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p66Shc: at the crossroad of oxidative stress and the genetics of aging
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p66Shc: at the crossroad of oxidative stress and the genetics of aging Sally Purdom1 and Qin M. Chen2 1 2
Interdisciplinary Graduate Program for Genetics and Genomics, University of Arizona, 1501 N. Campbell, Tucson, AZ 85724, USA Department of Pharmacology, University of Arizona, 1501 N. Campbell, Tucson, AZ 85724, USA
The biology of aging has been mysterious for centuries. Removal of the p66Shc gene, which encodes an adaptor protein for cell signaling, extends lifespan by , 30% in mice and confers resistance to oxidative stress. The absence of p66Shc correlates with reduced levels of apoptosis. Oxidants induce phosphorylation of serine36 on p66Shc, contributing to inactivation of members of the Forkhead transcription factor family, some of which appear to regulate the expression of antioxidant genes. The expression of p66Shc is regulated by the methylation status of its promoter. This leads us to hypothesize that increased methylation of the p66Shc promoter might contribute to the absence of its expression and therefore extended longevity in particular individuals. Aging is an inevitable consequence of life. With the development of better healthcare, most countries will be experiencing an increase in the population above the age of 65. A striking phenomenon of aging is an increased risk of most forms of neurodegenerative and life-threatening diseases. The incidence of cardiovascular disease or cancer rises exponentially with age in the population over 45-years old [1]. Alzheimer’s and Parkinson’s diseases are known as ‘diseases of old age’. Why are these diseases prone to the aging population instead of being dispersed evenly throughout all ages? Understanding the biology of aging becomes essential in addressing this question. Aging is a process of getting old or reaching terminal stages of maximal lifespan. Although longevity is a measurement of maximal lifespan of certain species or individuals, mortality rate indicates the frequency or number of deaths in proportion to a population. Regardless of these terminologies, several experimental models have been developed to study the biology of aging, with which an extension of the maximal lifespan has been a popular approach. These models include rodents and the invertebrate systems of Caenorhabditis elegans and Drosophila melanogaster. The short life cycle and well-defined morphological features of aging make these invertebrate systems advantageous in studying the biology of aging. Recent findings reinstate the vitality of the Free Radical Theory of Aging. Numerous evidence indicates the association of elevated oxidative damage in the aging tissue of humans and experimental models [2]. At the molecular level, an increased expression of stress Corresponding author: Qin M. Chen ([email protected]).
response genes has been observed with normal aging, indicating the imprint of oxidative stress [3]. Although the results of genetic manipulation of antioxidant enzymes are somewhat controversial or inconsistent between species or experimental designs, overexpressing the genes encoding the antioxidant enzymes superoxide dismutase and catalase has been shown to extend the lifespan of Drosophila [4], whereas disruption of the gene encoding superoxide dismutase resulted in a shortening of lifespan in this species [5]. Molecules that mimic these antioxidant enzymes can increase the longevity of C. elegans [6]. Based on these observations, it has been hypothesized that effectively eliminating oxidants might be a potential ‘fountain of youth’. However, every one of us lives with aerobic metabolism and presumably experiences similar or equal rates of oxidative stress from endogenous sources. Individual or populational differences in longevity argue for additional factors perhaps related to genetics in the determination of maximal lifespan. The genetic components of longevity have already been demonstrated in several models of aging. Extreme examples supporting the genetic theory of aging in humans include premature aging syndromes, such as Werner’s syndrome and Hutchinson-Gilford syndrome. Mouse strains with prolonged life spans are usually known to be associated with specific genetic factors. For example, a long-lived mouse strain, the Ames dwarf mouse, carries a mutation in the gene encoding Prophet-of-Pit-1 (PROP), a pituitary transcription factor that regulates the secretion of growth hormone and insulin production [7]. An important effort among several laboratories involved in aging research has been to identify gerontogenes, which are genes that affect aging. Nine classes of gerontogenes have been identified in C. elegans [8]. On the surface, it might appear that oxidative stress and genetics are two independent determinants of aging. However, more and more evidence suggests that these two aspects are intrinsically linked. Gene knockout studies and functional analyses reveal that the gene encoding the p66Shc (Src-homology-containing) protein in mammals might be one of the convergent points linking oxidative stress and the genetics of aging. p66Shc knockout: a pleasant surprise Few reliable means exist to extend the lifespan of rodents. Two of these are caloric restriction (CR) and knocking out the p66Shc gene. Caloric restriction without malnutrition
