Gene mutations in invertebrates have been identified that extend life span and enhance resistance to environmental stresses such as ultraviolet light or reactive oxygen species. In mammals, the mechanisms that regulate stress response are poorly understood and no genes are known to increase individual life span. Here we report that targeted mutation of the mouse p66shc gene induces stress resistance and prolongs life span. p66shc is a splice variant of p52shc/p46shc (ref. 2), a cytoplasmic signal transducer involved in the transmission of mitogenic signals from activated receptors to Ras. We show that: (1) p66shc is serine phosphorylated upon treatment with hydrogen peroxide (H2O2) or irradiation with ultraviolet light; (2) ablation of p66shc enhances cellular resistance to apoptosis induced by H2O2 or ultraviolet light; (3) a serine-phosphorylation defective mutant of p66shc cannot restore the normal stress response in p66shc-/- cells; (4) the p53 and p21 stress response is impaired in p66shc-/- cells; (5) p66shc-/- mice have increased resistance to paraquat and a 30% increase in life span. We propose that p66shc is part of a signal transduction pathway that regulates stress apoptotic responses and life span in mammals.

The reactive oxygen species that are generated by mitochondrial respiration, including hydrogen peroxide (H2O2), are potent inducers of oxidative damage and mediators of ageing. It is not clear, however, whether oxidative stress is the result of a genetic programme or the by-product of physiological processes. Recent findings demonstrate that a fraction of mitochondrial H2O2, produced by a specialized enzyme as a signalling molecule in the pathway of apoptosis, induces intracellular oxidative stress and accelerates ageing. We propose that genes that control H2O2 production are selected determinants of lifespan.

Reactive oxygen species (ROS) are potent inducers of oxidative damage and have been implicated in the regulation of specific cellular functions, including apoptosis. Mitochondrial ROS increase markedly after proapoptotic signals, though the biological significance and the underlying molecular mechanisms remain undetermined. P66Shc is a genetic determinant of life span in mammals, which regulates ROS metabolism and apoptosis. We report here that p66Shc is a redox enzyme that generates mitochondrial ROS (hydrogen peroxide) as signaling molecules for apoptosis. For this function, p66Shc utilizes reducing equivalents of the mitochondrial electron transfer chain through the oxidation of cytochrome c. Redox-defective mutants of p66Shc are unable to induce mitochondrial ROS generation and swelling in vitro or to mediate mitochondrial apoptosis in vivo. These data demonstrate the existence of alternative redox reactions of the mitochondrial electron transfer chain, which evolved to generate proapoptotic ROS in response to specific stress signals.

The 66-kilodalton isoform of the growth factor adapter Shc (p66Shc) translates oxidative damage into cell death by acting as reactive oxygen species (ROS) producer within mitochondria. However, the signaling link between cellular stress and mitochondrial proapoptotic activity of p66Shc was not known. We demonstrate that protein kinase C beta, activated by oxidative conditions in the cell, induces phosphorylation of p66Shc and triggers mitochondrial accumulation of the protein after it is recognized by the prolyl isomerase Pin1. Once imported, p66Shc causes alterations of mitochondrial Ca2+ responses and three-dimensional structure, thus inducing apoptosis. These data identify a signaling route that activates an apoptotic inducer shortening the life span and could be a potential target of pharmacological approaches to inhibit aging.

Shc proteins are targets of activated tyrosine kinases and are implicated in the transmission of activation signals to Ras. The p46shc and p52shc isoforms share a C‐terminal SH2 domain, a proline‐ and glycine‐rich region (collagen homologous region 1; CH1) and a N‐terminal PTB domain. We have isolated cDNAs encoding for a third Shc isoform, p66shc. The predicted amino acid sequence of p66shc overlaps that of p52shc and contains a unique N‐terminal region which is also rich in glycines and prolines (CH2). p52shc/p46shc is found in every cell type with invariant reciprocal relationship, whereas p66shc expression varies from cell type to cell type. p66shc differs from p52shc/p46shc in its inability to transform mouse fibroblasts in vitro. Like p52shc/p46shc, p66shc is tyrosine‐phosphorylated upon epidermal growth factor (EGF) stimulation, binds to activated EGF receptors (EGFRs) and forms stable complexes with Grb2. However, unlike p52shc/p46shc it does not increase EGF activation of MAP kinases, but inhibits fos promoter activation. The isolated CH2 domain retains the inhibitory effect of p66shc on the fos promoter. p52shc/p46shc and p66shc, therefore, appear to exert different effects on the EGFR‐MAP kinase and other signalling pathways that control fos promoter activity. Regulation of p66shc expression might, therefore, influence the cellular response to growth factors.

