physiology, Dalton Cardiovascular Center, University of Missouri-Columbia, Columbia, MO 65211, USA Received 14 May 2004; received in revised form 9 July 2004; accepted 20 August 2004 Available online 11 September 2004
Abstract Evidence implies that satellite cells could play some limiting role in aged muscle undergoing repair or maintenance of mass, which is of potential clinical concern as this could contribute to sarcopenia. Further, insufficient information is available concerning the cellular mechanisms responsible for the lower rat satellite cell proliferation in old animals. Thus, it was hypothesized that the following proteins would be increased in nuclei of satellite cells from old rat skeletal muscle: the cyclin-dependent kinase (CDK) inhibitors p21WAF1/CIP1 and p27Kip1 as well as the transcription factors p53 and Forkhead box, subgroup O1 (FOXO1). In addition, the NADC-dependent histone deacetylase SIRT1, the mammalian ortholog of the yeast SIR2 (silence information regulator 2) and a member of the Sirtuin family, was hypothesized to decrease in satellite cell nuclei of old rats. Old satellite cells (30-months old) exhibited a lesser number of BrdU-positive cells as compared to satellite cells (3-months old) from young growing animals. Western blot analysis demonstrated that nuclei of old satellite cells accumulated the cell cycle inhibitors p21WAF1/CIP1 and p27Kip1. In addition, nuclear p53 and FOXO1 proteins were also higher in old satellite cells than in cells from young growing animals. These data indicated both p53/p21WAF1/CIP1- and FOXO1/p27Kip1-dependent pathways might contribute to the age-associated decrease in satellite cell proliferation. Cytoplasmic manganese superoxide dismutase (MnSOD), a gene driven by FOXO1, was higher in old satellite cells. Unexpectedly, nuclear SIRT1 was also increased in old satellite cells compared with satellite cells from young growing animals. The physiological significance of enhanced nuclear SIRT1 expression in old satellite cells remains elusive at this time. In summary, satellite cells in old rats have nuclear accumulation of proteins inhibiting the cell cycle as compared to young, growing animals. q 2004 Elsevier Inc. All rights reserved. Keywords: Forkhead; p21; p27; p53; Sir2
1. Introduction Aged skeletal muscle has impaired capacities of regeneration (Brooks and Faulkner, 1990; Grounds, 1998) and regrowth (Chakravarthy et al., 2000b). Conboy et al. (2003) attribute the dramatic age-related decline in myoblast generation in response to injury an impairment
* Corresponding author. Address: Department of Biomedical Sciences, University of Missouri-Columbia, E102 Veterinary Medical Building, 1600 East Rollins Road, Columbia, MO 65211, USA. Tel.: C1-573-882-6652; fax: C1-573-884-6890. E-mail address: [email protected] (F.W. Booth). 0531-5565/$ - see front matter q 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.exger.2004.08.009
of activation of satellite cells. Satellite cells are the myogenic cells responsible for post-natal growth, as well as regeneration and repair of injured muscle (Hawke and Garry, 2001). Satellite cell replication has been reported to be limiting in skeletal muscle growth. Elimination of proliferation by satellite cells with low-level g-irradiation in young animals limits muscle hypertrophy produced by mechanical overload (Rosenblatt and Parry, 1992; Adams et al., 2002). A second example is that myoblast transfer in gene therapy is limited by history of satellite cell proliferation. The replication of human satellite cells for an additional 15 divisions in culture resulted in a 6-fold
1522
S. Machida, F.W. Booth / Experimental Gerontology 39 (2004) 1521–1525
reduction in the number of myoblasts that were incorporated into muscle fibers (Cooper et al., 2003). Cooper et al. (2003) explained that their result substantiates the notion that the approach to replicative senescence for satellite cells, and therefore, the loss of replicative potential is important for successful host cell muscle regeneration after myoblast transplantation. However, insufficient cellular information is available to explain the decreased satellite cell proliferation in old animals/humans. The hypothesis of the current study was that proteins involved in cell cycle arrest would be elevated in nuclei of satellite cells isolated from old rats as compared to those from young, growing rats since the proliferation of old satellite cells is less. An experimental strategy was to follow-up previously published experiments on young, growing and old rats. Skeletal muscles in old rats showed no regrowth from limb immobilization whereas young muscles completely regrew (Pattison et al., 2003). The notion was then that proteins that inhibit the cell cycle would be less in young, growing than in old satellite cells. We therefore selected for study proteins with inhibitory actions on the cell cycle. p21WAF1/CIP1 and p27Kip1 induce cell cycle arrest (Coqueret, 2003), so a sub-hypothesis was made that nuclear level of p21WAF1/CIP1 and p27Kip1 proteins would be higher in satellite cells from old as compared