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extends the lifespan of experimental rodents and primates by 20% or more [9,10]. For example, the longevity of laboratory mice on a normal diet is an average of 28 months. Mice fed 53%, 31% or 25% of normal calories live an average of 33, 45 or 47 months, respectively [10]. A lifespan extension of ,30% was observed in mice by knocking out the p66Shc gene, although no alteration in food intake or weight gain has been reported with these transgenic animals [11]. In this experiment, wild-type mice have an average lifespan of 761 days, whereas the heterozygous (p66Shcþ/2 ) or homozygous (p66Shc2/2 ) mice have an average lifespan of 815 or 973 days, respectively [11]. Gene profiling revealed that calorie-restricted aging mice have reduced levels of oxidative-stress-inducible genes when compared to ad lib fed counterparts, consistent with the postulation that caloric restriction leads to reduced levels of oxidative stress [3,12]. Calorically restricted aging mice also expressed higher levels of genes involved in metabolic detoxification and DNA repair when compared with normal aged counterparts [3]. This supports the notion that genetic variation that leads to decreased levels of oxidants or increased damage repair capacity might confer an increase in lifespan. Do p66Shc knockout mice share a similar mechanism of lifespan extension with caloric restriction? Although the gene array data have not yet been reported, functional studies of the p66Shc gene provide a positive indication to this question. Similar to calorie-restricted mice, enhanced resistance to oxidative stress in p66Shc null mice is associated with the nature of increased longevity [11]. When exposed to the oxidant-generating compound paraquot, p66Shc2/2 mice had a 40% longer survival time compared to wild-type littermates [11]. In other words, normal mice die within 48 h in response to intraperitoneal injection of paraquot. By contrast, . 50% of p66Shc2/2 mice can survive the same dose of paraquot for 72 h or more. Therefore the transgenic animals are better equipped to handle oxidative stress than their wild-type counterparts. At the cellular level, p66Shc plays a crucial role in the regulation of oxidative stress response and apoptosis. Mouse embryonic fibroblasts (MEFs) derived from p66Shc2/2 rodents were shown to have normal baseline levels of intracellular oxidative stress compared to wild type [13]. However, unlike wild-type MEFs, the p66Shc2/2 cells are resistant to producing reactive oxygen species in response to nutrient deprivation or serum starvation [13]. When subjected to oxidative stress through either H2O2 or UV treatment, p66Shc2/2 cells are reluctant to undergo apoptosis compared to wild-type cells [11]. Expression of a plasmid containing wild-type p66Shc in the null cells was able to return the cells to levels of normal sensitivity [11]. Overexpression of a wild-type p66Shc transgene in normal MEFs resulted in increased sensitivity to apoptosis under the same conditions [11]. These reports support the theory that p66Shc might participate in the regulation of the aging process through regulating oxidant generation and inducing apoptosis.
proteins are present throughout various tissues in human and mouse except the brain or neurons, where the proteins encoded by ShcB or ShcC are expressed [14 –16]. The ShcA gene encodes two mRNA species: p66Shc and p46/p52Shc. The transcriptional start site for p46/p52Shc mRNA is , 3900 base pairs upstream of that for p66Shc mRNA. Because of alternative translation start sites, two proteins, p46Shc and p52Shc, are derived from the same mRNA [17]. Each ShcA protein harbors three identical functional domains: an N-terminal phosphotyrosine-binding domain (PTB), which is slightly truncated in the p46 isoform, a central proline-rich domain (CH1), and a carboxy terminal Src homology 2 (SH2) domain [14]. p66Shc differs from p46 or p52Shc by an additional N-terminal proline-rich domain (CH2) [14]. Although the structural homology seems to indicate a functional overlap between these isoforms, experimental evidence has demonstrated several differences. All three ShcA proteins (p46, p52 and p66) participate in mitogenic signaling and oncogenesis by regulating receptor tyrosine kinase signaling. Whereas the SH2 domain of Shc is important for certain receptor interactions, such as epidermal growth factor (EGF) receptor and ErbB-2, the PTB domain can bind to phospholipids, implying a role of phosphatidylinositol 3-kinase (PI3K) in activation of Shc [18– 20]. Indeed, insulin induces tyrosine phosphorylation of Shc via a PI3K-dependent mechanism involving the PTB domain, whereas the interaction between the SH2 domain and EGF receptor tyrosine kinases plays an important role in phosphorylation of tyrosines in the CH1 domain of Shc [18 –20]. Tyrosine phosphorylation of p46 and p52 enables them to bind to the adaptor Grb2 protein, which then recruits the guanine nucleotide exchange factor SOS, causing activation of Ras and subsequently the mitogen-activated protein kinase (MAPK) cascade [18,19,21]. Grb2 has been known to activate the Ras signaling pathway without interacting with p46 or p52Shc. The participation of p46 or p52Shc is therefore thought to enhance certain weak signals from growth factor receptors or G-protein coupled receptors [18,22]. No evidence yet indicates that p66Shc activates the Ras signaling pathway [21]. Instead, p66Shc competes with p46 or p52Shc for Grb2 binding [19]. This suggests that p66Shc serves as a dominant negative regulator of p46 or p52Shc-mediated Ras signaling. The p66Shc protein contains two serine phosphorylation sites, Ser36 in its CH2 domain and Ser138 in its PTB domain. The first site is unique to p66Shc and is important for oxidative stress response (see next section). The second site is homologous to Ser29 in p52Shc. This shared serine/threonine phosphorylation site is regulated by protein kinase C (PKC) and an upstream MAPK kinase MEK1 [22–25]. Two functional consequences of PTB domain serine phosphorylation have been reported: binding to the sequestering protein 14–3-3 and binding to the protein tyrosine phosphatase PTP-PEST. These interactions might contribute to the role of Shc in regulating apoptosis.
