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Increased nuclear proteins in muscle satellite cells in aged animals as compared to young growing animals
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 p21(WAF1/CIP1) and p27(Kip1) as well as the transcription factors p53 and Forkhead box, subgroup O1 (FOXO1). In addition, the NAD(+)-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 p21(WAF1/CIP1) and p27(Kip1). 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/p21(WAF1/CIP1)- and FOXO1/p27(Kip1)-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.
... The limited data on senescence in models of insulin resistance suggest that obese tissue has increased expression of cell cycle inhibitors (Kumar and Dey, 2002;Mordier and Iynedjian, 2007;Musi and Goodyear, 2003) and senescence marker proteins, resembling that of aged tissue (Machida and Booth, 2004;Rathbone et al., 2008). Furthermore, premature senescence in skeletal muscle is not well understood, even less as it relates to ceramide-induced insulin resistance. ...
... Unlike normal, non-cancerous cells, inducing senescence in cancer cells is advantageous to limiting growth. Although, limiting the proliferative potential, promoting senescence, and/or reducing metabolic flexibility of skeletal muscle cells can be detrimental to adaptation and growth (Machida and Booth, 2004;Nehlin et al., 2011;Rathbone et al., 2008). Accordingly, staining for bgalactosidase, a well-established marker of cell senescence, was evident in over 90% of the cells treated with ceramide, strongly supporting our model of senescence. ...
... However, the role of p53 in skeletal muscle is even more complex. Skeletal muscle expression of p53 and p21 are elevated in aging (senescent) skeletal muscle (Liu et al., 2008;Machida and Booth, 2004;Zwetsloot et al., 2013). Treatment of middle-aged mice with a p53 inhibitor, pifithrin-m, promoted mitochondrial DNA accumulation and antioxidant protein expression (He et al., 2012). ...
... In particular, subcellular location comes into play as a major regulator of p27 Kip1 s function in the context of aging. It has been demonstrated that ectopic expression of p27 Kip1 in satellite cells reduces proliferative capacity [101] and that satellite cells isolated from old muscle proliferate significantly less than cells from young growing muscles [16]. Although mRNA expression of p27 Kip1 is lower in aged satellite cells [95], there is higher accumulation of p27 Kip1 protein in the nucleus [16], where its ability to promote cell survival is limited indicating that nuclear p27 Kip1 may play a role in decreased satellite cell function in old muscles. ...
... It has been demonstrated that ectopic expression of p27 Kip1 in satellite cells reduces proliferative capacity [101] and that satellite cells isolated from old muscle proliferate significantly less than cells from young growing muscles [16]. Although mRNA expression of p27 Kip1 is lower in aged satellite cells [95], there is higher accumulation of p27 Kip1 protein in the nucleus [16], where its ability to promote cell survival is limited indicating that nuclear p27 Kip1 may play a role in decreased satellite cell function in old muscles. ...
Once believed to solely function as a cyclin-dependent kinase inhibitor, p27Kip1 is now emerging as a critical mediator of autophagy, cytoskeletal dynamics, cell migration and apoptosis. During periods of metabolic stress, the subcellular location of p27Kip1 largely dictates its function. Cytoplasmic p27Kip1 has been found to be promote cellular resilience through autophagy and anti-apoptotic mechanisms. Nuclear p27Kip1, however, inhibits cell cycle progression and makes the cell susceptible to quiescence, apoptosis, and/or senescence. Cellular location of p27Kip1 is regulated, in part, by phosphorylation by various kinases, including Akt and AMPK. Aging promotes nuclear localization of p27Kip1 and a predisposition to senescence or apoptosis. Here, we will review the role of p27Kip1 in healthy and aging cells with a particular emphasis on the interplay between autophagy and apoptosis.
... In addition, the mTOR-raptor complex can also regulate p27 Kip1 phosphorylation and cellular localization through the serum and glucocorticoid-inducible kinase (SGK) (Hong et al., 2008). In aged MuSC, there is less mRNA expression of p27 Kip1 (Chakkalakal et al., 2012), yet protein expression is greater in the nuclei where it can serve as cyclin inhibitor (Machida and Booth, 2004) with minimal effect on cell survival. In addition, p27 Kip1 expression associates with maintenance of satellite cell populations that proliferate less frequently, but have long-term self-renewal capacity (Chakkalakal et al., 2014). ...
... We observed the anti-apoptotic capacity of AMPK is dependent on phosphorylation and subsequent cytoplasmic translocation of p27 Kip1 . Machida and Booth (2004) reported aged MuSCs to have a greater expression of nuclear p27 Kip1 ; we confirmed these findings. However, our data suggest cellular location of p27 Kip1 is dynamic, shifting from the nucleus to the cytoplasm as the cell un-dergoes activation. ...
... In addition, the mTOR-raptor complex can also regulate p27 Kip1 phosphorylation and cellular localization through the serum and glucocorticoid-inducible kinase (SGK) (Hong et al., 2008). In aged MuSC, there is less mRNA expression of p27 Kip1 (Chakkalakal et al., 2012), yet protein expression is greater in the nuclei where it can serve as cyclin inhibitor (Machida and Booth, 2004) with minimal effect on cell survival. In addition, p27 Kip1 expression associates with maintenance of satellite cell populations that proliferate less frequently, but have long-term self-renewal capacity (Chakkalakal et al., 2014). ...
... We observed the anti-apoptotic capacity of AMPK is dependent on phosphorylation and subsequent cytoplasmic translocation of p27 Kip1 . Machida and Booth (2004) reported aged MuSCs to have a greater expression of nuclear p27 Kip1 ; we confirmed these findings. However, our data suggest cellular location of p27 Kip1 is dynamic, shifting from the nucleus to the cytoplasm as the cell un-dergoes activation. ...
Skeletal muscle stem cell (MuSC) function declines with age and contributes to impaired muscle regeneration in older individuals. Acting through AMPK/p27Kip1, we have identified a pathway regulating the balance between autophagy, apoptosis, and senescence in aged MuSCs. While p27Kip1 is implicated in MuSC aging, its precise role and molecular mechanism have not been elucidated. Age-related MuSC dysfunction was associated with reduced autophagy, increased apoptosis, and hypophosphorylation of AMPK and its downstream target p27Kip1. AMPK activation or ectopic expression of a phosphomimetic p27Kip1 mutant was sufficient to suppress in vitro apoptosis, increase proliferation, and improve in vivo transplantation efficiency of aged MuSCs. Moreover, activation of the AMPK/p27Kip1 pathway reduced markers of cell senescence in aged cells, which was, in part, dependent on p27Kip1 phosphorylation. Thus, the AMPK/p27Kip1 pathway likely regulates the autophagy/apoptosis balance in aged MuSCs and may be a potential target for improving muscle regeneration in older individuals.
... If trauma occurs to the fibers, muscle regeneration and repair begins with the activation of the progenitor cells, known as satellite cells. [89][90][91] These mononuclear cells are present in all types of muscle, and in skeletal muscle they reside between the basal lamina and the myofiber it encapsulates. While they are not always found in the same density in all skeletal muscles, they are often found spaced throughout the myofiber with which they reside. ...
... These mononuclear cells are present in skeletal, smooth, and cardiac muscles. In skeletal muscle specifically, the mononuclear satellite cells reside between the myofiber and the basal lamina and are able to move freely.[88][89][90][91] When trauma to the musculature occurs, available satellite cells become activated and migrate to the site of the defect, where they proliferate. ...
... Satellite cells were isolated from the major hind-limb muscles of rats, according to methods described in Machida et al. (27). Briefly, animals were anesthetized with pentobarbital sodium (50 mg/kg). ...
... Cultures were pulse-labelled with 10 lM BrdUrd in medium for the final 1 h, 24 h after stimulation with IL-6, and numbers of BrdUrd-positive cells were determined using immunocytochemistry analyses as previously described (27). Activation and proliferation were indicated by percentage of cells BrdUrd + . ...
Objectives: To determine whether interleukin-6 (IL-6) stimulates rat muscle satellite cell proliferation in culture, and if so, to clarify the signalling mechanisms. Materials and methods: Primary satellite cells were isolated from thirty male F344 rats, 11 weeks of age. IL-6 at concentrations of 0.01, 0.1, 1, 10 or 100 ng/ml was added to culture media. Results: IL-6 at 0.01–1 ng/ml induced dose-dependent increase in cell proliferation. After treatment with 1 ng/ml IL-6, cell proliferation increased by 31%, and p-STAT3 + /MyoD + cells increased in number compared to those in control media (P < 0.05). Inhibitors of JAK2 (AG 490) and STAT3 (STAT3 peptide) blocked the increase in BrdUrd + cell numbers at 6 h post stimulation with 1 ng/ml IL-6 (P < 0.05). Furthermore, cyclin D1 mRNA expression and cyclin D1 + /MyoD + cell numbers significantly increased in cultures treated with 1 ng/ml IL-6 compared to those in control media (P < 0.05). In contrast, treatment with 10 and 100 ng/ml IL-6 did not stimulate cell proliferation. Treatment with 10 ng/ml IL-6 induced greater SOCS3 mRNA expression than with 1 ng/ml IL-6 and control media. Moreover, co-localization of SOCS3 and myogenin was observed after treatment with 10 ng/ml IL-6. Conclusions: IL-6 induced dose-dependent increase in satellite cell proliferation by activating the JAK2/STAT3/cyclin D1 pathway.
... These cells merge with the existing muscle fibers to carry out muscle repair and hypertrophy. The decline in satellite cell function with age has been identified as one of the reasons for the decline in the ability of muscles to recover with age 21,22) . ...
... This hinders the intranuclear translocation of atrogenes and suppresses the breakdown of muscle protein 25,26) . However, FOXO proteins have been shown to accumulate in the nuclei of satellite cells in old age, suggesting that they are involved in the decline in growth functions 22) . ...
The occurrence of sarcopenia and muscular dystrophy with aging has attracted attention. Many factors are reported as causes of sarcopenia, such as the functional decline of a digestive organ occurring with aging and malnutrition due to a decrease in food intake. Also, a decrease in growth hormone and an increase in cytokines are also considered to be causes of sarcopenia. Meanwhile, the differentiation between sarcopenia and disuse muscle atrophy is not clear. It will be important in future studies to clearly define the differences between sarcopenia and disuse muscle atrophy. Recently, the diagnostic criteria of sarcopenia have been defined according to a large-scale investigation. In the future, an easier sarcopenia diagnostic method should be developed. It is necessary to design specific treatment strategies more closely correlated to the clinical condition of individual patients, because the causes of sarcopenia vary widely. In this review, we summarize the characteristics of the clinical condition, diagnosis, and treatment of sarcopenia.
... Satellite cells were isolated from the major hind-limb muscles of rats, according to methods described in Machida et al. (27). Briefly, animals were anesthetized with pentobarbital sodium (50 mg/kg). ...
... Cultures were pulse-labelled with 10 lM BrdUrd in medium for the final 1 h, 24 h after stimulation with IL-6, and numbers of BrdUrd-positive cells were determined using immunocytochemistry analyses as previously described (27). Activation and proliferation were indicated by percentage of cells BrdUrd + . ...
To determine whether interleukin-6 (IL-6) stimulates rat muscle satellite cell proliferation in culture, and if so, to clarify the signalling mechanisms.
Primary satellite cells were isolated from thirty male F344 rats, 11 weeks of age. IL-6 at concentrations of 0.01, 0.1, 1, 10 or 100 ng/ml was added to culture media.
IL-6 at 0.01-1 ng/ml induced dose-dependent increase in cell proliferation. After treatment with 1 ng/ml IL-6, cell proliferation increased by 31%, and p-STAT3(+) /MyoD(+) cells increased in number compared to those in control media (P < 0.05). Inhibitors of JAK2 (AG 490) and STAT3 (STAT3 peptide) blocked the increase in BrdUrd(+) cell numbers at 6 h post stimulation with 1 ng/ml IL-6 (P < 0.05). Furthermore, cyclin D1 mRNA expression and cyclin D1(+) /MyoD(+) cell numbers significantly increased in cultures treated with 1 ng/ml IL-6 compared to those in control media (P < 0.05). In contrast, treatment with 10 and 100 ng/ml IL-6 did not stimulate cell proliferation. Treatment with 10 ng/ml IL-6 induced greater SOCS3 mRNA expression than with 1 ng/ml IL-6 and control media. Moreover, co-localization of SOCS3 and myogenin was observed after treatment with 10 ng/ml IL-6.