Correlative evidence links stress, accumulation of oxidative cellular damage and ageing in lower organisms and in mammals. We investigated their mechanistic connections in p66Shc knockout mice, which are characterized by increased resistance to oxidative stress and extended life span. We report that p66Shc acts as a downstream target of the tumour suppressor p53 and is indispensable for the ability of stress-activated p53 to induce elevation of intracellular oxidants, cytochrome c release and apoptosis. Other functions of p53 are not in¯uenced by p66Shc expression. In basal conditions, p66Shc7/7 and p537/7 cells have reduced amounts of intracellular oxidants and oxidation-damaged DNA. We propose that steady-state levels of intracellular oxidants and oxidative damage are genetically determined and regulated by a stress-induced signal transduction pathway involving p53 and p66Shc.

P66Shc regulates life span in mammals and is a critical component of the apoptotic response to oxidative stress. It functions as a downstream target of the tumor suppressor p53 and is indispensable for the ability of oxidative stress-activated p53 to induce apoptosis. The molecular mechanisms underlying the apoptogenic effect of p66Shc are unknown. Here we report the following three findings. (i) The apoptosome can be properly activated in vitro in the absence of p66Shc only if purified cytochrome c is supplied. (ii) Cytochrome c release after oxidative signals is impaired in the absence of p66Shc. (iii) p66Shc induces the collapse of the mitochondrial trans-membrane potential after oxidative stress. Furthermore, we showed that a fraction of cytosolic p66Shc localizes within mitochondria where it forms a complex with mitochondrial Hsp70. Treatment of cells with ultraviolet radiation induced the dissociation of this complex and the release of monomeric p66Shc. We propose that p66Shc regulates the mitochondrial pathway of apoptosis by inducing mitochondrial damage after dissociation from an inhibitory protein complex. Genetic and biochemical evidence suggests that mitochondria regulate life span through their effects on the energetic metabolism (mitochondrial theory of aging). Our data suggest that mitochondrial regulation of apoptosis might also contribute to life span determination.

Background-Enhanced production of reactive oxygen species (ROS) has been recognized as the major determinant of age-related endothelial dysfunction. The p66 shc protein controls cellular responses to oxidative stress. Mice lacking p66 shc (p66 shcϪ/Ϫ ) have increased resistance to ROS and a 30% prolonged life span. The present study investigates age-dependent changes of endothelial function in this model. Methods and Results-Aortic rings from young and old p66shcϪ/Ϫ or wild-type (WT) mice were suspended for isometric tension recording. Nitric oxide (NO) release was measured by a porphyrinic microsensor. Expression of endothelial NO synthase (eNOS), inducible NOS (iNOS), superoxide dismutase, and nitrotyrosine-containing proteins was assessed by Western blotting. Nitrotyrosine residues were also identified by immunohistochemistry. Superoxide (O 2 Ϫ ) production was determined by coelenterazine-enhanced chemiluminescence. Endothelium-dependent relaxation in response to acetylcholine was age-dependently impaired in WT mice but not in p66shcϪ/Ϫ mice. Accordingly, an age-related decline of NO release was found in WT but not in p66shcϪ/Ϫ mice. The expression of eNOS and manganese superoxide dismutase was not affected by aging either in WT or in p66shcϪ/Ϫ mice, whereas iNOS was upregulated only in old WT mice. It is interesting that old WT mice displayed a significant increase of O 2 Ϫ production as well as of nitrotyrosine expression compared with young animals. Such age-dependent changes were not found in p66shcϪ/Ϫ mice. Conclusions-We report that inactivation of the p66shc gene protects against age-dependent, ROS-mediated endothelial dysfunction. These findings suggest that the p66 shc is part of a signal transduction pathway also relevant to endothelial integrity and may represent a novel target to prevent vascular aging. Key Words: aging Ⅲ endothelium Ⅲ free radicals Ⅲ nitric oxide Ⅲ genes S hc proteins are adaptor proteins that exist in 3 different isoforms with relative molecular masses of 46, 52, and 66 kDa. P52shc /p46 shc is involved in the transmission of mitogenic signals from tyrosine kinases to Ras. 1 p66 shc has the same modular structure of p52 shc /p46 shc (SH2-CH1-PTB) and contains a unique N-terminal region (CH2); however, it is not involved in Ras regulation but rather functions in the intracellular pathway that converts oxidative signals into apoptosis. Indeed, embryo fibroblasts from mice carrying a targeted mutation of p66 shc (p66 shcϪ/Ϫ ) are more resistant to oxidative stress-induced apoptosis. 2 p66 shcϪ/Ϫ mice have an approximately 30% increase in life span and reduced early atherogenesis after long-term consumption of a high-fat diet, 3 suggesting that p66shc is implicated in aging and in the pathogenesis of aging-associated diseases in mammals. The biochemical function of p66 shc remains, however, largely unknown. Recent reports demonstrated that p66 shc acts as a downstream target of the tumor suppressor p53 and is indispensable to the ability of activated p53 to induce elevation of intra...