A matter of Shc isoforms Three Shc genes have been found in the mammalian system: ShcA, ShcB (Sli) and ShcCðRaiÞ [14]. ShcA
The missing link between p66Shc and apoptosis Phosphorylation of the unique Ser36 site of p66Shc appears to influence intracellular oxidative stress and apoptosis.
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UV irradiation and oxidants have been shown to induce phosphorylation at Ser36 [13,24]. Insulin presumably also induces phosphorylation of this site [26]. A p66Shc transgene with mutated Ser36 failed to restore apoptosis sensitivity in p66Shc null cells [11], suggesting the importance of Ser36 in regulating apoptosis. An important downstream target of Ser36-phosphorylated p66Shc is Forkhead transcription factors, which belong to a family containing at least 80 members and which have complex functions in regulating apoptosis. Activated p66Shc contributes to phosphorylation of Forkhead mediated by Akt [13,27,28]. Phosphorylation of Forkhead causes the protein to translocate to the cytosol and to become inactive. In lymphocytes, cytokines can activate the Forkead transcription factor FKHR-L1, which contributes to the loss of mitochondrial membrane potential by regulating the expression of the proapoptotic protein Bim and therefore apoptosis [29,30]. In this scenario, inactivation of Forkhead transcription factors downstream of Akt because of growth factor stimulation appears to be a cell survival mechanism [29]. However in other cell types, including neuronal cells and fibroblasts, Forkhead transcription factors have been reported to regulate the expression of several antioxidant enzymes, including superoxide dismutase and catalase [13,31]. Studies by Nemoto and Finkel show that after oxidative stress, FKHR-L1 could preferentially induce antioxidant or survival genes, but this activity is blocked by p66Shc [13]. Consistent with this hypothesis, oxidative stress causes a greater increase in Forkhead activity in cells lacking p66Shc, which are reluctant to undergo apoptosis [13]. The apparent contradictory findings regarding Forkhead transcription factors either enhancing or preventing apoptosis could be related to a cell-type effect or gene-dosing effect. It has been shown that either too much or too little Forkhead protein contributes to cell death [27,32]. Longevity studies of nematodes support the Forkhead gene-dosing hypothesis. In C. elegans, the Forkhead transcription factor family member Daf16 plays a central role in longevity. Daf16 is involved in dauer formation (Daf), a developmental option available to C. elegans larvae when they sense overpopulation, heat stress and/or starvation [33 – 35]. Daf16 is downregulated by the insulinlike receptor homologue Daf2 and its subsequent signaling pathway resembling phosphoinositide 3-kinase and Akt in the mammalian system [35]. Although there is no evidence that a p66Shc-like gene is present in this invertebrate system and regulates Daf16, mutating any number of constituents releasing the suppression of Daf16 leads to increased longevity [35]. Consistent with these results, mutating Daf16 itself causes a moderately shortened lifespan in nematodes whereas overexpressing Daf16 causes a moderate increase in lifespan [36]. In this model, it appears that small increases in Daf16 can slightly increase longevity, whereas too much Daf16 can result in growth arrest [36]. This supports the hypothesis that a moderate increase in the activity of Forkhead transcription factors, such as in the case of p66Shc knockout mice, can contribute to increased longevity. Although the effect of Forkhead transcription factors in aging appear to be conserved throughout evolution from http://tmm.trends.com
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nematodes to mammals, an interesting paradox remains: why did mammals gain p66Shc, a possibly harmful regulatory gene, during evolution? One could argue that p66Shc is necessary as a tumor suppressor, but p66Shc knockout mice were not significantly more prone to neoplasia than wild-type mice [11]. Understanding the evolutionary importance of p66Shc could answer many questions related to the mechanisms of aging among different experimental systems. Methylation of the p66 promoter: fountain of youth? An interesting aspect of the ShcA gene is its differential regulation of expression. Lack of p66Shc expression can contribute to a minor activation of Forkhead transcription factors and therefore increased expression of antioxidant enzymes as described above. The promoter of p66Shc gene contains a relatively high GC