to young, growing muscles, and thus be associated with decreased satellite cell proliferation. In response to various types of stress, including increased oxidative stress, p53 accumulates in the nucleus, where it transcriptionally activates genes that are involved in cell cycle arrest, such as p21WAF1/CIP1 (el-Deiry et al., 1993), and thus p53 induces cell cycle arrest (Donehower, 2002; Vogelstein et al., 2000). Therefore, an additional sub-hypothesis was generated that p53 protein would be more in nuclei of satellite cells from old than young, growing animals. The rationale was that oxidative stress is higher in old skeletal muscle (Ji, 2001), so p53 could be lower in the nuclei of young, growing satellite cells. The Forkhead box, subgroup O1, (FOXO1) transcription factor is of interest to aging studies because it binds to promoters of specific genes, stimulating cell cycle arrest by upregulating p27Kip1 (Dijkers et al., 2000b; Medema et al., 2000) and stimulates manganese superoxide dismutase (MnSOD) expression. Increased MnSOD activity leads to decreased cell growth due to prolonged cell cycle transition times in G1 and S phases without significant changes in G2/M phase (Kim et al., 2004), so an increase in MnSOD in old satellite cells could contribute to their decreased proliferation, as well as to minimize oxidative stress. Consequently, another sub-hypothesis was that FOXO1 and MnSOD proteins would be higher in nuclei from satellite cells of old than young, growing animals. NADC-dependent histone deacetylase Sir2 (silence information regulator 2) (designated as SIRT1 (sirtuin 1) in the current paper) overexpression in yeast and C. elegans extends lifespan and has been linked to longevity regulation
(Lin et al., 2000; Tissenbaum and Guarente, 2001). Previously we have shown that satellite cells isolated from old rats have decreased longevity in culture (Chakravarthy et al., 2000b); therefore, our final sub-hypothesis is that SIRT1 would be less in old satellite cell nuclei.
2. Materials and methods 2.1. Materials Cell culture medium and reagents were purchased from Life Technologies, Inc. (Rockville, MD). Antibodies against anti- p21WAF1/CIP1 (Catalog # 05-345), p27Kip1 (#06-445), MnSOD (#06-984) and Sir2 (#07-131) were obtained from Upstate Biotechnology (Lake Placid, NY). Mouse monoclonal antibody to p53 (#2524) and polyclonal rabbit anti-FKHR (FOXO1) (#9462) were purchased from Cell Signaling (Beverly, MA). Anti-paxillin (#P13520) was purchased from Transduction Laboratories (Lexington, KY). D3 mouse monoclonal anti-desmin was acquired from the Developmental Studies Hybridoma Bank (Iowa City, IA). MoAb5.8A mouse monoclonal anti-MyoD antibody was purchased from Pharmingen (San Diego, CA). 5-bromo-2 0 -deoxyuridine-5 0 -monophosphate (BrdU) was obtained from Sigma (St Louis, MO) and mouse monoclonal antibody to BrdU from Roche Diagnostics (Indianapolis, IN). 2.2. Animals Pathogen-free, F1 generation of Fisher 344! Brown Norway male rats (3- and 30-months old) were obtained from Harlan Labs (National Institute of Aging). Since old rats exhibit defects in muscle regrowth from atrophy (Chakravarthy et al., 2000b), age comparisons were selected between young, growing rats and old rats experiencing muscle loss in order to determine differences in inhibitory cell cycle proteins in nuclei of satellite cells. All the animals were housed at 21 8C in a 12-h light/12-h dark cycle. Rat chow and water were provided ad libitum. The animals were allowed to acclimatize to their new surrounding for 2 weeks before the cell isolation procedures were performed. All animal experimental protocols were approved by the University of Missouri Institutional Animal Care and Use Committee. 2.3. Isolation of satellite cells Animals were anesthetized with a single intraperitoneal injection of a cocktail (2.0 ml/kg body weight) consisting of 75 mg/ml ketamine, 3 mg/ml xylazine, and 5 mg/ml acepromazine. Satellite cells were isolated from the gastrocnemius, plantaris, tibialis anterior, extensor digitorum longus, and quadriceps muscles of individual rats according to the previously described methods used in our
S. Machida, F.W. Booth / Experimental Gerontology 39 (2004) 1521–1525
lab (Machida et al., 2004). No-passage, isolated cells from a single rat for every observation were seeded separately for immunocytochemistry, for BrdU, and for Westerns. To determine the percentage of myogenic cells present, the isolated cells were stained with the MyoD antibody (dilution; 1:100) and desmin antibody (dilution; 1:3) by immunocytochemical analysis performed as previously described by us (Machida et al., 2004) with the following modifications. Cells were seeded at 5000 cells per well. For the visualization of the primary antibody binding, the diaminobenzidine (DAB) substrate kit (Vector Laboratories, California) for peroxidase was used. At least a total of 200 cells were counted. The isolated cells of each age group contained more than 90% both MyoD- and desmin-positive cells (data not shown). All cultures were maintained at 37 8C in a humid air atmosphere containing 6% CO2.