IL-6 induced dose-dependent increase in satellite cell proliferation by activating the JAK2/STAT3/cyclin D1 pathway.
... Due to the small size of 1-month old rats, muscles from three animals were combined to make one sample for 1month old MPC isolations. We have previously published that our MPC isolation technique provides greater than 90% isolated cells express, thus stain positively, for both MyoD and desmin (18)(19)(20)(21)(22). ...
... After 2-h pre-plating, cells were plated on Matrigel (BD Biosciences, San Jose, CA, USA)-coated plates (0.01 mg/ml Matrigel, 60 min at 37°C) and cultured in growth media (GM; containing 10% rat serum, 100 units/ml penicillin, 100 lg/ml streptomycin, 40 lg/ml gentamicin in Ham's F-10) in a humidified incubator, 5% O 2 and 5% CO 2 at 37°C (HERAcell, Thermo Scientific, Waltham, MA, USA). Greater than 90% of all cells contain desmin-and MyoD-positive immunoreactivity (markers for MPCs) using this protocol for MPC isolation (18)(19)(20)(21)(22). All experiments in the current study were performed on non-passaged (passage 0; P-0) MPCs and GM was refreshed every 24 h after plating. ...
Objectives:
The systemic environment and satellite cell dysfunction have been proposed as important contributors in the development of sarcopenia and impaired skeletal muscle regrowth with ageing. In the present study, we investigated effects of serum age on proliferation of muscle precursor cells (MPCs) isolated from skeletal muscles of young and old rats.
Materials and methods:
We examined proliferation and subsequent differentiation of non-passaged MPCs isolated from skeletal muscles of 1-, 3- and 32-month old rats over a 72-h time course, using a serum cross-over design.
Results and conclusions:
We found no effect of serum age on MPC proliferation, but we did discover that MPCs isolated from skeletal muscle of 32-month old rats had delayed onset of, and exit from proliferation, compared to MPCs isolated from skeletal muscle of 1-month old rats. Delayed proliferation of MPCs from 32-month old rats was associated with delayed p38 MAPK phosphorylation, and MyoD and p21(Cip1) protein expression. We also demonstrate that MPCs from 32-month old rats exhibited lower levels of muscle creatine kinase mRNA compared to 1-month old rats, but elevated levels of myogenin mRNA, when stimulated to differentiate after 36 h proliferation. These findings suggest that delayed entry and exit of the cell cycle observed in MPCs from 32-month old rats may compromise their ability to respond to differentiation stimuli and subsequently impair myogenic potential of 32-month old skeletal muscle, in this model.
... In mammals, the cell cycle regulatory network is primarily controlled by a set of 20 kinases known as cyclin-dependent protein kinases (CDKs) [58]. It has been previously discovered that the proliferation of myosatellite cells and chondrocytes is significantly influenced by several CDKs [59,60]. ...
Objectives
Accumulating evidence indicates a strong link between knee osteoarthritis (KOA) and sarcopenia. However, the mechanisms involved have not yet been elucidated. This study primarily aims to explore the molecular mechanisms that explain the connection between these 2 disorders.
Methods
The gene expression profiles for KOA and sarcopenia were obtained from the Gene Expression Omnibus database, specifically from GSE55235, GSE169077, and GSE1408. Various bioinformatics techniques were employed to identify and analyze common differentially expressed genes (DEGs) across the 3 datasets. The techniques involved the analysis of Gene Ontology and pathways to enhance understanding, examining protein-protein interaction (PPI) networks, and identifying hub genes. In addition, we constructed the network of interactions between transcription factors (TFs) and genes, the co-regulatory network of TFs and miRNAs for hub genes, and predicted potential drugs.
Results
In total, 14 common DEGs were found between KOA and sarcopenia. Detailed information on biological processes and signaling pathways of common DEGs was obtained through enrichment analysis. After performing PPI network analysis, we discovered 4 hub genes (FOXO3, BCL6, CDKN1A, and CEBPB). Subsequently, we developed coregulatory networks for these hub genes involving TF-gene and TF-miRNA interactions. Finally, we identified 10 potential chemical compounds.
Conclusions
By conducting bioinformatics analysis, our study has successfully identified common gene interaction networks between KOA and sarcopenia. The potential of these findings to offer revolutionary understanding into the common development of these 2 conditions could lead to the identification of valuable targets for therapy.
... Therefore, the activation of muscle satellite cells has been shown to be an important step in the regeneration of injured skeletal muscle tissues. However, age-related decreases in the number of muscle satellite cells have been reported [1], as well as decreases in self-replication functions [4], and proliferative capacity [5]. Moreover, satellite cell abnormalities are related to the pathology of Duchenne muscular dystrophy (DMD) in mdx mice, an animal model of DMD [6]. ...
Mesenchymal stem cells (MSCs) have been transplanted directly into lesions or injected intravenously. The administration of MSCs using these delivery methods requires specialized knowledge, techniques, and facilities. Here, we describe intrarectal systemic administration of MSCs, a simple, non-invasive route for homing to the injury sites to promote the regeneration of skeletal muscle injuries. Using a cardiotoxin (CTX)-induced rabbit skeletal muscle injury model, homing to the site of muscle injury was confirmed by intrarectal administration of MSCs; the time required for homing after intrarectal administration was approximately 5 days. In addition, the C-X-C chemokine ligand 12 (CXCL12)/C-X-C chemokine receptor-4 (CXCR4) axis was found to be involved in the homing process. Histopathological examinations showed that skeletal muscle regeneration was promoted in the MSCs-administered group compared to the CTX-only group. Myosin heavy polypeptide 3 (Myh3) expression, an indicator of early muscle regeneration, was detected earlier in the intrarectal MSCs group compared to the CTX-only group. These findings indicate that intrarectal administration of MSCs is effective in homing to the injured area, where they promote injury repair. Since intrarectal administration is a simple and non-invasive delivery route, these findings may be valuable in future research on stem cell therapy.
... With regard to the physiological mechanism of the age-related changes in MT and EI, sarcopenia, unlike muscle atrophy due to disuse, involves not only muscle fiber atrophy but also a decrease in the number of muscle fibers. It is considered that one of the reasons for this is that the number and proliferation function of muscle satellite cells involved in muscle regeneration decrease with age (Hawke and Garry 1985;Machida and Booth 2004). Furthermore, since muscle Table 9 The linear mixed effect models for EI of each muscle among the age groups ...
PurposeThe objective of this study was to investigate the age-related changes in muscle thickness and muscle echo intensity of trunk in subjects including wide range of age groups.Methods
The subjects were 112 healthy women (age range 20–60s). The rectus abdominis, external oblique, internal oblique, transversus abdominis, erector spinae, and lumbar multifidus muscles were examined. To confirm the differences among the age groups, the linear mixed effect models were performed.ResultsThere were significant decreases in muscle thickness of the rectus abdominis and external oblique muscles in the 50s and 60s age groups compared to those in the 20s age group, and a significant decrease in muscle thickness of the erector spinae muscle in the 60s age group compared to those in the 20s age group. However, there was no significant difference among the age groups in muscle thickness of other trunk muscles. There were significant increases in echo intensity of the abdominal muscles in other age groups compared to those in the 20s age group, and significant increases in echo intensity of the back muscles in the age groups over 40 compared to those in the 20s group.Conclusion
Our study revealed that muscle quality may be more affected by age than muscle quantity and the effects of aging differ among muscles.
... Cellular senescence promoted without p16 increment has been previously described [16]. An increased accumulation of p21 and p53 proteins has been previously described in satellite cells from old animals compared to young animals, contributing to a lower proliferation rate of these cells [42]. Cellular senescence has been identified as a mechanism involved in the genesis of the myopathy associated with muscular dystrophy mouse model [43] and related to premature muscle wasting in young mice deficient in the protein Bmi1 [44] or mutant mice carrying BubR1 hypomorphic alleles [45]. ...
In mammalians, advancing age is associated with sarcopenia, the progressive and involuntary loss of muscle mass and strength. Hyperphosphatemia is an aging-related condition involved in several pathologies. The aim of this work was to assess whether hyperphosphatemia plays a role in the age-related loss of mass muscle and strength by inducing cellular senescence in murine myoblasts and to explore the intracellular mechanism involved in this effect. Cultured mouse C2C12 cells were treated with 10 mM beta-glycerophosphate (BGP] at different periods of time to induce hyperphosphatemia. BGP promoted cellular senescence after 24 h of treatment, assessed by the increased expression of p53, acetylated-p53 and p21 and senescence associated β-galactosidase activity. In parallel, BGP increased ILK expression and activity, followed by mTOR activation and autophagy reduction. Knocking-down ILK expression increased autophagy and protected cells from senescence induced by hyperphosphatemia. BGP also reduced the proliferative capacity of cultured myoblasts. Old mice (24-months-old] presented higher serum phosphate concentration, lower forelimb strength, higher expression of p53 and ILK and less autophagy in vastus muscle than young mice (5-months-old]. In conclusion, we propose that hyperphosphatemia induces senescence in cultured myoblasts through ILK overexpression, reducing their proliferative capacity, which could be a mechanism involved in the development of sarcopenia, since old mice showed loss of muscular strength correlated with high serum phosphate concentration and increased levels of ILK and p53.
... In the same way, the circadian clock controls intracellular NAD + levels by a transcriptional feedback loop [123]. Moreover, SIRT1 is also able to promote proliferation of muscle cell precursors by blocking the expression of cell cycle inhibitors and hindering their transformation into fully differentiated myocytes [111,124,125]. This observation has been made in cultured murine myotubes and has been associated with SIRT1-mediated repression of MYOD, a major muscle transcriptional regulator, and of MEF2 [114,126]. ...
Fighting diseases and controlling the signs of ageing are the major goals of biomedicine. Sirtuins, enzymes with mainly deacetylating activity, could be pivotal targets of novel preventive and therapeutic strategies to reach such aims. Scientific proofs are accumulating in experimental models, but, to a minor extent, also in humans, that the ancient practice of calorie restriction could prove an effective way to prevent several degenerative diseases and to postpone the detrimental signs of ageing. In the present review, we summarize the evidence about the central role of sirtuins in mediating the beneficial effects of calorie restriction in skeletal and cardiac muscle since these tissues are greatly damaged by diseases and advancing years. Moreover, we entertain the possibility that the identification of sirtuin activators that mimic calorie restriction could provide the benefits without the inconvenience of this dietary style.
... Equal amounts of enriched nuclear extract protein were separated by SDS-PAGE and subsequently transferred to nitrocellulose membranes. Membranes were stained with Ponceau S (Sigma), and the image was quantified to verify equal loading [P ϭ 0.576; data not shown (19)]. -Catenin protein was analyzed by Western blot and detected with a primary antibody from Cell Signaling Technology (no. ...
... The reason for that is the decrease of the stimulatory effect of insulin and IGF-1 in the skeletal muscle of old rats, which is twice as severe in the young [34]. Aging-caused sarcopenia and glucocorticoid-caused myopathy both develop as a result of the decrease and damage of satellite cells [35,36]. In old myopathic muscle, degradation of contractile proteins doubles [15]. ...
Muscle weakness in corticosteroid myopathy is mainly the result of the destruction and atrophy of the myofibrillar compartment of fast-twitch muscle fibers. Decrease of titin and myosin, and the ratio of nebulin and MyHC in myopathic muscle, shows that these changes of contractile and elastic proteins are the result of increased catabolism of the abovementioned proteins in skeletal muscle. Slow regeneration of skeletal muscle is in good correlation with a decreased number of satellite cells under the basal lamina of muscle fibers. Aging causes a reduction of AMP-activated protein kinase (AMPK) activity as the result of the reduced function of the mitochondrial compartment. AMPK activity increases as a result of increased functional activity. Resistance exercise causes anabolic and anticatabolic effects in skeletal muscle: muscle fibers experience hypertrophy while higher myofibrillar proteins turn over. These changes are leading to the qualitative remodeling of muscle fibers. As a result of these changes, possible maximal muscle strength is increasing. Endurance exercise improves capillary blood supply, increases mitochondrial biogenesis and muscle oxidative capacity, and causes a faster turnover rate of sarcoplasmic proteins as well as qualitative remodeling of type I and IIA muscle fibers. The combination of resistance and endurance exercise may be the fastest way to prevent or decelerate muscle atrophy due to the anabolic and anticatabolic effects of exercise combined with an increase in oxidative capacity. The aim of the present short review is to assess the role of myofibrillar protein catabolism in the development of glucocorticoid-caused myopathy from aging and physical activity aspects.