content, although not enough to qualify as a CpG island [17]. DNA methylation in CpG islands is well characterized to regulate gene silencing because of stabilization of chromatin structure and inaccessibility for the transcription apparatus. Several cell lines derived from various human tumor tissues have been tested for the promoter methylation status and the expression level of the p66Shc gene [17]. Apparently, cell lines with the least amounts of methylation of the cytosines in the promoter have the highest levels of p66Shc expression, and vice versa. Treatment of highly methylated cell lines with demethylating agents resulted in renewed transcription of p66Shc [17], arguing for the importance of promoter methylation in silencing the p66Shc gene. The finding that p66Shc expression is regulated by its promoter methylation status raises the possibility that p66Shc promoter methylation status could be linked to human aging. The difference in methylation status of the p66Shc promoter among several cell lines indicates the possibility that the same phenomenon can be reproduced in human individuals. If this is true, then perhaps differences in p66Shc promoter methylation status among individuals contribute to the epigenetic basis of variations in longevity. It would be intriguing to determine the methylation status of the p66Shc promoter among various aging populations. Do centenarians have a hypermethylated p66Shc promoter and lower levels of p66Shc proteins in their cells than other human populations? For those who have a hypomethylated p66Shc promoter and more p66Shc protein, are they prone to develop aging-associated diseases at a younger age? Several studies have linked changes in DNA methylation status with aging and aging-related diseases, particularly cancer [37 – 39]. Aged mice and human tissues have been found to have global hypomethylation of DNA [38]. By contrast, at the individual gene level, several genes increase methylation of their promoters during the process of aging [37]. Examples of these genes include those encoding c-fos [40], estrogen receptor a [41], collagen a1(I) [42] and E-cadherin [43]. These lines of evidence support the hypothesis that the methylation status of the p66Shc promoter can change during the process of aging and there is probably a difference in the p66Shc promoter methylation status among individuals.
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Conclusions Is aging a process of wearing and tearing? The answer is probably ‘yes’. However, this wearing and tearing process appears to be influenced by certain genes, some of which can accelerate the process whereas others delay the process. We now know one magic way to extend the lifespan in mice, which is to knock out the p66Shc gene. These mice can tolerate oxidative stress and have reduced levels of oxidants in response to stress. At the cellular level, p66Shc participates in signaling pathways regulating apoptosis. An important function of p66Shc is repression of Forkhead transcription factors, some of which have been shown to regulate the expression of superoxide dismutase and catalase genes. Animal studies and cell culture experiments point to the necessity for human studies to determine the expression level of p66Shc protein and the promoter methylation status of the p66Shc gene among groups with extended longevity. However, if lack of p66Shc expression indeed correlates with increased longevity, it is unimaginable that changing p66Shc promoter methylation status or p66Shc protein expression is a simple task in human beings with current technology, nutritional and pharmacological supplements. Reducing oxidative stress therefore remains a key point in the goal of delaying aging or aging-associated diseases. One issue we have not addressed here is the relationship between p66Shc and insulin signaling pathways in the biology of mammalian aging. A recent report demonstrates that knocking out the insulin receptor in adipose tissue results in healthy lean mice that have normal rates of food intake but live an average of 134 days longer than usual, equivalent to an 18% increase in longevity [44]. Is p66Shc a mediator linking insulin receptor and aging? This is one of many questions that remain unanswered in the study of the biology of aging. Acknowledgements Work from our laboratory has been supported by Burroughs Wellcome New Investigator Award, American Federation for Aging Research, American Heart Association Beginning-Grant-In-Aid, Arizona Disease Control Commission Research Grant, NIH R03 AG17688 and NIH R01 ES10826 (to Q.M.C.).