1523
Fig. 1. Nuclear proteins isolated from satellite cells of young (Y) and old (O) rats do not contain detectable paxillin in Western blots. Cytoplasmic proteins from the young satellite cells was loaded as a positive control. All lanes were loaded with equal protein (30 mg). MW stands for the lane containing the molecular weight markers (Right-hand arrows indicate position of markers).
2.4. Western blots No-passage, isolated cells from a single rat were seeded in a 100-mm collagen-coated dish at a density of 1.5–2!105 cells/dish and cultured 3–4 days in the growth medium until cells for both young- and old-derived cells were 60–70% confluent. Five rats composed each age group. Cells were lysed and scraped in ice-cold hypotonic buffer (HB) (25 mM Tris (pH 7.5), 1 mM MgCl2, 5 mM KCl, 0.05% NP-40, 10% glycerol, 1 mM Na3VO4, 1 mM benzamidine, 1 mM DTT, 10 mg/ml leupeptin, 10 mg/ml aprotinin, 10 mg/ml pepstatin, 10 mg/ml tosyl-L-phenylalanine chloromethyl ketone, 10 mg/ml Na-tosyl-L-lysine chloromethyl ketone, and 2 mM Pefabloc SC Plus). The scraped cells were lysed while slowly rotated for 10 min at 4 8C. Nuclei were pelleted by centrifugation at 500 g for 5 min, washed two times in HB, and lysed in extract buffer (50 mM HEPES (pH 7.5), 300 mM NaCl, 5 mM EDTA, 0.065% NP-40, 10% glycerol, plus inhibitors described above). The nuclei were lysed while slowly rotated for 30 min at 4 8C. Nuclear protein was pelleted by centrifugation at 15,000 g for 10 min. The separation of the nuclear fraction was confirmed by the absence of paxillin in Western blotting (Fig. 1). Paxillin is a cytoplasmic protein that localizes to focal adhesions at the sarcolemma (Schaller, 2001). Protein concentrations were determined by Bradford assay (Biorad). Equal amounts of total protein from young and old (nZ5/age group) were resolved on the same SDS-PAGE gel and subsequently transferred to a nitrocellulose membrane. All blots were then incubated with Ponceau S (Sigma) and verified equal loading in all lanes (data not shown). The membranes were then blocked with 5% nonfat dry milk in Tris-buffer saline with 0.1% Tween (TBST) for p53, FOXO1 and Sir2 antibodies, 3% milk in PBS for p27Kip1, p21WAF1/CIP1 and MnSOD, and 2.5% milk and 1% bovine serum albumin (BSA) in TBST for paxillin. All membranes were then probed overnight with the appropriate antibody. Antibodies were used at the following
concentrations: p53 (1:2000 in 5% BSA-TBST), Sir2 (1:10,000 in 5% BSA-TBST), FOXO1 (1:1000 in 5% BSA-TBST), p27Kip1 (1:5000 in 3% milk-PBS), p21WAF1/CIP1(1:2000 in 3% milk-PBS), MnSOD (1:2000 in 3% milk-PBS), and paxillin (1:10,000 in 2.5% milk and 1% BSA-TBST). The membranes, except for p53 protein, were washed and then incubated for 1 h with horseradish peroxidase-conjugated rabbit or mouse secondary antibody (1:2000 or 1:3000 rabbit, 1:1000 or 1:2000 mouse, Amersham Pharmacia Biotech, Piscataway, NJ. For p53 protein detection, the membrane was incubated with biotinylated anti-mouse IgG (1:200; Vector Laboratories, Burlingame, CA) in the blocking buffer for 30 min. The blot was then washed and incubated with the VECTASTAIN elite ABC reagent (1:50; Vector Laboratories) in the blocking buffer for an additional 30 min. Immunocomplexes were visualized using the enhanced chemiluminescence reagent (NEN Life Science Products, Boston, MA). The membrane was exposed to Hyperfilme ECL (Amersham Pharmacia Biotech, Piscataway, NJ) with the exposure time adjusted to keep the integrated optical densities (IOD) within a linear and non-saturated range. The signal bands were scanned utilizing the Personal Densitometer SI (Molecular Dynamics, Sunnyvale, CA) and quantified using ImageQuant software (Molecular Dynamics). Linear responses to loaded protein concentrations of 50 mg have been shown for p21WAF1/CIP1, p27Kip1, and SIRT1 (unpublished observations). 