... Moreover, overexpression of SIRT1 can promote muscle precursor cell proliferation and cell cycle progression [11]. Associated with the SIRT1-mediated proliferation of C2C12 cells were the bidirectional decreases and increases in the expression of the cyclin-dependent kinase inhibitors p21 (Waf1, Cip1) and p27Kip1, respectively [12]. Although SIRT1 can improve the myoblast proliferation, the specific mechanism(s) underlying remains to be clarified. ...
Sirtuin type 1 (SIRT1) is a potent NAD + dependent deacetylase that deacetylates histone and nonhistone proteins to regulate gene expression and protein activity. Emerging evidences have indicated that SIRT1 plays a significant role in diverse cellular processes including cell growth, differentiation, development, and physiological function in muscle cells; however the signaling mechanisms involved remain to be established. In order to investigate its potential role in muscle biological processes, we administrated C2C12 cells which were isolated from skeletal muscle tissue of dystrophic mice with SIRT1 activator resveratrol (REV), inhibitor nicotinamide (NAM) and Wnt inhibi-tor FH535. By CCK-8, BrdU assay, real-time PCR and Western blot, we investigated whether the SIRT1 has a function in C2C12 cells by promoting β-catenin accumulation. Our results demonstrate that SIRT1 increases cell proliferation of C2C12 myoblast in a SIRT1-dependent manner. And SIRT1 significantly up-regulates the expression of cyclin D1, C-myc and Dvl2 in vitro as well as stimulates the accumulation of the Wnt/β-catenin. In conclusion, this study indicates that SIRT1 promotes the proliferation of C2C12 myoblast cells, at least partly via Wnt signaling pathway.
... Moreover, overexpression of SIRT1 can promote muscle precursor cell proliferation and cell cycle progression [11]. Associated with the SIRT1-mediated proliferation of C2C12 cells were the bidirectional decreases and increases in the expression of the cyclin-dependent kinase inhibitors p21 (Waf1, Cip1) and p27Kip1, respectively [12]. Although SIRT1 can improve the myoblast proliferation, the specific mechanism(s) underlying remains to be clarified. ...
Sirtuin type 1 (SIRT1) is a potent NAD+ dependent deacetylase that deacetylates histone and nonhistone proteins to regulate gene expression and protein activity. Emerging evidences have indicated that SIRT1 plays a significant role in diverse cellular processes including cell growth, differentiation, development, and physiological function in muscle cells,cells; however the signaling mechanisms involved remain to be established. In order to investigate its potential role in muscle biological processes, we administrated C2C12 cells which were isolated from skeletal muscle tissue of dystrophic mice with SIRT1 activator resveratrol (REV), inhibitor nicotinamide (NAM) and Wnt inhibitor FH535. By CCK-8, BrdU assay, real-time PCR and Western Blotblot, we investigated whether the SIRT1 has a function in C2C12 cells by promoting β-catenin accumulation. Our results demonstrate that SIRT1 increases cell proliferation of C2C12 myoblast in a SIRT1-dependent manner. And SIRT1 significantly up-regulates the expression of cyclin D1 ,D1, C-myc and Dvl2 in vitro as well as stimulates the accumulation of the Wnt/β-catenin. In conclusion, this study indicates that SIRT1 promotes the proliferation of C2C12 myoblast cells, at least partly via Wnt signaling pathway.
... These mononuclear cells are present in skeletal, smooth and cardiac muscles. In skeletal muscle specifically, they reside between the basal lamina and the existing myofiber it encapsulates.[32][33][34] While the cells appear to be associated with the specific myofiber, this is not the case, as is shown by the presence of a gap between the plasma membrane of the myofiber and the membrane of the satellite cell. ...
... These findings suggest that SIRT-1 may have an important role in prolonging or enhancing proliferation of satellite cells. However, while satellite cell function is reduced with aging, and thus, lower SIRT-1 protein levels might be expected in muscles of old animals, it is interesting to note that increased levels of SIRT-1 have been reported in satellite cells isolated from old rats [30], although the significance of this is not clear. One possibility to explain this complex role of SIRT-1 in skeletal muscle is that SIRT-1 activity and not the abundance of this protein may be more important for determining the downstream function in sarcopenic muscle as it is in muscles of young animals [31]. ...
Accumulation of reactive oxygen species (ROS) in skeletal muscles and the resulting decline in muscle performance are hallmarks of sarcopenia. However, the precise mechanism by which ROS results in a decline in muscle performance is unclear. We demonstrate that isometric-exercise concomitantly increases the activities of Silent information regulator 1 (SIRT-1) and Poly [ADP-ribose] polymerase (PARP-1), and that activated SIRT-1 physically binds with and inhibits PARP-1 activity by a deacetylation dependent mechanism in skeletal muscle from young mice. In contrast, skeletal muscle from aged mice displays higher PARP-1 activity and lower SIRT-1 activity due to decreased intracellular NAD+ content, and as a result reduced muscle performance in response to exercise. Interestingly, injection of PJ34, a PARP-1 inhibitor, in aged mice increased SIRT-1 activity by preserving intracellular NAD+ content, which resulted in higher skeletal muscle mitochondrial biogenesis and performance. We found that the higher activity of PARP-1 in H2O2-treated myotubes or in exercised-skeletal muscles from aged mice is due to an elevated level of PARP-1 acetylation by the histone acetyltransferase General control of amino acid synthesis protein 5-like 2 (GCN-5). These results suggest that activation of SIRT-1 and/or inhibition of PARP-1 may ameliorate skeletal muscle performance in pathophysiological conditions such as sarcopenia and disuse-induced atrophy in aging.
... It is therefore not surprising that the allelic status of p53 might influence myoblasts differentiation, thereby correlating with the risk of developing sarcopenia, as we show in the present paper. In fact, it has been demonstrated that, in the presence of differentiation-promoting stimuli, p53-defective myoblasts exit from the cell cycle but do not differentiate into myocytes and myotubes, thereby implying a role for p53 in skeletal muscle differentiation [36,37]. Moreover, p53 is involved in the NF-B and PI3 K/Akt pathways that are central in controlling muscle size, promoting protein synthesis, and blocking degradation [38][39][40][41]. ...
Background and aim:
p53 activity plays a role in muscle homeostasis and skeletal muscle differentiation; all pathways that lead to sarcopenia are related to p53 activities. We investigate the allelic frequency of the TP53 codon 72 in exon 4 polymorphism in the Italian female population and the association with appendicular skeletal muscle mass index in normal weight (NW), normal weight obese (NWO), and preobese-obese (Preob-Ob) subjects.
Methods:
We evaluated anthropometry, body composition, and p53 polymorphism in 140 women distinguished in NW, NWO, and Preob-Ob.
Results:
*Arg/*Arg genotype increases sarcopenia risk up to 20% (*Arg/*Arg genotype OR = 1.20; 95% CI = 0.48-2.9; *proallele carriers OR = 0.83; 95% CI = 0.83-2.06). The risk of being sarcopenic for *Arg/*Arg genotype in NWO and Preob-Ob is 31% higher than NW carriers of *proallele (RR = 0,31, 95% CI = 0,15-0,66, P = 0,0079). We developed a model able to predict sarcopenia risk based on age, body fat, and p53 polymorphism.
Conclusion:
Our study evidences that genotyping TP53 polymorphism could be a useful new genetic approach, in association with body composition evaluations, to assess sarcopenia risk.
... Nevertheless, together the data suggest that resveratrol might lower excessively high ROS levels, and this would be expected to improve satellite cell function and/or prevent loss of some of the activated satellite cells in responses to stressors (e.g., loading), that otherwise might be lost (destroyed) in a very high ROS environment such as aging and loading, although it probably has modest effects on quiescent satellite cells that are attached to sarcopenic muscles. It is noteworthy that increased levels of Sirt1 have been reported in satellite cells isolated from old rats, although the significance of this is not clear (Machida and Booth, 2004). Thus, it is possible that when Sirt1 was elevated in satellite cells of old animals, it may not have been active and therefore unable to produce the anticipated benefits that have been associated with Sirt1 in aged muscles. ...
The mechanisms contributing to sarcopenia include reduced satellite cell (myogenic stem cell) function that is impacted by the environment (niche) of these cells. Satellite cell function is affected by oxidative stress, which is elevated in aged muscles, and this along with changes in largely unknown systemic factors, likely contribute to the manner in which satellite cells respond to stressors such as exercise, disuse, or rehabilitation in sarcopenic muscles. Nutritional intervention provides one therapeutic strategy to improve the satellite cell niche and systemic factors, with the goal of improving satellite cell function in aging muscles. Although many elderly persons consume various nutraceuticals with the hope of improving health, most of these compounds have not been thoroughly tested, and the impacts that they might have on sarcopenia and satellite cell function are not clear. This review discusses data pertaining to the satellite cell responses and function in aging skeletal muscle, and the impact that three compounds: resveratrol, green tea catechins, and β-Hydroxy-β-methylbutyrate have on regulating satellite cell function and therefore contributing to reducing sarcopenia or improving muscle mass after disuse in aging. The data suggest that these nutraceutical compounds improve satellite cell function during rehabilitative loading in animal models of aging after disuse (i.e., muscle regeneration). While these compounds have not been rigorously tested in humans, the data from animal models of aging provide a strong basis for conducting additional focused work to determine if these or other nutraceuticals can offset the muscle losses, or improve regeneration in sarcopenic muscles of older humans via improving satellite cell function.
... Regenerative capacity of skeletal muscle in response to oxidative stress or aging might require activation of quiescent muscle precursor cells (65)(66)(67). Activation of satellite cells is essential for regeneration, maintenance and repair of adult skeletal muscle damage due to aging and/or chronic stress states. Therefore, we analyzed the protein expression for PAX7 (stem cell marker) and MyoD (muscle differentiation factor) by immunoblotting. ...
Skeletal muscle redox homoeostasis is transcriptionally regulated by nuclear erythroid-2-p45-related factor-2 (Nrf2). We recently demonstrated that age-associated stress impairs Nrf2-ARE (antioxidant response element) transcriptional signaling. Here, we hypothesize that age-dependent decline or genetic ablation of Nrf2 leads to accelerated apoptosis and skeletal muscle degeneration. Under basal-physiological conditions, disruption of Nrf2 significantly down regulates antioxidants and causes oxidative stress. Surprisingly, Nrf2-null mice had enhanced antioxidant capacity identical to wild-type (WT) upon acute endurance exercise stress (AEES), suggesting activation of Nrf2-independent mechanisms (i.e. PGC1α) against oxidative stress. Analysis of pro-survival pathways under the basal state reveals decreased Akt levels, while pp53, a repressor of Akt, was increased in Nrf2-null versus WT mice. Upon AEES, Akt and p-Akt levels were significantly (p<0.001) increased (>10 fold) along with profound down regulation of pp53 (p<0.01) in Nrf2-null versus WT skeletal muscle, indicating the onset of pro-survival mechanisms to compensate the loss of Nrf2 signaling. However, we found a decreased stem cell population (Pax7) and MyoD expression (differentiation) along with profound activation of ubiquitin and apoptotic pathways in Nrf2- null versus WT mice upon AEES, suggesting that compensatory pro-survival mechanisms failed to overcome the programed cell death and degeneration in skeletal muscle. Further, the impaired regeneration was sustained in Nrf2-null vs. WT mice after 1 week of post-AEES recovery. In an age-associated oxidative stress condition, ablation of Nrf2 results in induction of apoptosis and impaired muscle regeneration.
... Recently, many efforts have relied on the characterization of skeletal or heart muscle-specific stem and/or satellite cells that, once recruited, may contribute to repairing injured tissues. Since it has been proposed that SirT1 may also influence the lineage/cell-fate decisions of stem cells by sensing redox status (Machida and Booth, 2004; Mantel and Broxmeyer, 2008), it is conceivable that SirT1 may also influence the regenerative potential of skeletal muscle and ...