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40 Choi, E.K. et al. (1996) Alterations of c-fos gene methylation in the processes of aging and tumorigenesis in human liver. Mutat. Res. 354, 123– 128 41 Post, W.S. et al. (1999) Methylation of the estrogen receptor gene is associated with aging and atherosclerosis in the cardiovascular system. Cardiovasc. Res. 43, 985– 991 42 Takatsu, M. et al. (1999) Age-dependent alterations in mRNA level and promoter methylation of collagen a1(I) gene in human periodontal ligament. Mech. Ageing Dev. 110, 37– 48 43 Bornman, D.M. et al. (2001) Methylation of the E-cadherin gene in bladder neoplasia and in normal urothelial epithelium from elderly individuals. Am. J. Pathol. 159, 831 – 835 44 Bluher, M. et al. (2003) Extended longevity in mice lacking the insulin receptor in adipose tissue. Science 299, 572– 574
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p66Shc: at the crossroad of oxidative stress and the genetics of aging Sally Purdom1 and Qin M. Chen2 1 2
Interdisciplinary Graduate Program for Genetics and Genomics, University of Arizona, 1501 N. Campbell, Tucson, AZ 85724, USA Department of Pharmacology, University of Arizona, 1501 N. Campbell, Tucson, AZ 85724, USA
The biology of aging has been mysterious for centuries. Removal of the p66Shc gene, which encodes an adaptor protein for cell signaling, extends lifespan by , 30% in mice and confers resistance to oxidative stress. The absence of p66Shc correlates with reduced levels of apoptosis. Oxidants induce phosphorylation of serine36 on p66Shc, contributing to inactivation of members of the Forkhead transcription factor family, some of which appear to regulate the expression of antioxidant genes. The expression of p66Shc is regulated by the methylation status of its promoter. This leads us to hypothesize that increased methylation of the p66Shc promoter might contribute to the absence of its expression and therefore extended longevity in particular individuals. Aging is an inevitable consequence of life. With the development of better healthcare, most countries will be experiencing an increase in the population above the age of 65. A striking phenomenon of aging is an increased risk of most forms of neurodegenerative and life-threatening diseases. The incidence of cardiovascular disease or cancer rises exponentially with age in the population over 45-years old [1]. Alzheimer’s and Parkinson’s diseases are known as ‘diseases of old age’. Why are these diseases prone to the aging population instead of being dispersed evenly throughout all ages? Understanding the biology of aging becomes essential in addressing this question. Aging is a process of getting old or reaching terminal stages of maximal lifespan. Although longevity is a measurement of maximal lifespan of certain species or individuals, mortality rate indicates the frequency or number of deaths in proportion to a population. Regardless of these terminologies, several experimental models have been developed to study the biology of aging, with which an extension of the maximal lifespan has been a popular approach. These models include rodents and the invertebrate systems of Caenorhabditis elegans and Drosophila melanogaster. The short life cycle and well-defined morphological features of aging make these invertebrate systems advantageous in studying the biology of aging. Recent findings reinstate the vitality of the Free Radical Theory of Aging. Numerous evidence indicates the association of elevated oxidative damage in the aging tissue of humans and experimental models [2]. At the molecular level, an increased expression of stress Corresponding author: Qin M. Chen ([email protected]).
response genes has been observed with normal aging, indicating the imprint of oxidative stress [3]. Although the results of genetic manipulation of antioxidant enzymes are somewhat controversial or inconsistent between species or experimental designs, overexpressing the genes encoding the antioxidant enzymes superoxide dismutase and catalase has been shown to extend the lifespan of Drosophila [4], whereas disruption of the gene encoding superoxide dismutase resulted in a shortening of lifespan in this species [5]. Molecules that mimic these antioxidant enzymes can increase the longevity of C. elegans [6]. Based on these observations, it has been hypothesized that effectively eliminating oxidants might be a potential ‘fountain of youth’. However, every one of us lives with aerobic metabolism and presumably experiences similar or equal rates of oxidative stress from endogenous sources. Individual or populational differences in longevity argue for additional factors perhaps related to genetics in the determination of maximal lifespan. The genetic components of longevity have already been demonstrated in several models of aging. Extreme examples supporting the genetic theory of aging in humans include premature aging syndromes, such as Werner’s syndrome and Hutchinson-Gilford syndrome. Mouse strains with prolonged life spans are usually known to be associated with specific genetic factors. For example, a long-lived mouse strain, the Ames dwarf mouse, carries a mutation in the gene encoding Prophet-of-Pit-1 (PROP), a pituitary transcription factor that regulates the secretion of growth hormone and insulin production [7]. An important effort among several laboratories involved in aging research has been to identify gerontogenes, which are genes that affect aging. Nine classes of gerontogenes have been identified in C. elegans [8]. On the surface, it might appear that oxidative stress and genetics are two independent determinants of aging. However, more and more evidence suggests that these two aspects are intrinsically linked. Gene knockout studies and functional analyses reveal that the gene encoding the p66Shc (Src-homology-containing) protein in mammals might be one of the convergent points linking oxidative stress and the genetics of aging. p66Shc knockout: a pleasant surprise Few reliable means exist to extend the lifespan of rodents. Two of these are caloric restriction (CR) and knocking out the p66Shc gene. Caloric restriction without malnutrition