2.5. Pulse labeling proliferation index To evaluate proliferation, BrdU incorporation was examined Briefly, the isolated cells were seeded at 10,000 cells per well on the collagen-coated glass chamber slides (Lab-Tek II, Nunc) in 600 (L of F-10 with high serum condition. After a 48-h-incubation, cultures were pulse labeled for 1 h with 10 mM bromodeoxyuridine
1524
S. Machida, F.W. Booth / Experimental Gerontology 39 (2004) 1521–1525
(BrdU), followed by immunocytochemistry for detection of BrdU according to the manufacturer’s directions (Roche Diagnostics, Indianapolis, IN). For the visualization of the BrdU antibody binding, the diaminobenzidine (DAB) substrate kit (Vector Laboratories, California) for peroxidase was used. At least a total of 200 cells were counted. The percentage of BrdU labeled cells was used as an indicator of proliferating cells. 2.6. Statistics The data are shown as mean G SE and were analyzed by Student’s t-test. P!0.05 was considered statistically significant.
3. Results 3.1. Pulse labeling proliferation index of cultured satellite cells, protein concentrations in satellite cell nuclei, and cytoplasmic MnSOD protein Immunohistochemical detection of BrdU-positive cells demonstrated that the percentage of labeled satellite cells was decreased by 61% in old as compared to young, growing muscles (Table 1). Nuclear p21WAF1/CIP1 and p27Kip1 proteins were 114 and 782%, respectively, higher in isolated satellite cells from old than young, growing rats (Table 1, Fig. 2). Nuclear p53 protein was 68% higher in primary satellite cells from old than young, growing rats (Table 1, Fig. 2). The nuclear expression of FOXO1 was increased by 131% in old satellite cells compared with young, growing satellite cells (Table 1, and Fig. 2). Nuclear SIRT1 protein was 75% higher in old satellite cells than young, growing satellite cells (Table 1, and Fig. 2). Nuclear levels of GSK-3b protein did not differ (pZ0.39) between young and old rats (data not shown), suggesting that the aforementioned increases in proteins in the nucleus were not a generalized response. Cytoplasmic MnSOD protein was
Table 1 Pulse labeling proliferation index and protein concentrations Measurement
Age a
BrDU incorporation Nuclear p21WAF1/CIP1b Nuclear p27Kip1b Nuclear p53b Nuclear FOXO1b Cytoplasmic MnSODb Nuclear SIRT1b †
3-months old
30-months old
31.3G2.32 320G38.4 0.0365G0.008 132G8.01 152G44.4 938G128 663G109
12.2G0.97‡ 684G98.2‡ 0.322G0.09‡ 222G25.5‡ 351G30.2‡ 1594G225† 1157G176†
p!0.05; ‡p!0.01. Values are the means G SEM. nZ5 per group. a % Positive cells. b Arbitrary densitometry units.
Fig. 2. Representative blots of nuclear p21WAF1/CIP1, p27Kip1, p53, FOXO1 and SIRT1 proteins and of cytoplasmic MnSOD protein in satellite cells isolated from young (Y) and old (O) rats. The isolated satellite cells were seeded in 100-mm collagen-coated dishes at a density 2!105 cells/dish. Nuclear and cytoplasmic extracts were isolated at 60–70% confluence without passage. All young and old samples were assayed on the same blot. See Table 1 for summary of results.
70% higher in satellite cells from old than young, growing rats (Table 1, Fig. 2).