Why is SIRT1 considered to be a 'longevity' gene? SirT1 is the mammalian ortholog of yeast Sir2, an enzyme that is involved in protein deacetylation, which was first characterized as an important regulator of life span in this organism, and subsequently in higher eukaryotes (Longo and Kennedy, 2006). However, whether SirT1 is associated with an extension of the life span of human cells is a matter of some debate (Michishita et al., 2005). SirT1 substrates and transcriptional/epigenetic co-factors make up an impressive and constantly growing list, including, among others, PGC-1a, Westerheide et al., 2009). SirT1 influences numerous processes that are crucial to cell viability, such as gene silencing or activation, apoptosis, stress resistance, senescence, energy balance, and lipid and glucose metabolism (Fig. 1). Recent elegant work on SirT1 knockout mouse embryonic fibroblasts (MEFs) and embryonic stem cells showed that SirT1 activity impacts functionally on the circadian clock (Asher et al., 2008; Nakahata et al., 2008) and on genome (chromatin) stability (Oberdoerffer et al., 2008; Wang et al., 2008), and an integrated picture of SirT1-dependent anti-cancer and anti-aging effects is just emerging (Fig. 1) (Jung-Hynes and Ahmad, 2009; Liu et al., 2009). Several mechanisms that are capable of activating sirtuin enzymatic activity have been shown to increase life span. Classical activators of SirT1 include the polyphenol resveratrol (contained in red grapes and green tea) (Howitz et al., 2003), as well as a regimen of caloric restriction (CR) (Cohen et al., 2004). CR, defined in mice as a reduction in food intake of 30%-50% compared with animals fed ad libitum, is a very well-known intervention that enhances longevity in laboratory animals (Fig. 1). CR may actually increase life span by triggering a complex interplay of signaling molecules, including not only SirT1, but also AMP-activated protein kinase (AMPK), forkhead box O transcription factors (FOXOs), mammalian target of rapamycin (mTOR), and the ratio of NAD + to NADH (Cantó and Auwerx, 2009b). Similarly, resveratrol impacts on additional cellular pathways, probably owing to its chemical nature as a protein-binding polyphenol. Owing to the pleiotropic positive effects of SirT1 on the health of organisms, the pharmaceutical industry have shown a growing interest in developing compounds that are able to modulate SirT1 activity (Lavu et al., 2008). In this review, we will narrow our focus on the role of SirT1 activity in two striated muscle tissues of embryonic mesodermic origin, skeletal and heart muscle, which govern fundamental processes such as glucose and lipid metabolism, physical activity, and propulsion of blood around the circulatory system, with a particular attention to relevant SirT1-specific mouse models. Readers who are interested in other sirtuins (SirT2-SirT7) or in other tissue-specific SirT1 mice models are referred to the recent excellent reviews by Finkel et al. and Guarente (Guarente, 2007; Finkel et al., 2009). Given the technologies available to manipulate the mouse genome (van der Weyden et al., 2003), and the high degree of homology between murine and human genomes, the mouse is considered the premier organism for modeling human pathologies.
... The question is whether this activity occurs at the level of the organism in vivo as well. The finding that there is an increase in the levels of p53, FOXO1, and SIRT1 in the nuclei of cells of old animals [110] may be indicative of a complex setting in mammals. In sporadic inclusion-body myositis, which is an agingassociated muscle disease, it was found that deacetylation of p53 was decreased [111]. ...
Abstract Reactive oxygen species (ROS) are continuously generated during metabolism. ROS are involved in redox signalling but, in significant concentrations they can greatly elevate oxidative damage leading to neurodegeneration. Because of the enhanced sensitivity of brain to ROS, it is especially important to maintain a normal redox state in brain and spinal cord cell types. The complex effects of exercise benefit brain function, including functional enhancement as well as preventive and therapeutic roles. Exercise can induce neurogenesis via neurotrophic factors, increase capillarization, decrease oxidative damage, and enhance repair of oxidative damage. Exercise is also effective in attenuating age-associated loss in brain function, which suggests that physical activity-related complex metabolic and redox changes are important for a healthy neural system.
... To examine the hypothesis, we measured the amount of cell cycle inhibiters, which are known to be increased because of the association with cellular senescence and organismal aging. [12][13][14] We also examined the effect of an angiotensin II receptor blocker (ARB) on cardiac function and expression of senescence-associated molecules in the hearts of diabetic animals. ...
Purpose: Thyroid hormone (T4) induces physiological cardiac hypertrophy in animals. Activation of phosphoinositide 3-kinase (PI3K) is shown to be necessary and sufficient to promote physiological hypertrophy in mice. We assessed the hypothesis that PI3K is involved in T4 induced cardiac hypertrophy. Method: To examine if T4 activates effectors of PI3K, T4 or saline was intraperitoneally administered to mice. The mice were sacrificed 1, 4, 8, or 24 hours after the injection. The amount of phosphorylated Akt, p70 ribosomal S6kinase1 (S6K1), ribosomal S6 Protein (S6) in the heart tissue was analyzed by Western blotting. To examine if PI3K is necessary for T4 induced cardiac hypertrophy, T4 was daily administered to transgenic mice expressing dominant-negative form of PI3K in the heart (dnPI3K mice) or non-transgenic mice (NTg mice) for 14 days. After the echocardiographic examination, the mice were sacrificed for analysis. Result: The amount of phosphorylated Akt was increased at 1, 4, and 8 hours after T4 injection. The S6K1 phosphorylation increased at 4 and 8 hours. The S6 phosphorylation increased at 8 hours. Heart weight/body weight of T4 treated NTg mice was increase by 27% compared with the saline treated NTg mice. In contrast, T4 treated dnPI3K mice was increased by 9% compared with saline treated dnPI3K mice. Conclusion: Thyroid hormone induced physiological cardiac hypertrophy in a PI3K dependent manner.
... Myostatin activates FOXO1, while FOXO1 in turn increases myostatin expression, resulting in a feed-forward loop between myostatin and FOXO and a subsequent increase in protein degradation (Allen and Unterman 2007). In addition, FOXO1 also inhibits IGF-1-mediated satellite cell proliferation (Machida and Booth 2004). However, it has been shown that myostatin over-expression in rat tibialis anterior muscle was unable to alter the activity of proteasome components, and treatment of Eur J Appl Physiol (2010) 110:665–694 679 human skeletal muscle cells with myostatin even resulted in a decrease in the expression of E3 ligases (Amirouche et al. 2008; Trendelenburg et al. 2009). ...
... The question is whether this activity occurs at the level of the organism in vivo as well. The finding that there is an increase in the levels of p53, FOXO1, and SIRT1 in the nuclei of cells of old animals [110] may be indicative of a complex setting in mammals. In sporadic inclusion-body myositis, which is an agingassociated muscle disease, it was found that deacetylation of p53 was decreased [111]. ...
The consequence of decreased nicotinamide adenine dinucleotide (NAD+) levels as a result of oxidative challenge is altered activity of sirtuins, which, in turn, brings about a wide range of modifications in mammalian cellular metabolism.Sirtuins, especially SIRT1, deacetylate important transcription factors such as p53, forkheadhomeobox type O (FOXO)s, nuclear factor kappa B (NF-κB) or peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α which controls the transcription of pro- and anti-oxidant enzymes, by which cellular redox state is affected). The role of SIRT1 in DNA repair is enigmatic, since it activates Ku70 to cope with double-strand breaks, but deacetylation of apurinic/apyrimidinic endonuclease-1 and probably of 8-oxoguanine-DNA glycosylase 1 decreases the activity of these DNA repair enzyme. The protein-stabilizing effects of the NAD+-dependent lysine deacetylases are readily related to housekeeping and redox regulation. The role of sirtuins in caloric restriction (CR)-related longevity in yeast is currently under debate. However, in mammals, it seems certain that sirtuins are involved in many cellular processes that mediate longevity, and disease prevention via the effects of CR through the vascular, neuronal and muscular systems. Regular physical exercise-mediated health promotion also involves sirtuin-regulated pathways including the antioxidant-, macromolecular damage repair-, energy-, mitochondrial function- and neuronal plasticity-associated pathways. The present review critically evaluates the findings and points out the age-associated role of sirtuins.
... Probably the most important observation from these in vitro experiments is that, in the case of both aged myoblasts and aged BMSCs, myostatin consistently suppressed cell proliferation. Aged muscle precursors have an impaired capacity for proliferation (Conboy et al., 2003;Machida and Booth, 2004), as do aged BMSCs (Kretlow et al., 2008). Blocking myostatin function in aged animals may therefore have significant potential for improving the repair and regeneration of both muscle and bone by improving the proliferative capacity of both myo-and osteo-progenitor cells. ...
The activin A-myostatin-follistatin system is thought to play an important role in the regulation of muscle and bone mass throughout growth, development, and aging; however, the effects of these ligands on progenitor cell proliferation and differentiation in muscle and bone are not well understood. In addition, age-associated changes in the relative expression of these factors in musculoskeletal tissues have not been described. We therefore examined changes in protein levels of activin A, follistatin, and myostatin (GDF-8) in both muscle and bone with age in C57BL6 mice using ELISA. We then investigated the effects of activin A, myostatin and follistatin on the proliferation and differentiation of primary myoblasts and mouse bone marrow stromal cells (BMSCs) in vitro. Myostatin levels and the myostatin:follistatin ratio increased with age in the primarily slow-twitch mouse soleus muscle, whereas the pattern was reversed with age in the fast-twitch extensor digitorum longus muscle. Myostatin levels and the myostatin: follistatin ratio increased significantly (+75%) in mouse bone marrow with age, as did activin A levels (+17%). Follistatin increased the proliferation of primary myoblasts from both young and aged mice, whereas myostatin increased proliferation of younger myoblasts but decreased proliferation of older myoblasts. Myostatin reduced proliferation of both young and aged BMSCs in a dose-dependent fashion, and activin A increased mineralization in both young and aged BMSCs. Together these data suggest that aging in mice is accompanied by changes in the expression of activin A and myostatin, as well as changes in the response of bone and muscle progenitor cells to these factors. Myostatin appears to play a particularly important role in the impaired proliferative capacity of muscle and bone progenitor cells from aged mice.
... p53 is implicated in stem cell regulation (Cicalese et al, 2009; Hong et al, 2009; Kawamura et al, 2009; Li et al, 2009; Marion et al, 2009; TeKippe et al, 2003). We and others have demonstrated a role for p53 in the regulation of skeletal muscle stem cells, muscle homeostasis, and aging (Chung & Ng, 2006; Machida & Booth, 2004; Schwarzkopf et al, 2006; Siu & Alway, 2005). We show here that activated p53 levels increase with age and in the Ku80 heterozygous and null muscle indicating the presence of age-related stress signalling. ...
Muscle wasting is a major cause of morbidity in the elderly. Ku80 is required for DNA double strand repair and is implicated in telomere maintenance. Complete loss-of-function leads to reduced post-natal growth and severe progeria in mice. We examined the role of Ku80 in age-related skeletal muscle atrophy. While complete loss of Ku80 leads to pronounced aging in muscle as expected, accompanied by accumulation of DNA damage, loss of a single allele is sufficient to accelerate aging in skeletal muscle although post-natal growth is normal. Ku80 heterozygous muscle shows no DNA damage accumulation but undergoes premature telomere shortening that alters stem cell self-renewal through stress response pathways including p53. These data reveal an unexpected requirement for both Ku80 alleles for optimal progenitor function and prevention of early onset aging in muscle, as well as providing a useful model for therapeutic approaches.
... These changes together may be one of the reasons for sarcopenia (Figure 1). The mechanisms responsible for sarcopenia in aged skeletal muscle are largely unknown, but muscle satellite cells required for the repair of fibers certainly exhibit impaired activation [50] and proliferation [51] compared to young muscle. Autografting of skeletal muscle has been used as a model of muscle regeneration. ...
Changes in skeletal muscle quantity and quality lead to disability in the aging population. Physiological changes in aging skeletal muscle are associated with a decline in mass, strength, and inability to maintain balance. Glucocorticoids, which are in wide exploitation in various clinical scenarios, lead to the loss of the myofibrillar apparatus, changes in the extracellular matrix, and a decrease in muscle strength and motor activity, particularly in the elderly. Exercise therapy has shown to be a useful tool for the prevention of different diseases, including glucocorticoid myopathy and muscle unloading in the elderly. The purpose of the paper is to discuss the possibilities of using exercise therapy in the prevention of glucocorticoid caused myopathy and unloading in the elderly and to describe relationships between the muscle contractile apparatus and the extracellular matrix in different types of aging muscles.