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extends the lifespan of experimental rodents and primates by 20% or more [9,10]. For example, the longevity of laboratory mice on a normal diet is an average of 28 months. Mice fed 53%, 31% or 25% of normal calories live an average of 33, 45 or 47 months, respectively [10]. A lifespan extension of ,30% was observed in mice by knocking out the p66Shc gene, although no alteration in food intake or weight gain has been reported with these transgenic animals [11]. In this experiment, wild-type mice have an average lifespan of 761 days, whereas the heterozygous (p66Shcþ/2 ) or homozygous (p66Shc2/2 ) mice have an average lifespan of 815 or 973 days, respectively [11]. Gene profiling revealed that calorie-restricted aging mice have reduced levels of oxidative-stress-inducible genes when compared to ad lib fed counterparts, consistent with the postulation that caloric restriction leads to reduced levels of oxidative stress [3,12]. Calorically restricted aging mice also expressed higher levels of genes involved in metabolic detoxification and DNA repair when compared with normal aged counterparts [3]. This supports the notion that genetic variation that leads to decreased levels of oxidants or increased damage repair capacity might confer an increase in lifespan. Do p66Shc knockout mice share a similar mechanism of lifespan extension with caloric restriction? Although the gene array data have not yet been reported, functional studies of the p66Shc gene provide a positive indication to this question. Similar to calorie-restricted mice, enhanced resistance to oxidative stress in p66Shc null mice is associated with the nature of increased longevity [11]. When exposed to the oxidant-generating compound paraquot, p66Shc2/2 mice had a 40% longer survival time compared to wild-type littermates [11]. In other words, normal mice die within 48 h in response to intraperitoneal injection of paraquot. By contrast, . 50% of p66Shc2/2 mice can survive the same dose of paraquot for 72 h or more. Therefore the transgenic animals are better equipped to handle oxidative stress than their wild-type counterparts. At the cellular level, p66Shc plays a crucial role in the regulation of oxidative stress response and apoptosis. Mouse embryonic fibroblasts (MEFs) derived from p66Shc2/2 rodents were shown to have normal baseline levels of intracellular oxidative stress compared to wild type [13]. However, unlike wild-type MEFs, the p66Shc2/2 cells are resistant to producing reactive oxygen species in response to nutrient deprivation or serum starvation [13]. When subjected to oxidative stress through either H2O2 or UV treatment, p66Shc2/2 cells are reluctant to undergo apoptosis compared to wild-type cells [11]. Expression of a plasmid containing wild-type p66Shc in the null cells was able to return the cells to levels of normal sensitivity [11]. Overexpression of a wild-type p66Shc transgene in normal MEFs resulted in increased sensitivity to apoptosis under the same conditions [11]. These reports support the theory that p66Shc might participate in the regulation of the aging process through regulating oxidant generation and inducing apoptosis.
proteins are present throughout various tissues in human and mouse except the brain or neurons, where the proteins encoded by ShcB or ShcC are expressed [14 –16]. The ShcA gene encodes two mRNA species: p66Shc and p46/p52Shc. The transcriptional start site for p46/p52Shc mRNA is , 3900 base pairs upstream of that for p66Shc mRNA. Because of alternative translation start sites, two proteins, p46Shc and p52Shc, are derived from the same mRNA [17]. Each ShcA protein harbors three identical functional domains: an N-terminal phosphotyrosine-binding domain (PTB), which is slightly truncated in the p46 isoform, a central proline-rich domain (CH1), and a carboxy terminal Src homology 2 (SH2) domain [14]. p66Shc differs from p46 or p52Shc by an additional N-terminal proline-rich domain (CH2) [14]. Although the structural homology seems to indicate a functional overlap between these isoforms, experimental evidence has demonstrated several differences. All three ShcA proteins (p46, p52 and p66) participate in mitogenic signaling and oncogenesis by regulating receptor tyrosine kinase signaling. Whereas the SH2 domain of Shc is important for certain receptor interactions, such as epidermal growth factor (EGF) receptor and ErbB-2, the PTB domain can bind to phospholipids, implying a role of phosphatidylinositol 3-kinase (PI3K) in activation of Shc [18– 20]. Indeed, insulin induces tyrosine phosphorylation of Shc via a PI3K-dependent mechanism involving the PTB domain, whereas the interaction between the SH2 domain and EGF receptor tyrosine kinases plays an important role in phosphorylation of tyrosines in the CH1 domain of Shc [18 –20]. Tyrosine phosphorylation of p46 and p52 enables them to bind to the adaptor Grb2 protein, which then recruits the guanine nucleotide exchange factor SOS, causing activation of Ras and subsequently the mitogen-activated protein kinase (MAPK) cascade [18,19,21]. Grb2 has been known to activate the Ras signaling pathway without interacting with p46 or p52Shc. The participation of p46 or p52Shc is therefore thought to enhance certain weak signals from growth factor receptors or G-protein coupled receptors [18,22]. No evidence yet indicates that p66Shc activates the Ras signaling pathway [21]. Instead, p66Shc competes with p46 or p52Shc for Grb2 binding [19]. This suggests that p66Shc serves as a dominant negative regulator of p46 or p52Shc-mediated Ras signaling. The p66Shc protein contains two serine phosphorylation sites, Ser36 in its CH2 domain and Ser138 in its PTB domain. The first site is unique to p66Shc and is important for oxidative stress response (see next section). The second site is homologous to Ser29 in p52Shc. This shared serine/threonine phosphorylation site is regulated by protein kinase C (PKC) and an upstream MAPK kinase MEK1 [22–25]. Two functional consequences of PTB domain serine phosphorylation have been reported: binding to the sequestering protein 14–3-3 and binding to the protein tyrosine phosphatase PTP-PEST. These interactions might contribute to the role of Shc in regulating apoptosis.