4. Discussion In order to provide further insight into why satellite cell proliferation is lower in older animals Schultz and Lipton (1982) selected for comparison 6- and 30-months old rats, a similar evaluation strategy as employed in our microarray study (Pattison et al., 2003). The notion was that to enhance the detection of those factors responsible for loss of cell growth in old age, the comparison would be to muscles in growing rats. Changes in four of these molecules (p53, p21WAF1/CIP1, FOXO1, and p27Kip1) are in the direction known to inhibit the cell cycle. p21WAF1/CIP1and p27Kip1, which bind and inhibit cyclindependent kinase complexes, block cell cycle progression (Coqueret, 2003). p21WAF1/CIP1-null satellite cells display increased cell number and enhanced cell cycle progression compared with wild-type satellite cells (Hawke et al., 2003). Ectoptic expression of p27Kip1 decreases satellite cell proliferation (Chakravarthy et al., 2000a). Therefore, the higher levels of p21WAF1/CIP1and p27Kip1 protein in satellite cells isolated from old as compared to young, growing animals suggests that their increases could be playing some role in the lower satellite cell proliferation seen in old skeletal muscle. p53, perhaps the single most important human tumor suppressor, is commonly mutated in human cancers
S. Machida, F.W. Booth / Experimental Gerontology 39 (2004) 1521–1525
(Grimberg, 2000). p21WAF1/CIP1 is one of a family of genes transactivated by p53 (el-Deiry, 1998) so its increase in satellite cells from old as compared young, growing muscles likely contributes to activation of the p21WAF1/CIP1 promoter. FOXO1 also is a transcription factor driving expression of a family of genes, including p27Kip1 and MnSOD (Birkenkamp and Coffer, 2003). The higher FOXO1 protein in the nuclei of satellite cells from old as compared to young, growing animals probably plays a role in the higher protein levels of p27Kip1 and MnSOD. It has been speculated that FOXO1’s upregulation of MnSOD counters some of the greater oxidative stress (Kops et al., 2002a). Finally, nuclear SIRT1 expression increased in old satellite cells. Interestingly while this article was under review, SIRT1’s deacetylation of FOXO1 was shown to enhance FOXO1’s transactivation of the MnSOD and p27Kip1 genes, which Daitoku et al. (2004) suggested is an event that could prolong lifespan. In summary, nuclei in satellite cells isolated from old as compared to young, growing rats exhibit changes in protein levels consistent with the diminished proliferation observed. Acknowledgements We would like to thank Tsghe Abraha for expert assistance and Dr Simon Lees for editorial suggestions. This work was supported by NIH grant AG18780 (FB). References Adams, G.R., Caiozzo, V.J., Haddad, F., Baldwin, K.M., 2002. Cellular and molecular responses to increased skeletal muscle loading after irradiation. Am. J. Physiol. Cell Physiol. 283, C1182–C1195. Birkenkamp, K.U., Coffer, P.J., 2003. Regulation of cell survival and proliferation by the FOXO (Forkhead box, class O) subfamily of Forkhead transcription factors. Biochem. Soc. Trans. 31 (Pt 1), 292–297. Brooks, S.V., Faulkner, J.A., 1990. Contraction-induced injury: recovery of skeletal muscles in young and old mice. Am. J. Physiol. 258 (3 Pt 1), C436–C442. Chakravarthy, M.V., Abraha, T.W., Schwartz, R.J., Fiorotto, M.L., Booth, F.W., 2000a. Insulin-like growth factor-I extends in vitro replicative life span of skeletal muscle satellite cells by enhancing G1/S cell cycle progression via the activation of phosphatidylinositol 3 0 kinase/Akt signaling pathway. J. Biol. Chem. 275, 35942–35952. Chakravarthy, M.V., Davis, B.S., Booth, F.W., 2000b. IGF-I restores satellite cell proliferative potential in immobilized old skeletal muscle. J. Appl. Physiol. 89, 1365–1379. Conboy, I.M., Conboy, M.J., Smythe, G.M., Rando, T.A., 2003. Notchmediated restoration of regenerative potential to aged muscle. Science 302, 1575–1577. Cooper, R.N., Thiesson, D., Furling, D., Di Santo, J.P., ButlerBrowne, G.S., Mouly, V., 2003. Extended amplification in vitro and replicative senescence: key factors implicated in the success of human myoblast transplantation. Hum. Gene Ther. 14, 1169–1179. Coqueret, O., 2003. New roles for p21 and p27 cell-cycle inhibitors: a function for each cell compartment? Trends Cell Biol. 13, 65–70.