... For instance, p53 is up-regulated in immobilized muscle [21], during unloading-induced muscle atrophy [42] , and in ageing skeletal muscle [2]. Specifically, the p53/p21 pathway is involved in age-associated loss of satellite cell proliferative capacity [22]. We have reported a decrease in satellite cell number in p53 deficient mice [45], however satellite cell number is not affected in p53+/mt mice, indicating that minimal p53 activity is necessary to sustain satellite cells but satellite cell number is not affected by gene dosage. ...
4) Université Pierre et Marie Curie-Paris6, UMR S 787, 75634 Paris, France; (5) Current address: Abstract p53 tumor suppressor activity has been proposed to regulate the rate of ageing in part by suppressing postnatal stem cell numbers. Severe and rapid skeletal muscle atrophy is a hallmark of cachexia whereas muscle atrophy that occurs during aging (sarcopenia) has a slower rate of progression. Despite these differences, these two forms of muscle atrophy share many features although it remains unclear whether these processes are regulated by the same key regulatory factors. We demonstrated a requirement for p53 function in mediating severe and rapid cachexia induced by tumor load. Furthermore, the p53 target gene, PW1, activates p53 providing a potential positive feedback loop whereby a stress response is amplified in muscle cells. In the presence of TNF, a p53/PW1-dependent pathway mediates the block of myogenic differentiation in vitro and in vivo. To further characterize how p53 and PW1 mediate muscle atrophy, we analyzed a mouse model in which chronic p53 hyperactivation leads to early onset aging. We demonstrate that p53 hyperactivity is sufficient to induce muscle atrophy consistent with sarcopenia in ageing muscle. We observe that this process is accompanied by alterations in the distribution, but not in the number of muscle stem cells. Finally, we demonstrate that p53 upregulates PW1 expression in muscle in vitro and in vivo. Taken together, our data demonstrate that p53 and PW1 activities are required in promoting muscle atrophy induced by cytokines.
... Interestingly, protein levels of the cdk inhibitors p21 Waf/Cip1 and p27 Kip1 are higher in MPCs from old skeletal muscle that have decreased proliferation. 15 Because FoxO3a has been shown to increase the abundance of the cell-cycle inhibitors p27 Kip1 20 and p21 Waf/Cip1 24 in non-muscle cell types, and given the cell context specificity of FoxO transcription factors, we aimed to determine the effect of increasing FoxO3a on MPC proliferation and on p27 Kip1 and p21 Waf/Cip1 . ...
Previous work has demonstrated that forkhead transcription factors, which include the FoxO subfamily, play a critical role in muscle atrophy by inducing expression of the atrophy-related ubiquitin ligases. The proliferation of muscle precursor cells (MPC) is also essential for skeletal muscle mass. The hypothesis was tested that the FoxO forkhead transcription factor FoxO3a hinders MPC proliferation. The present studies were designed to determine the effects of overexpression of FoxO3a on in vitro proliferation of MPCs. MPCs infected with an adenovirus for wild-type FoxO3a had decreased DNA synthesis as detected by the incorporation of 5-bromo-2′ deoxyuridine. In general, cyclin-dependent kinase inhibitors, including p27Kip1and p21Waf/Cip1, inhibit cell proliferation. Associated with the impaired MPC proliferation, we found an increase in the promoter activity and protein levels of the cyclin-dependent kinase inhibitor p27Kip1, whereas there was no effect and a decrease in the promoter activity and protein levels of p21Waf/Cip1. FoxO3a overexpression had no effect on either the phosphorylation of retinoblastoma protein (ser780) or cyclin D1 protein levels, suggesting that FoxO3a does not effect the early phase of the G1–S transition. In addition to its ability to induce muscle atrophy, these studies identify FoxO3a as a negative regulator of MPC proliferation. Our findings suggest that attenuating increased FoxO3a may restore MPC proliferation to prevent atrophy and improve the regenerative capacity of skeletal muscle. Muscle Nerve, 2007
A normal somatic cell undergoes cycles of finite cellular divisions. The presence of surveillance checkpoints arrests cell division in response to stress inducers: oxidative stress from excess free radicals, oncogene-induced abnormalities, genotoxic stress, and telomere attrition. When facing such stress when undergoing these damages, there is a brief pause in the cell cycle to enable repair mechanisms. Also, the nature of stress determines whether the cell goes for repair or permanent arrest. As the cells experience transient or permanent stress, they subsequently choose the quiescence or senescence stage, respectively. Quiescence is an essential stage that allows the arrested/damaged cells to go through appropriate repair mechanisms and then revert to the mainstream cell cycle. However, senescent cells are irreversible and accumulate with age, resulting in inflammation and various age-related disorders. In this review, we focus on senescence-associated pathways and therapeutics understanding cellular senescence as a cascade that leads to aging, while discussing the recent details on the molecular pathways involved in regulating senescence and the benefits of therapeutic strategies against accumulated senescent cells and their secretions. ARTICLE HISTORY
The age-associated decline in muscle mass has become synonymous with physical frailty among the elderly due to its major contribution in reduced muscle function. Alterations in protein and redox homeostasis along with chronic inflammation, denervation and hormonal dysregulation are all hallmarks of muscle wasting and lead to clinical sarcopenia in older adults. Reduction in skeletal muscle mass has been observed and reported in the scientific literature for nearly two centuries; however, identification and careful examination of molecular mediators of age-related muscle atrophy have only been possible for roughly three decades. Here we review molecular targets of recent interest in age-related muscle atrophy and briefly discuss emerging small molecule therapeutic treatments for muscle wasting in sarcopenic susceptible populations.
In this study, we identified a previously uncharacterized skeletal satellite cell-secreted protein, R3h domain containing-like (R3hdml). Expression of R3hdml increases during skeletal muscle development and differentiation in mice. Body weight and skeletal muscle mass of R3hdml knockout (KO) mice are lower compared to control mice. Expression levels of cell cycle-related markers, phosphorylation of Akt, and expression of insulin-like growth factor within the skeletal muscle are reduced in R3hdml KO mice compared to control mice. Expression of R3hdml increases during muscle regeneration in response to cardiotoxin (CTX)-induced muscle injury. Recovery of handgrip strength after CTX injection was significantly impaired in R3hdml KO mice, which is rescued by R3hdml. Our results indicate that R3hdml is required for skeletal muscle development, regeneration, and, in particular, satellite cell proliferation and differentiation.
Changes in histone acetylation and methylation status with aging affect gene expression and phenotype in several tissues; however, age-related changes in histone modification in the skeletal muscle have not been elucidated yet. This study investigated age-related global changes in histone modification in rat gastrocnemius muscle. Male Wistar rats (n = 28) were assigned to one of four age groups (n = 7 per group) corresponding to different life stages: 3 months old (3-mo; young), 6 months old (adult), 12 months old (12-mo; middle-aged), and 24 months old (24-mo; old). The gastrocnemius muscle was removed and global histone modification (acetylation and tri-methylation) at K9 and K27 was evaluated by western blotting. Relative muscle mass decreased in the 12- and 24-mo rats accompanied with reduction in type IIb myosin heavy chain isoforms and Myh4 (MHC IIB) mRNA expression. Histone H3 acetylation decreased in an age-dependent manner, with lower levels in 12- and 24-mo groups than in the 3-mo group. K9 and K27 acetylation decreased with age. Although there was no significant change in K27 tri-methylation, K9 tri-methylation showed an age-dependent decline. Histone modification status (acetylation at K9 and K27 and tri-methylation at K9) was positively associated with relative gastrocnemius muscle weight, the percentage of type IIb myosin heavy chain isoform, myosin heavy chain type IIb protein expression, and the level of Myh4 mRNA. Thus, global histone H3 methylation and acetylation decrease with age, and the latter might be associated with age-related muscle atrophy of rat gastrocnemius muscle.
Primary human muscle progenitor cells (hMPCs) are commonly used to understand skeletal muscle biology, including the regenerative process. Variability from unknown origin in hMPC expansion capacity occurs independent of disease, age, or sex of the donor. We sought to determine the transcript profile that distinguishes hMPC cultures with greater expansion capacity and to identify biological underpinnings of these transcriptome profile differences. Sorted (CD56+/CD29+) hMPC cultures were clustered using unbiased K means cluster analysis into FAST and SLOW based on growth parameters (saturation density and population doubling time). FAST had greater expansion capacity indicated by significantly reduced population doubling time (-60%) and greater saturation density (+200%), nuclei area under the curve (AUC)(+250%), and confluency AUC (+120%) as well as fewer % dead cells AUC (-44%, p<0.05). RNAseq was conducted on RNA extracted during the expansion phase. Principal component analysis distinguished FAST and SLOW based on the transcript profile. There were 2205 differentially expressed genes (DEgenes) between FAST and SLOW (q value {less than or equal to} 0.05), and 362 DEgenes that met a more stringent cut-off (q value {less than or equal to} 0.001 and 2.0 fold-change). DEgene enrichment suggested FAST (vs. SLOW) had promotion of the cell cycle, reduced apoptosis and cellular senescence, and enhanced DNA replication. Novel (RABL6, IRGM1, and AREG) and known (FOXM1, CDKN1A, Rb) genes emerged as regulators of identified functional pathways. Collectively the data suggest that variation in hMPC expansion capacity occurs independent of age and sex and is driven, in part, by intrinsic mechanisms that support the cell cycle.
Mitochondria are the central hubs of cellular metabolism, equipped with their own mitochondrial DNA (mtDNA) blueprints to direct part of the programming of mitochondrial oxidative metabolism and thus reactive oxygen species (ROS) levels. In stem cells, many stem cell factors governing the intricate balance between self-renewal and differentiation have been found to directly regulate mitochondrial processes to control stem cell behaviors during tissue regeneration and aging. Moreover, numerous nutrient-sensitive signaling pathways controlling organismal longevity in an evolutionarily conserved fashion also influence stem cell-mediated tissue homeostasis during aging via regulation of stem cell mitochondria. At the genomic level, it has been demonstrated that heritable mtDNA mutations and variants affect mammalian stem cell homeostasis and influence the risk for human degenerative diseases during aging. Because such a multitude of stem cell factors and signaling pathways ultimately converge on the mitochondria as the primary mechanism to modulate cellular and organismal longevity, it would be most efficacious to develop technologies to therapeutically target and direct mitochondrial repair in stem cells, as a unified strategy to combat aging-related degenerative diseases in the future.
The forkhead transcription factor FoxO1 has been studied as the most downstream targets of the signaling pathway in mammals, and played a key role in adipogeneic, cell cycle progression, and energy metabolism and so on. The aim of this study was to investigate the tissue-specific and weight-dependent expression patterns of FoxO1 gene in porcine omental adipose tissue and its relation to adipose deposition. Sixty female Duroc × Landrace × Yorkshire pigs in 5 groups, each group containing twelve pigs weighing 1, 20, 40, 60, and 90 kg were used to study developmental expression of FoxO1 gene by means of semi-quantitative RT-PCR. The results showed that FoxO1 mRNA was expressed at a higher level in heart, brain, omental adipose tissue, skeletal muscle, liver and spleen, at a moderate level in kidney, lung, and peritoneal adipose tissue, and at a less level in duodenum, subcutaneous adipose tissue and pancreas. In addition, our data also showed that the expression levels of FoxO1 gene was lowest in the pigs at 20 kg, and continuously increased from 20 to 90 kg body weight (P<0.05). Furthermore, a close positive correlation between the expression levels of FoxO1 gene and the adipose deposition rate was found in pigs (r=0.84, P<0.05).
A decline in adult stem cell function is one of the features of ageing, which is associated with impaired tissue maintenance and reduced regenerative capacity. Telomere shortening occurs in the vast majority of human tissues during ageing and is also observed in ageing stem cell compartments. Studies of somatic cells have shown that telomere dysfunction induces DNA damage checkpoints, which lead to cell cycle arrest (senescence) or apoptosis. Whether similar checkpoint responses occur in stem cells in response to telomere shortening and whether this contributes to impaired organ maintenance during ageing remains to be investigated. In addition to the activation of cell intrinsic checkpoints, telomere shortening can also induce cell extrinsic, environmental alterations that limit stem cell function. Understanding of adult stem cell ageing and could ultimately lead to the identification of targets for therapies aiming to improve organ maintenance during ageing. In this chapter we summarize our current knowledge on checkpoint responses and environmental alteration induced by telomere dysfunction.