A matter of Shc isoforms Three Shc genes have been found in the mammalian system: ShcA, ShcB (Sli) and ShcCðRaiÞ [14]. ShcA
The missing link between p66Shc and apoptosis Phosphorylation of the unique Ser36 site of p66Shc appears to influence intracellular oxidative stress and apoptosis.
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UV irradiation and oxidants have been shown to induce phosphorylation at Ser36 [13,24]. Insulin presumably also induces phosphorylation of this site [26]. A p66Shc transgene with mutated Ser36 failed to restore apoptosis sensitivity in p66Shc null cells [11], suggesting the importance of Ser36 in regulating apoptosis. An important downstream target of Ser36-phosphorylated p66Shc is Forkhead transcription factors, which belong to a family containing at least 80 members and which have complex functions in regulating apoptosis. Activated p66Shc contributes to phosphorylation of Forkhead mediated by Akt [13,27,28]. Phosphorylation of Forkhead causes the protein to translocate to the cytosol and to become inactive. In lymphocytes, cytokines can activate the Forkead transcription factor FKHR-L1, which contributes to the loss of mitochondrial membrane potential by regulating the expression of the proapoptotic protein Bim and therefore apoptosis [29,30]. In this scenario, inactivation of Forkhead transcription factors downstream of Akt because of growth factor stimulation appears to be a cell survival mechanism [29]. However in other cell types, including neuronal cells and fibroblasts, Forkhead transcription factors have been reported to regulate the expression of several antioxidant enzymes, including superoxide dismutase and catalase [13,31]. Studies by Nemoto and Finkel show that after oxidative stress, FKHR-L1 could preferentially induce antioxidant or survival genes, but this activity is blocked by p66Shc [13]. Consistent with this hypothesis, oxidative stress causes a greater increase in Forkhead activity in cells lacking p66Shc, which are reluctant to undergo apoptosis [13]. The apparent contradictory findings regarding Forkhead transcription factors either enhancing or preventing apoptosis could be related to a cell-type effect or gene-dosing effect. It has been shown that either too much or too little Forkhead protein contributes to cell death [27,32]. Longevity studies of nematodes support the Forkhead gene-dosing hypothesis. In C. elegans, the Forkhead transcription factor family member Daf16 plays a central role in longevity. Daf16 is involved in dauer formation (Daf), a developmental option available to C. elegans larvae when they sense overpopulation, heat stress and/or starvation [33 – 35]. Daf16 is downregulated by the insulinlike receptor homologue Daf2 and its subsequent signaling pathway resembling phosphoinositide 3-kinase and Akt in the mammalian system [35]. Although there is no evidence that a p66Shc-like gene is present in this invertebrate system and regulates Daf16, mutating any number of constituents releasing the suppression of Daf16 leads to increased longevity [35]. Consistent with these results, mutating Daf16 itself causes a moderately shortened lifespan in nematodes whereas overexpressing Daf16 causes a moderate increase in lifespan [36]. In this model, it appears that small increases in Daf16 can slightly increase longevity, whereas too much Daf16 can result in growth arrest [36]. This supports the hypothesis that a moderate increase in the activity of Forkhead transcription factors, such as in the case of p66Shc knockout mice, can contribute to increased longevity. Although the effect of Forkhead transcription factors in aging appear to be conserved throughout evolution from http://tmm.trends.com
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nematodes to mammals, an interesting paradox remains: why did mammals gain p66Shc, a possibly harmful regulatory gene, during evolution? One could argue that p66Shc is necessary as a tumor suppressor, but p66Shc knockout mice were not significantly more prone to neoplasia than wild-type mice [11]. Understanding the evolutionary importance of p66Shc could answer many questions related to the mechanisms of aging among different experimental systems. Methylation of the p66 promoter: fountain of youth? An interesting aspect of the ShcA gene is its differential regulation of expression. Lack of p66Shc expression can contribute to a minor activation of Forkhead transcription factors and therefore increased expression of antioxidant enzymes as described above. The promoter of p66Shc gene contains a relatively high GC content, although not enough to qualify as a CpG island [17]. DNA methylation in CpG islands is well characterized to regulate gene silencing because of stabilization of chromatin structure and inaccessibility for the transcription apparatus. Several cell lines derived from various human tumor tissues have been tested for the promoter methylation status and the expression level of the p66Shc gene [17]. Apparently, cell lines with the least amounts of methylation of the cytosines in the promoter have the highest levels of p66Shc expression, and vice versa. Treatment of highly methylated cell lines with demethylating agents resulted in renewed transcription of p66Shc [17], arguing for the importance of promoter methylation in silencing the p66Shc gene. The finding that p66Shc expression is regulated by its promoter methylation status raises the possibility that p66Shc promoter methylation status could be linked to human aging. The difference in methylation status of the p66Shc promoter among several cell lines indicates the possibility that the same phenomenon can be reproduced in human individuals. If this is true, then perhaps differences in p66Shc promoter methylation status among individuals contribute to the epigenetic basis of variations in longevity. It would be intriguing to determine the methylation status of the p66Shc promoter among various aging populations. Do centenarians have a hypermethylated p66Shc promoter and lower levels of p66Shc proteins in their cells than other human populations? For those who have a hypomethylated p66Shc promoter and more p66Shc protein, are they prone to develop aging-associated diseases at a younger age? Several studies have linked changes in DNA methylation status with aging and aging-related diseases, particularly cancer [37 – 39]. Aged mice and human tissues have been found to have global hypomethylation of DNA [38]. By contrast, at the individual gene level, several genes increase methylation of their promoters during the process of aging [37]. Examples of these genes include those encoding c-fos [40], estrogen receptor a [41], collagen a1(I) [42] and E-cadherin [43]. These lines of evidence support the hypothesis that the methylation status of the p66Shc promoter can change during the process of aging and there is probably a difference in the p66Shc promoter methylation status among individuals.