1525
Daitoku, H., Hatta, M., Matsuzaki, H., Aratani, S., Ohshima, T., Miyagishi, M., Nakajima, T., Fukamizu, A., 2004. Silent information regulator 2 potentiates Foxo1-mediated transcription through its deacetylase activity. Proc. Natl Acad. Sci. USA 25. Epub ahead of print. Dijkers, P.F., Medema, R.H., Pals, C., Banerji, L., Thomas, N.S., Lam, E.W., Burgering, B.M., Raaijmakers, J.A., Lammers, J.W., Koenderman, L., Coffer, P.J., 2000. Forkhead transcription factor FKHR-L1 modulates cytokine-dependent transcriptional regulation of p27(KIP1). Mol. Cell Biol. 20, 9138–9148. Donehower, L.A., 2002. Does p53 affect organismal aging? J. Cell Physiol. 192, 23–33. el-Deiry, W.S., 1998. Regulation of p53 downstream genes. Semin. Cancer Biol. 8, 345–357. el-Deiry, W.S., Tokino, T., Velculescu, V.E., Levy, D.B., Parsons, R., Trent, J.M., Lin, D., Mercer, W.E., Kinzler, K.W., Vogelstein, B., 1993. WAF1, a potential mediator of p53 tumor suppression. Cell 75, 817– 825. Grimberg, A., 2000. P53 and IGFBP-3: apoptosis and cancer protection. Mol. Genet. Metab. 70, 85–98. Grounds, M.D., 1998. Age-associated changes in the response of skeletal muscle cells to exercise and regeneration. Ann. NY Acad. Sci. 854, 78– 91. Hawke, T.J., Garry, D.J., 2001. Myogenic satellite cells: physiology to molecular biology. J. Appl. Physiol. 91, 534–551. Hawke, T.J., Jiang, N., Garry, D.J., 2003. Absence of p21CIP rescues myogenic progenitor cell proliferative and regenerative capacity in Foxk1 null mice. J. Biol. Chem. 278, 4015–4020. Ji, L.L., 2001. Exercise at old age: does it increase or alleviate oxidative stress?. Ann. NY Acad. Sci. 928, 236–247. Kim, A., Zhong, W., Oberley, T.D., 2004. Reversible modulation of cell cycle kinetics in NIH/3T3 mouse fibroblasts by inducible overexpression of mitochondrial manganese superoxide dismutase. Antioxid. Redox. Signal 6, 489–500. Kops, G.J., Dansen, T.B., Polderman, P.E., Saarloos, I., Wirtz, K.W., Coffer, P.J., Huang, T.T., Bos, J.L., Medema, R.H., Burgering, B.M., 2002a. Forkhead transcription factor FOXO3a protects quiescent cells from oxidative stress. Nature 419, 316–321. Lin, S.J., Defossez, P.A., Guarente, L., 2000. Requirement of NAD and SIR2 for life-span extension by calorie restriction in Saccharomyces cerevisiae. Science 289, 2126–2128. Machida, S., Spangenburg, E.E., Booth, F.W., 2004. Primary rat muscle progenitor cells have decreased proliferation and myotube formation during passages. Cell Prolif. 37, 267–277. Medema, R.H., Kops, G.J., Bos, J.L., Burgering, B.M., 2000. AFX-like Forkhead transcription factors mediate cell-cycle regulation by Ras and PKB through p27Kip1. Nature 404, 782–787. Pattison, J.S., Folk, L.C., Madsen, R.W., Childs, T.E., Booth, F.W., 2003. Transcriptional profiling identifies extensive downregulation of extracellular matrix gene expression in sarcopenic rat soleus muscle. Physiol. Genomics 15, 34–43. Rosenblatt, J.D., Parry, D.J., 1992. Gamma irradiation prevents compensatory hypertrophy of overloaded mouse extensor digitorum longus muscle. J. Appl. Physiol. 73, 2538–2543. Schaller, M.D., 2001. Paxillin: a focal adhesion-associated adaptor protein. Oncogene 20, 6459–6472. Schultz, E., Lipton, B.H., 1982. Skeletal muscle satellite cells: changes in proliferation potential as a function of age. Mech. Ageing Dev. 20, 377– 383. Tissenbaum, H.A., Guarente, L., 2001. Increased dosage of a sir-2 gene extends lifespan in Caenorhabditis elegans. Nature 410, 227–230. Vogelstein, B., Lane, D., Levine, A.J., 2000. Surfing the p53 network. Nature 408, 307–310.