In the last 50 years, the number of individuals over the age of 65 years in the United States has doubled. A further doubling is expected by 2030, dramatically increasing the number of adults at risk of sarcopenia, a condition characterized by an age-related loss of muscle mass with an associated reduction in physical function. A reduction in muscle mass and functional capacity is typically viewed as an undesirable, yet inevitable, consequence of aging, and in its early stages, may be easily masked by subtle lifestyle adaptations. However, advanced sarcopenia is synonymous with physical frailty and is associated with an increased likelihood of falls and impairments in the ability to perform routine activities of daily living. In many instances, the progression of sarcopenia is mirrored by a decrease in physical activity, which feeds into a vicious cycle of disuse and negative outcomes, including impaired insulin action, accelerated loss of muscle and bone mass, fatigue, impaired motor control and functional capacity, and increased morbidity and mortality.
Intramuscular adipose tissue (IMAT) formation is observed in some pathological conditions such as Duchenne muscular dystrophy (DMD) and sarcopenia. Several studies have suggested that IMAT formation is not only negatively correlated with skeletal muscle mass but also causes decreased muscle contraction in sarcopenia. In the present study, we examined whether adipocytes affect myogenesis. For this purpose, skeletal muscle progenitor cells were transfected with siRNA of PPARγ (siPPARγ) in an attempt to inhibit adipogenesis. Myosin heavy chain (MHC)-positive myotube formation was promoted in cells transfected with siPPARγ compared to that of cells transfected with control siRNA. To determine whether direct cell-to-cell contact between adipocytes and myoblasts is a prerequisite for adipocytes to affect myogenesis, skeletal muscle progenitor cells were cocultured with pre- or mature adipocytes in a Transwell coculture system. MHC-positive myotube formation was inhibited when skeletal muscle progenitor cells were cocultured with mature adipocytes, but was promoted when they were cocultured with preadipocytes. Similar effects were observed when pre- or mature adipocyte-conditioned medium was used. These results indicate that preadipocytes play an important role in maintaining skeletal muscle mass by promoting myogenesis; once differentiated, the resulting mature adipocytes negatively affect myogenesis, leading to the muscle deterioration observed in skeletal muscle pathologies.
The importance of oxidation in vascular disease was established in the mid-1980s with the emergence of the “LDL modification
hypothesis” of atherosclerosis, initially referring to the vessel wall damage that occurs subsequent to oxidation of low-density
lipoproteins deposited in the vascular wall. Oxygen radicals and derived reactive species not only accelerate the development
or affect the stability of atherosclerotic plaque but are also involved in regulation many aspects of vascular diseases or
conditions associated with hypertension, diabetes, ischemia-reperfusion or vascular injury. In recent years, the term oxidative stress has been used to describe the manifestations of changes in redox enzyme activity in vascular cells, as well as the process
of oxidation of cellular components caused by the intracellular excess of free radicals. The cellular response attributed
to these reactive species can be as diverse as cell dysfunction, alteration of cell proliferation or migration, modification
of extracellular matrix composition or cell death by apoptosis. In addition, there is growing evidence showing that the long-term
and insidious action of oxygen radicals may irreversibly damage genomic or mitochondrial DNA. Accumulation of nucleic acid
lesions due to oxidant stress may accelerate the natural processes of vascular cells aging. In addition, DNA damage may interfere
with the cell cycle and cell survival by activating ROS-sensitive transcription factors and activating DNA damage-dependent
signalling pathways. This chapter will describe the evidence for oxidative stress in vascular disease, the sources of reactive
species in the vascular wall, and their relevance in vascular pathologies Largely focusing on atherosclerosis.
KeywordsOxidative stress-Atherosclerosis-Hypertension-Vascular remodeling-Aging-Telomeres-DNA damage
Blood pressure increases with age in men and women. Whether and how oxidative stress plays a role in mediating hypertension
has been under intense study for the past 10 years. Although most animal studies have shown that oxidative stress will increase
blood pressure, the clinical trials using antioxidants to treat hypertension in humans have not been successful. One theory
for how oxidative stress increases blood pressure is that oxidants cause an increase in renal vasoconstriction that increases
sodium and water reabsorption by the kidney. However, whether the blood pressure in males and females responds the same to
increases in oxidative stress is not clear. There are several problems with regard to the study of oxidative stress: the lack
of consistent measurements of oxidative stress, lack of specific inhibitors of oxidants or scavengers of reactive oxygen species,
and the lack of specific, selective ways to increase oxidative stress in animal models. Problems in human studies contribute
additional roadblocks to answering the questions of whether and how oxidative stress contributes to hypertension, such as
genetic differences in the population, lack of power to take into consideration that men and women may respond differently
to antioxidants, and lack of separation of genders in analyses of the data. This review will address these issues.
KeywordsHypertension-F2-isoprostanes-Menopause-Oxidative stress-Sexual dimorphism
In many species including humans, females live longer than males. We and others have observed that mitochondria from females
of Wistar rats and of OF1 mice produce half the amount of peroxide produced by males. We attributed this to a change in the
expression of antioxidant, longevity-related genes. We have found that in those species in which females live longer than
males, estrogens activate longevity-related genes, particularly antioxidant ones. It should be emphasized that estrogens do
not act as antioxidants because of their phenolic ring but rather they act indirectly; that is, they behave as hormones and
bind to estrogen receptors, which eventually leads to the upregulation of the expression of antioxidant genes. The pathway
by which estrogens activate the expression of these genes has been elucidated, and we have traced it to the activation of
the mitogen activated proteins (MAP) kinase pathway. It is remarkable that estrogens activate proliferation genes (related
to their feminizing function and also to their cancer-promoting effects) by binding to estrogen receptor alpha whereas the
longevity-related genes, in particular the antioxidant ones, are mediated by binding to estrogen receptor beta. Phytoestrogens,
which in their vast majority bind to estrogen receptor beta, promote longevity-related genes without increasing the rate of
cell division or promoting feminization. Thus, a practical approach discussed here is that administration of phytoestrogens
may be very beneficial for longevity because they bind very preferentially to estrogen receptor beta and promote the upregulation
of longevity-related genes.
KeywordsAging-Mitochondria-Estrogens-Phytoestrogens-Longevity genes
Methylglyoxal (MG) is a highly reactive product of mainly glucose metabolism. Reactive oxygen species (ROS) are produced during
various biochemical reactions, including mitochondrial electron transport. An excess of MG production can increase ROS production
and oxidative stress. MG can also form advanced glycation end products (AGEs) by reacting with proteins, DNA, and other biomolecules.
The process of aging is multifactorial and involves changes at the cellular, tissue, organ, and whole-body levels that lead
to decreased functioning, development of diseases, and death. MG, ROS, and AGEs are all associated with the aging process
and age-related diseases such as cardiovascular complications of diabetes, neurodegenerative diseases, and connective tissue
disorders. Specific and effective scavengers and cross-link breakers of MG and AGEs are being developed to slow the aging
process and prevent many diseases.
KeywordsMethylglyoxal-Oxidative stress-Advanced glycation end products-Aging
Aging is characterized by a time-dependent loss of function, generally agreed to be the result of an increased incidence of
damage produced by free radicals and other reactive oxygen species and a decreased ability of the animal to mount sufficient
protective defenses to maintain adequate function. In many species, imposing food calorie restriction is effective at bolstering
these defenses and extending life span. At the same time, caloric restriction induces increased activation of one or more
regulatory enzymes, the sirtuins. Sirtuins are NAD+-dependent deacetylases that act on a wide array of protein substrates to connect metabolic state – as reflected in the NAD+ concentration – with regulation of many key cellular and physiologic processes including apoptosis, DNA repair, inflammation,
immunity, transformation, and autophagy. Although increasing the expression of a single sirtuin in a few simpler and shorter-lived
animals can significantly extend life span, the effects on mammalian life span of any of the seven mammalian sirtuins are
much less robust. Whereas overexpression of sirtuins in mammals typically improves physiology without increasing life span,
reduction often results in progeria, tissue degeneration and dysfunction, shortened life span, or outright lethality. Changes
in sirtuin expression or activity are often found to play key roles in a variety of age-associated degenerative diseases in
both humans and in animal models; treatments that appropriately modulate sirtuin activity often ameliorate these conditions.
Thus there is accelerating interest in advancing understanding not only of the roles sirtuins normally play in regulating
and maintaining homeostasis but also in how to modulate their activities so as to treat age-related degenerative diseases
more successfully.
KeywordsSirtuins-Aging-Mammals
The Forkhead transcription factors AFX, FKHR and FKHR-L1 are orthologues of DAF-16, a Forkhead factor that regulates longevity in Caenorhabditis elegans. Here we show that overexpression of these Forkhead transcription factors causes growth suppression in a variety of cell lines, including a Ras-transformed cell line and a cell line lacking the tumour suppressor PTEN. Expression of AFX blocks cell-cycle progression at phase G1, independent of functional retinoblastoma protein (pRb) but dependent on the cell-cycle inhibitor p27kip1. Indeed, AFX transcriptionally activates p27kip1, resulting in increased protein levels. We conclude that AFX-like proteins are involved in cell-cycle regulation and that inactivation of these proteins is an important step in oncogenic transformation.
Interest is growing in methods to extend replicative life span of non-immortalized stem cells. Using the insulin-like growth factor I (IGF-I) transgenic mouse in which the IGF-I transgene is expressed during skeletal muscle development and maturation prior to isolation and during culture of satellite cells (the myogenic stem cells of mature skeletal muscle fibers) as a model system, we elucidated the underlying molecular mechanisms of IGF-I-mediated enhancement of proliferative potential of these cells. Satellite cells from IGF-I transgenic muscles achieved at least five additional population doublings above the maximum that was attained by wild type satellite cells. This IGF-I-induced increase in proliferative potential was mediated via activation of the phosphatidylinositol 3'-kinase/Akt pathway, independent of mitogen-activated protein kinase activity, facilitating G(1)/S cell cycle progression via a down-regulation of p27(Kip1). Adenovirally mediated ectopic overexpression of p27(Kip1) in exponentially growing IGF-I transgenic satellite cells reversed the increase in cyclin E-cdk2 kinase activity, pRb phosphorylation, and cyclin A protein abundance, thereby implicating an important role for p27(Kip1) in promoting satellite cell senescence. These observations provide a more complete dissection of molecular events by which increased local expression of a growth factor in mature skeletal muscle fibers extends replicative life span of primary stem cells than previously known.
Calorie restriction extends life-span in a wide variety of organisms. Although it has been suggested that calorie restriction
may work by reducing the levels of reactive oxygen species produced during respiration, the mechanism by which this regimen
slows aging is uncertain. Here, we mimicked calorie restriction in yeast by physiological or genetic means and showed a substantial
extension in life-span. This extension was not observed in strains mutant forSIR2 (which encodes the silencing protein Sir2p) orNPT1 (a gene in a pathway in the synthesis of NAD, the oxidized form of nicotinamide adenine dinucleotide). These findings suggest
that the increased longevity induced by calorie restriction requires the activation of Sir2p by NAD.
Interleukin-3 (IL-3), IL-5, and granulocyte-macrophage colony-stimulating factor regulate the survival, proliferation, and
differentiation of hematopoietic lineages. Phosphatidylinositol 3-kinase (PI3K) has been implicated in the regulation of these
processes. Here we investigate the molecular mechanism by which PI3K regulates cytokine-mediated proliferation and survival
in the murine pre-B-cell line Ba/F3. IL-3 was found to repress the expression of the cyclin-dependent kinase inhibitor p27KIP1 through activation of PI3K, and this occurs at the level of transcription. This transcriptional regulation occurs through
modulation of the forkhead transcription factor FKHR-L1, and IL-3 inhibited FKHR-L1 activity in a PI3K-dependent manner. We
have generated Ba/F3 cell lines expressing a tamoxifen-inducible active FKHR-L1 mutant [FKHR-L1(A3):ER*]. Tamoxifen-mediated
activation of FKHR-L1(A3):ER* resulted in a striking increase in p27KIP1 promoter activity and mRNA and protein levels as well as induction of the apoptotic program. The level of p27KIP1 appears to be critical in the regulation of cell survival since mere ectopic expression of p27KIP1 was sufficient to induce Ba/F3 apoptosis. Moreover, cell survival was increased in cytokine-starved bone marrow-derived stem
cells from p27KIP1 null-mutant mice compared to that in cells from wild-type mice. Taken together, these observations indicate that inhibition
of p27KIP1transcription through PI3K-induced FKHR-L1 phosphorylation provides a novel mechanism of regulating cytokine-mediated survival
and proliferation.