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Conclusions Is aging a process of wearing and tearing? The answer is probably ‘yes’. However, this wearing and tearing process appears to be influenced by certain genes, some of which can accelerate the process whereas others delay the process. We now know one magic way to extend the lifespan in mice, which is to knock out the p66Shc gene. These mice can tolerate oxidative stress and have reduced levels of oxidants in response to stress. At the cellular level, p66Shc participates in signaling pathways regulating apoptosis. An important function of p66Shc is repression of Forkhead transcription factors, some of which have been shown to regulate the expression of superoxide dismutase and catalase genes. Animal studies and cell culture experiments point to the necessity for human studies to determine the expression level of p66Shc protein and the promoter methylation status of the p66Shc gene among groups with extended longevity. However, if lack of p66Shc expression indeed correlates with increased longevity, it is unimaginable that changing p66Shc promoter methylation status or p66Shc protein expression is a simple task in human beings with current technology, nutritional and pharmacological supplements. Reducing oxidative stress therefore remains a key point in the goal of delaying aging or aging-associated diseases. One issue we have not addressed here is the relationship between p66Shc and insulin signaling pathways in the biology of mammalian aging. A recent report demonstrates that knocking out the insulin receptor in adipose tissue results in healthy lean mice that have normal rates of food intake but live an average of 134 days longer than usual, equivalent to an 18% increase in longevity [44]. Is p66Shc a mediator linking insulin receptor and aging? This is one of many questions that remain unanswered in the study of the biology of aging. Acknowledgements Work from our laboratory has been supported by Burroughs Wellcome New Investigator Award, American Federation for Aging Research, American Heart Association Beginning-Grant-In-Aid, Arizona Disease Control Commission Research Grant, NIH R03 AG17688 and NIH R01 ES10826 (to Q.M.C.).
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featuring The pathway to the cell and its organelles: one hundred years of the Golgi apparatus by M. Bentivoglio and P. Mazzarello Joseph Fourier, the ‘greenhouse effect’ and the quest for a universal theory of terrestrial temperatures by J.R. Fleming The hunt for red elixir: an early collaboration between fellows of the Royal Society by D.R. Dickson Art as science: scientific illustration 1490–1670 in drawing, woodcut and copper plate by C.M. Pyle The history of reductionism versus holistic approaches to scientific research by H. Andersen Reading and writing the Book of Nature: Jan Swammerdam (1637–1680) by M. Cobb Coming to terms with ambiguity in science: wave–particle duality by B.K. Stepansky The role of museums in history of science, technology and medicine by L. Taub The ‘internal clocks’ of circadian and interval timing by S. Hinton and W.H. Meck The troubled past and uncertain future of group selectionism by T. Shanahan A botanist for a continent: Ferdinand Von Mueller (1825–1896) by R.W. Home Rudolf Virchow and the scientific approach to medicine by L. Benaroyo Darwinism and atheism: different sides of the same coin? by M. Ruse Alfred Russel Wallace and the flat earth controversy by C. Garwood John Dalton: the world’s first stereochemist by Dennis H. Rouvray Forensic chemistry in 19th-century Britain by N.G. Coley Owen and Huxley: unfinished business by C.U.M. Smith Characteristics of scientific revolutions by H. Andersen and much, much more . . . Locate Endeavour in the BioMedNet Reviews collection. Log on to http://reviews.bmn.com, hit the ‘Browse Journals’ tab and scroll down to Endeavour http://tmm.trends.com