In Caenorhabditis elegans, mutations that reduce the activity of an insulin-like receptor (daf-2) or a phosphatidylinositol-3-OH kinase (age-1) favour entry into the dauer state during larval development and extend lifespan in adults. Downregulation of this pathway activates a forkhead transcription factor (daf-16), which may regulate targets that promote dauer formation in larvae and stress resistance and longevity in adults. In yeast, the SIR2 gene determines the lifespan of mother cells, and adding an extra copy of SIR2 extends lifespan. Sir2 mediates chromatin silencing through a histone deacetylase activity that depends on NAD (nicotinamide adenine dinucleotide) as a cofactor. We have surveyed the lifespan of C. elegans strains containing duplications of chromosomal regions. Here we report that a duplication containing sir-2.1-the C. elegans gene most homologous to yeast SIR2-confers a lifespan that is extended by up to 50%. Genetic analysis indicates that the sir-2.1 transgene functions upstream of daf-16 in the insulin-like signalling pathway. Our findings suggest that Sir2 proteins may couple longevity to nutrient availability in many eukaryotic organisms.
Reactive oxygen species are required for cell proliferation but can also induce apoptosis. In proliferating cells this paradox is solved by the activation of protein kinase B (PKB; also known as c-Akt), which protects cells from apoptosis. By contrast, it is unknown how quiescent cells that lack PKB activity are protected against cell death induced by reactive oxygen species. Here we show that the PKB-regulated Forkhead transcription factor FOXO3a (also known as FKHR-L1) protects quiescent cells from oxidative stress by directly increasing their quantities of manganese superoxide dismutase (MnSOD) messenger RNA and protein. This increase in protection from reactive oxygen species antagonizes apoptosis caused by glucose deprivation. In quiescent cells that lack the protective mechanism of PKB-mediated signalling, an alternative mechanism is induced as a consequence of PKB inactivity. This mechanism entails the activation of Forkhead transcription factors, the transcriptional activation of MnSOD and the subsequent reduction of reactive oxygen species. Increased resistance to oxidative stress is associated with longevity. The model of Forkhead involvement in regulating longevity stems from genetic analysis in Caenorhabditis elegans, and we conclude that this model also extends to mammalian systems.
Foxk1 is a forkhead/winged helix transcription factor that is restricted to myogenic progenitor cells in adult skeletal muscle. Mice lacking Foxk1 (Foxk1-/-) display growth retardation and a severe impairment in skeletal muscle regeneration following injury. Here we show that myogenic progenitor cells from Foxk1-/- mice are reduced in number and have perturbed cell cycle progression (G(0)/G(1) arrest). Molecular analysis of Foxk1-/- myogenic progenitor cells revealed increased expression of the cyclin-dependent kinase inhibitor, p21(CIP), independent of changes in other cell cycle inhibitors, including p53. Combinatorial mating of Foxk1-/- mice with p21(CIP)-/- mice, to generate double mutant progeny, resulted in a complete restoration of the growth deficit, skeletal muscle regeneration, myogenic progenitor cell number, and cell cycle progression that characterized the Foxk1-/- mice. We conclude that Foxk1 is essential for regulating cell cycle progression in the myogenic progenitor cell and that the cyclin-dependent kinase inhibitor, p21(CIP), may be a downstream target of Foxk1.
Recently, the FOXO (Forkhead box, class O) subfamily of Forkhead transcription factors has been identified as direct targets of phosphoinositide 3-kinase-mediated signal transduction. The AFX (acute-lymphocytic-leukaemia-1 fused gene from chromosome X), FKHR (Forkhead in rhabdomyosarcoma) and FKHR-L1 (FKHR-like 1) transcription factors are directly phosphorylated by protein kinase B, resulting in nuclear export and inhibition of transcription. This signalling pathway was first identified in the nematode worm Caenorhabditis elegans, where it has a role in regulation of the life span of the organism. Studies have shown that this evolutionarily conserved signalling module has a role in regulation of both cell-cycle progression and cell survival in higher eukaryotes. These effects are co-ordinated by FOXO-mediated induction of a variety of specific target genes that are only now beginning to be identified. Interestingly, FOXO transcription factors appear to be able to regulate transcription through both DNA-binding-dependent and -independent mechanisms. Our understanding of the regulation of FOXO activity, and defining specific transcriptional targets, may provide clues to the molecular mechanisms controlling cell fate decisions to divide, differentiate or die.
The direction of change in skeletal muscle mass differs between young and old individuals, growing in young animals and atrophying in old animals. The purpose of the experiment was to develop a statistically conservative list of genes whose expression differed significantly between young growing and old atrophying (sarcopenic) skeletal muscles, which may be contributing to physical frailty. Gene expression levels of >24,000 transcripts were determined in soleus muscle samples from young (3-4 mo) and old (30-31 mo) rats. Age-related differences were determined using a Student's t-test (alpha of 0.05) with a Bonferroni adjustment, which yielded 682 probe sets that differed significantly between young (n = 25) and old (n = 20) animals. Of 347 total decreases in aged/sarcopenic muscle relative to young muscles, 199 were functionally identified; the major theme being that 24% had a biological role in the extracellular matrix and cell adhesion. Three themes were observed from 213 of the 335 total increases in sarcopenic muscles whose functions were documented in databases: 1) 14% are involved in immune response; 2) 9% play a role in proteolysis, ubiquitin-dependent degradation, and proteasome components; and 3) 7% act in stress/antioxidant responses. A total of 270 differentially expressed genes and ESTs had unknown/unclear functions. By decreasing the sample sizes of young and old animals from 25 x 20 to 15 x 15, 10 x 10, and 5 x 5 observations, we observed 682, 331, 73, and 3 statistically different mRNAs, respectively. Use of large sample size and a Bonferroni multiple testing adjustment in combination yielded increased statistical power, while protecting against false positives. Finally, multiple mRNAs that differ between young growing and old, sarcopenic muscles were identified and may highlight new candidate mechanisms that regulate skeletal muscle mass during sarcopenia.
The limited success of human myoblast transplantation has been related to immune rejection, poor survival, and limited spread of injected myoblasts after transplantation. An important issue that has received little attention, but is nevertheless of fundamental importance in myoblast transplantation protocols, is the proliferative capacity of human satellite cells. Previous studies from our laboratory have demonstrated that the maximum number of divisions that a population of satellite cells can make decreases with age during the first two decades of life then stabilizes in adulthood. These observations indicate that when satellite cells are used as vectors in myoblast transplantation protocols it is important to consider donor age and the number of divisions that the cells have made prior to transplantation as limiting factors in obtaining an optimal number of donor derived muscle fibers. In this study, myoblasts derived from donors of different ages (newborn, 17 years old, and 71 years old) were isolated and amplified in culture. Their potential to participate in in vivo muscle regeneration in RAG2(-/-)/gamma(c)/C5 triple immunodeficient hosts after implantation was evaluated at 4 and 8 weeks postimplantation. Our results demonstrate that prolonged amplification in culture and the approach to replicative senescence are both important factors that may condition the success of myoblast transplantation protocols.
A hallmark of aging is diminished regenerative potential of tissues, but the mechanism of this decline is unknown. Analysis
of injured muscle revealed that, with age, resident precursor cells (satellite cells) had a markedly impaired propensity to
proliferate and to produce myoblasts necessary for muscle regeneration. This was due to insufficient up-regulation of the
Notch ligand Delta and, thus, diminished activation of Notch in aged, regenerating muscle. Inhibition of Notch impaired regeneration
of young muscle, whereas forced activation of Notch restored regenerative potential to old muscle. Thus, Notch signaling is
a key determinant of muscle regenerative potential that declines with age.
To study the mechanism(s) by which manganese-containing superoxide dismutase (MnSOD) mediates cellular growth inhibition, an inducible retroviral vector system regulated by the lac repressor was used to overexpress MnSOD protein in NIH/3T3 cells. Increased MnSOD activity led to decreased cell growth due to prolonged cell cycle transition times in G(1) and S phases without significant changes in G(2)/M phase. Changes in cell cycle transition time were reversible and tightly correlated with MnSOD levels. A transient increase of reactive oxygen species and concomitant decrease in mitochondrial membrane potential were documented following MnSOD induction. N-Acetyl-L-cysteine prevented growth inhibition by MnSOD. Our data suggest that MnSOD may serve a physiological function of regulating cell cycle progression through its prooxidant activity of generating hydrogen peroxide, resulting in coordination of mitochondrial redox state and cellular proliferation.
Longevity regulatory genes include the Forkhead transcription factor FOXO and the NAD-dependent histone deacetylase silent information regulator 2 (Sir2). Genetic studies demonstrate that Sir2 acts to extend lifespan in Caenorhabditis elegans upstream of DAF-16, a member of the FOXO family, in the insulin-like signaling pathway. However, the molecular mechanisms underlying the requirement of DAF-16 activity in Sir2-mediated longevity remain unknown. Here we show that reversible acetylation of Foxo1 (also known as FKHR), the mouse DAF-16 ortholog, modulates its transactivation function. cAMP-response element-binding protein (CREB)-binding protein binds and acetylates Foxo1 at the K242, K245, and K262 residues, the modification of which is involved in the attenuation of Foxo1 as a transcription factor. Conversely, Sir2 binds and deacetylates Foxo1 at residues acetylated by cAMP-response element-binding protein-binding protein. Sir2 is recruited to insulin response sequence-containing promoter and increases the expression of manganese superoxide dismutase and p27(kip1) in a deacetylase-activity-dependent manner. Our findings establish Foxo1 as a direct and functional target for Sir2 in mammalian systems.
One of the key factors responsible for the age-associated reduction in muscle mass may be that satellite cell proliferation potential (number of doublings contained within each cell) could become rate limiting to old muscle regrowth. No studies have tested whether repeated cycles of atrophy-regrowth in aged animals deplete the remaining capacity of satellite cells to replicate or what measures can be taken to prevent this from happening. We hypothesized that there would be a pronounced loss of satellite cell proliferative potential in gastrocnemius muscles of aged rats (25- to 30-mo-old FBN rats) subjected to three cycles of atrophy by hindlimb immobilization (plaster casts) with intervening recovery periods. Our results indicated that there was a significant loss in gastrocnemius muscle mass and in the proliferative potential of the resident satellite cells after just one bout of immobilization. Neither the muscle mass nor the satellite cell proliferation potential recovered from their atrophied values after either the first 3-wk or later 9-wk recovery period. Remarkably, application of insulin-like growth factor I onto the atrophied gastrocnemius muscle for an additional 2 wk after this 9-wk recovery period rescued ∼46% of the lost muscle mass and dramatically increased proliferation potential of the satellite cells from this muscle.
The p53 tumor suppressor is the most commonly mutated gene in human cancer. p53 protein is stabilized in response to different checkpoints activated by DNA damage, hypoxia, viral infection, or oncogene activation resulting in diverse biological effects, such as cell cycle arrest, apoptosis, senescence, differentiation, and antiangiogenesis. The stable p53 protein is activated by phosphorylation, dephosphorylation and acetylation yielding a potent sequence-specific DNA-binding transcription factor. The wide range of p53's biological effects can in part be explained by its activation of expression of a number of target genes including p21WAF1, GADD45, 14-3-3σ, bax, Fas/APO1, KILLER/ DR5, PIG3, Tsp1, IGF-BP3 and others. This review will focus on the transcriptiona l targets of p53, their regulation by p53, and their relative importance in carrying out the biological effects of p53.
This paper looks at the effects of aging on the response of skeletal muscle to exercise from the perspective of the behavior of muscle precursor cells (widely termed satellite cells or myoblasts) and regeneration. The paper starts by outlining the ways in which skeletal muscle can respond to damage resulting from exercise or other trauma. The age-related changes within skeletal muscle tissue and the host environment that may affect the proliferation and fusion of myoblasts in response to injury in old animals are explored. Finally, in vivo and in vitro data concerning the wide range of signaling molecules that stimulate satellite cells and other aspects of regeneration are discussed with respect to aging. Emphasis is placed on the important role of the host environment, inflammatory cells, growth factors and their receptors (particularly for FGF-2), and the extracellular matrix.
Mouse extensor digitorum longus (EDL) muscle was subjected to a dose of gamma irradiation that causes reproductive death of satellite cells and/or to chronic compensatory overload, achieved by removal of the distal portion of the tibialis anterior muscle. Four weeks later the mass, fiber type percentage, and fiber size of the EDL muscle were measured. Both the irradiated + overloaded and the irradiated only EDL muscles were significantly lighter and contained significantly smaller fibers than untreated muscle or muscle subjected to chronic overload only. Overload muscle, whether irradiated or not, had a larger percentage of type IIx fibers and a smaller percentage of type IIb fibers than muscle that had not been overloaded. The results confirm that satellite cell proliferation is a prerequisite for muscle hypertrophy induced by synergist incapacitation, but it appears not to be required for the maintenance of, or change in, normal muscle fiber myosin heavy chain phenotype expression.
We tested the hypothesis that after the same amount of contraction-induced injury, skeletal muscles in old mice regenerate less well than muscles in young mice. Extensor digitorum longus (EDL) muscles in young and old mice were exposed to 15 min of lengthening contractions. The amount of injury was evaluated at 3, 7, 14, 28, and 60 days by measurements of maximum isometric tetanic force (Po) and number of fibers per cross section. When values 3 days after lengthening contractions were expressed as a percentage of control values, the Po (approximately 34%) and fiber number (approximately 80%) for muscles in old mice were not different from those in young mice, suggesting that muscles in old and young mice were injured to the same degree. By 28 days, injured muscles in young mice regained control values for Po and fiber number. In contrast, at 28 days, injured muscles in old mice recovered approximately 84 and approximately 87% of control values for Po and fiber number, respectively, and deficits in Po persisted at 60 days. We conclude that injured muscles regenerate less well in old mice than in young mice.
Skeletal muscle satellite cells were isolated from soleus and extensor digitorum longus muscles of Sprague-Dawley rats at selected ages between 6 days and 30 months and grown in cell culture. Cells at each donor age were monitored individually to determine the number of progeny they were capable of producing. Under identical in vitro conditions, the average number of progeny produced was inversely proportional to donor age. Hence, the proliferation potential of satellite cells decreases with age.
The ability of p53 to activate transcription from specific sequences suggests that genes induced by p53 may mediate its biological role as a tumor suppressor. Using a subtractive hybridization approach, we identified a gene, named WAF1, whose induction was associated with wild-type but not mutant p53 gene expression in a human brain tumor cell line. The WAF1 gene was localized to chromosome 6p21.2, and its sequence, structure, and activation by p53 was conserved in rodents. Introduction of WAF1 cDNA suppressed the growth of human brain, lung, and colon tumor cells in culture. Using a yeast enhancer trap, a p53-binding site was identified 2.4 kb upstream of WAF1 coding sequences. The WAF1 promoter, including this p53-binding site, conferred p53-dependent inducibility upon a heterologous reporter gene. These studies define a gene whose expression is directly induced by p53 and that could be an important mediator of p53-dependent tumor growth suppression.
p53, perhaps the single most important human tumor suppressor, is commonly mutated in human cancers. Normally genotoxic stress and hypoxia activate p53, which, through DNA-specific transcription activation, transcriptional repression, and protein-protein interactions, triggers cell cycle arrest and apoptosis. One of the genes induced by p53 was identified as that encoding the insulin-like growth factor binding protein (IGFBP)-3. IGFBP-3 was originally defined by the somatomedin hypothesis as the principal carrier of IGF-I in the circulation and the primary regulator of the amount of free IGF-I available to interact with the IGF-1 receptor. However, there is accumulating evidence that IGFBP-3 can also cause apoptosis in an IGF-independent manner. Thus, IGFBP-3 induction by p53 constitutes a new means of cross-talk between the p53 and IGF axes, and suggests that the ultimate function of IGFBP-3 may be to serve a protective role against the potentially carcinogenic effects of growth hormone and IGF-I.
One of the key factors responsible for the age-associated reduction in muscle mass may be that satellite cell proliferation potential (number of doublings contained within each cell) could become rate limiting to old muscle regrowth. No studies have tested whether repeated cycles of atrophy-regrowth in aged animals deplete the remaining capacity of satellite cells to replicate or what measures can be taken to prevent this from happening. We hypothesized that there would be a pronounced loss of satellite cell proliferative potential in gastrocnemius muscles of aged rats (25- to 30-mo-old FBN rats) subjected to three cycles of atrophy by hindlimb immobilization (plaster casts) with intervening recovery periods. Our results indicated that there was a significant loss in gastrocnemius muscle mass and in the proliferative potential of the resident satellite cells after just one bout of immobilization. Neither the muscle mass nor the satellite cell proliferation potential recovered from their atrophied values after either the first 3-wk or later 9-wk recovery period. Remarkably, application of insulin-like growth factor I onto the atrophied gastrocnemius muscle for an additional 2 wk after this 9-wk recovery period rescued approximately 46% of the lost muscle mass and dramatically increased proliferation potential of the satellite cells from this muscle.
The p53 tumour-suppressor gene integrates numerous signals that
control cell life and death. As when a highly connected node in the Internet
breaks down, the disruption of p53 has severe consequences.
Adult skeletal muscle has a remarkable ability to regenerate following myotrauma. Because adult myofibers are terminally differentiated, the regeneration of skeletal muscle is largely dependent on a small population of resident cells termed satellite cells. Although this population of cells was identified 40 years ago, little is known regarding the molecular phenotype or regulation of the satellite cell. The use of cell culture techniques and transgenic animal models has improved our understanding of this unique cell population; however, the capacity and potential of these cells remain ill-defined. This review will highlight the origin and unique markers of the satellite cell population, the regulation by growth factors, and the response to physiological and pathological stimuli. We conclude by highlighting the potential therapeutic uses of satellite cells and identifying future research goals for the study of satellite cell biology.
Paxillin is a focal adhesion-associated, phosphotyrosine-containing protein that may play a role in several signaling pathways. Paxillin contains a number of motifs that mediate protein-protein interactions, including LD motifs, LIM domains, an SH3 domain-binding site and SH2 domain-binding sites. These motifs serve as docking sites for cytoskeletal proteins, tyrosine kinases, serine/threonine kinases, GTPase activating proteins and other adaptor proteins that recruit additional enzymes into complex with paxillin. Thus paxillin itself serves as a docking protein to recruit signaling molecules to a specific cellular compartment, the focal adhesions, and/or to recruit specific combinations of signaling molecules into a complex to coordinate downstream signaling. The biological function of paxillin coordinated signaling is likely to regulate cell spreading and motility.
Aging is associated with increased free radical generation in the skeletal muscle that can cause oxidative modification of protein, lipid, and DNA. Physical activity has many well-established health benefits, but strenuous exercise increases muscle oxygen flux and elicits intracellular events that can lead to increased oxidative injury. The paradox arises as to whether exercise would be advisable to aged population. Research evidence indicates that senescent organisms are more susceptible to oxidative stress during exercise because of the age-related ultrastructural and biochemical changes that facilitate formation of reactive oxygen species (ROS). Aging also increases the incidence of muscle injury, and the inflammatory response can subject senescent muscle to further oxidative stress. Furthermore, muscle repair and regeneration capacity is reduced at old age that could potentially enhance the accrual of cellular oxidative damage. Predeposition of certain age-related pathologic conditions may exacerbate the risks. In spite of these risks, the elderly who are physically active benefit from exercise-induced adaptation in cellular antioxidant defense systems. Improved muscle mechanics, strength, and endurance make them less vulnerable to acute injury and chronic inflammation. Many critical questions remain regarding the relationship of aging and exercise as we enter a new millennium. For example, how does aging alter exercise-induced intracellular and intercellular mechanisms that generate ROS? Can acute and chronic exercise modulate the declined gene expression of metabolic and antioxidant enzymes seen at old age? Does exercise prevent age-dependent muscle loss (sarcopenia)? What kinds of antioxidant supplementation, if any, do aged people who are physically active need? Answers to these questions require highly specific research in both animals and humans.
The p53 protein plays a critical role in the prevention of cancer. It responds to a variety of cellular stresses to induce either apoptosis, a transient cell cycle arrest, or a terminal cell cycle arrest called senescence. Senescence in cultured cells is associated with augmented p53 activity and abrogation of p53 activity may delay in vitro senescence. Increasing evidence suggests that p53 may also influence aspects of organismal aging. Several mutant mouse models that display alterations in longevity and aging-related phenotypes have defects in genes that alter p53 signaling. Recently, my laboratory has developed and characterized a p53 mutant mouse line that appears to have an enhanced p53 response. These p53 mutants exhibit increased cancer resistance, yet have a shortened longevity and display a number of early aging-associated phenotypes, suggesting a role for p53 in the aging process. The nature of the aging phenotypes observed in this p53 mutant line is consistent with a model in which aging is driven in part by a gradual depletion of stem cell functional capacity.
Irradiation of rat skeletal muscles before increased loading has been shown to prevent compensatory hypertrophy for periods of up to 4 wk, possibly by preventing satellite cells from proliferating and providing new myonuclei. Recent work suggested that stem cell populations exist that might allow irradiated muscles to eventually hypertrophy over time. We report that irradiation essentially prevented hypertrophy in rat muscles subjected to 3 mo of functional overload (OL-Ir). The time course and magnitude of changes in cellular and molecular markers of anabolic and myogenic responses were similar in the OL-Ir and the contralateral nonirradiated, overloaded (OL) muscles for the first 3-7 days. These markers then returned to control levels in OL-Ir muscles while remaining elevated in OL muscles. The number of myonuclei and amount of DNA were increased markedly in OL but not OL-Ir muscles. Thus it appears that stem cells were not added to the irradiated muscles in this time period. These data are consistent with the theory that the addition of new myonuclei may be required for compensatory hypertrophy in the rat.
Cell division relies on the activation of cyclins, which bind to cyclin-dependent kinases (CDKs) to induce cell-cycle progression towards S phase and later to initiate mitosis. Since uncontrolled cyclin-dependent kinase activity is often the cause of human cancer, their function is tightly regulated by cell-cycle inhibitors such as the p21 and p27 Cip/Kip proteins. Following anti-mitogenic signals or DNA damage, p21 and p27 bind to cyclin-CDK complexes to inhibit their catalytic activity and induce cell-cycle arrest. Interestingly, recent discoveries suggest that p21 and p27 might have new activities that are unrelated to their function as CDK inhibitors. The identification of new targets of Cip/Kip proteins as well as evidence of Cip/Kip cytoplasmic relocalization have revealed unexpected functions for these proteins in the control of CDK activation, in the regulation of apoptosis and in transcriptional activation. This article discusses recent insights into these possible additional functions of p21 and p27.
Adult skeletal muscle contains populations of satellite cells and muscle-derived stem cells that are capable of forming multinucleate myotubes. The purpose of this study was to determine the phenotype of cells isolated from a common satellite cell isolation and passaging procedure from whole skeletal muscle. To ascertain the characteristics of the cellular phenotype, the myogenic markers MyoD and desmin, the satellite-cell-specific marker Pax7, and the haemopoietic stem cell markers CD34 and CD45 were examined by immunohistochemical analysis. Immediately after isolation, > 90% myogenic marker-positive cells were positive for desmin, MyoD and Pax7. In contrast, approximately 10% of the isolated cells expressed only CD34 or CD45. After three passages, the percentage of cells that were positive for the myogenic markers desmin, MyoD and Pax7 was reduced to approximately 55%, while the population of CD34- or CD45-positive cells increased to approximately 30% after the third passage. Immunohistochemical detection of bromodeoxyuridine demonstrated that the number of proliferating cells decreased progressively after each passaging. Finally, after the third passage the percentage of nuclei in myotubes decreased from 46.7% to 12.5%. Since passaging of muscle progenitor cells is common practice, the results of the current report suggest that characterization of cell heterogeneity needs to be made frequently.
- S Machida
- F W Booth
S. Machida, F.W. Booth / Experimental Gerontology 39 (2004) 1521–1525
Silent information regulator 2 potentiates Foxo1-mediated transcription through its deacetylase activity Epub ahead of print Forkhead transcription factor FKHR-L1 modulates cytokine-dependent transcriptional regulation of
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Requirement of NAD and SIR2 for life-span extension by calorie restriction in Saccharomyces cerevisiae
- S J Lin
- P A Defossez
- L Guarente
Lin, S.J., Defossez, P.A., Guarente, L., 2000. Requirement of NAD and
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Requirement of NAD and SIR2 for life-span extension by calorie restriction in Saccharomyces cerevisiae
- Lin
WAF1, a potential mediator of p53 tumor suppression
- el-Deiry