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Loss in oxidative stress tolerance with aging linked to reduced extracellular signal-regulated kinase and Akt kinase activities ¹

Laboratory of Cellular and Molecular Biology, National Institute on Aging-IRP, Baltimore, Maryland 21224, USA

²Correspondence: Yale University School of Medicine, Section of Geriatrics, 333 Cedar St., PO Box 309, New Haven, CT 06520, USA. E-mail: holbrook{at}yale.edu

SPECIFIC AIMS

This study investigated the hypothesis that mammalian aging is associated with a reduced ability to activate prosurvival signaling pathways in response to oxidative stress, and that this in turn leads to increased vulnerability of aged cells to oxidant injury. The activities of two prosurvival kinases, extracellular signal-regulated kinase (ERK) and Akt, were evaluated and cell survival was assessed after hydrogen peroxide (H₂O₂) treatment of primary rat hepatocytes derived from young and aged rats fed either a standard ad libitum diet or subjected to caloric restriction (which is known to delay the onset of many aging effects).

PRINCIPAL FINDINGS

1. Aging is associated with increased sensitivity to toxic effects of H₂O₂
To determine the effect of donor age on susceptibility of primary rat hepatocytes to toxic effects of H₂O₂, cells derived from young (4–6 months), middle-aged (12–14 months), and old (24–26 months) male Fischer 344 rats were exposed to various concentrations of the oxidant and 24 h later assessed for cell viability using trypan blue dye exclusion. Although no cytotoxic effects were evident with H₂O₂ concentrations at or below 100 µM, higher concentrations (300–1000 µM) led to cell death in a dose-dependent manner. Middle age hepatocytes did not differ from young hepatocytes in their susceptibility to H₂O₂ treatment, but old hepatocytes were much more sensitive to the oxidant treatment (representative comparisons can be seen in Fig. 1 B and Fig. 2 B). H₂O₂-treated cells exhibited morphological features of apoptosis and DNA fragmentation visualized electrophoretically as DNA laddering.

View larger version (38K): [in this window] [in a new window]   Figure 1. Effect of ERK and Akt pathway inhibitors on H2O2-induced cell death. Primary hepatocytes from young and old rats were incubated with 20 µM PD98059 or 50 µM LY294002 for 45 min before addition of H2O2. A) Cells were lysed 60 min after H2O2 treatment, and ERK and Akt phosphorylation was determined by Western blot analysis. B) Upper panel: 24 h after treatment with 600 µM H2O2, cell viability was assessed by trypan blue dye exclusion. Values shown are the means ± SD for three independent experiments. *Significant differences between cells treated with either H2O2 alone or H2O2 plus one or more inhibitors (P<0.05); **Significant differences between survival of cells receiving combined treatment with PD98059 and LY294002 relative to those treated with either inhibitor alone. Lower panel: DNA fragmentation was assessed 24 h after treatment with 600 µM H2O2 in the presence or absence of PD98059 and/or LY294002.

View larger version (42K): [in this window] [in a new window]   Figure 2. Effect of caloric restriction on kinase activation and survival of H2O2-treated hepatocytes. Hepatocytes derived from young and old ad libitum (AL) -fed and calorie-restricted (CR) rats were compared for responsiveness to 600 µM H2O2 treatment. A) ERK and Akt phosphorylations were monitored by Western blot analysis using the respective phospho-specific antibodies. ERK was examined 60 min after treatment; Akt, 30 min post-treatment. B) Cell survival was determined 24 h after H2O2 treatment using trypan blue dye exclusion to quantitate viable cells. For statistical analysis, all treatment groups were compared with young AL rats. *Significant difference (P<0.05) comparing young and old AL rats. Note that old CR do not differ significantly from young AL rats.

2. Aging is associated with decreased activation of ERK and Akt
ERK and Akt can be activated in response to oxidative stress and, in certain situations, can play an important role in promoting cell survival. To investigate the possibility that differential activation of ERK and Akt might account for age-related differences in the tolerance of hepatocytes to oxidative stress, we examined their phosphorylation in young and old cells after treatment with H₂O₂. ERK phosphorylation occurred in a time- and dose-dependent fashion, maximum activation being achieved within 30 min after treatment with 300–600 µM H₂O₂. However, the magnitude of activation was significantly lower (<threefold less) in old vs. young cells. Akt also underwent time- and concentration-dependent phosphorylation in response to H₂O₂ treatment, with maximum activation in young cells occurring 30 min after treatment with 600 µM H₂O₂. As seen for ERK, this response was significantly attenuated in old cells. Representative experiments showing differences in ERK and Akt activation in young and old cells are shown in Fig. 1A and Fig. 2A .

3. Pharmacologic inhibitors of ERK and Akt activation enhance the sensitivity of hepatocytes to H₂O₂
To determine whether activations of ERK and Akt are important for survival of H₂O₂-treated hepatocytes, specific pharmacologic inhibitors of these pathways were used. Pretreatment of cells with 20 µM PD98059 (a selective inhibitor of MEK1/2, the upstream kinase responsible for ERK phosphorylation) completely inhibited ERK activation by H₂O₂ (Fig. 1A , upper panel) and significantly increased cell death (Fig. 1B ). Likewise, treatment of cells with the PI3-kinase inhibitor LY294002 blocked Akt activation (Fig. 1A , lower panel) and sensitized cells to killing by the oxidant (Fig. 1B ). The consequences of inhibiting these kinases were more profound in young cells relative to old cells. The combined effectiveness of these ERK and Akt pathway inhibitors was greater than for either inhibitor alone. The effect of inhibiting ERK and Akt activation on survival of H₂O₂-treated cells was also evident by DNA fragmentation analysis, where the extent of DNA laddering was enhanced in the presence of the inhibitors (Fig. 1B , lower panel).

4. Caloric restriction prevents the age-related decline in ERK and Akt activation in response to oxidant injury and improves cell survival of H₂O₂-treated cells
A 30–40% reduction in caloric intake (caloric restriction) can increase longevity in many animal species and retards the accumulation of oxidative damage in mammalian cells. Therefore, we investigated whether caloric restriction would prevent the age-related loss in responsiveness of hepatocytes to oxidative stress. Caloric restriction did not alter the response to H₂O₂ treatment in young cells. However, compared with their ad libitum-fed counterparts, hepatocytes derived from old long-term calorie-restricted rats exhibited higher activation of ERK and Akt (with levels essentially the same as those of young animals) and showed increased survival after treatment with H₂O₂ (Fig. 2 A , B , respectively).

CONCLUSIONS AND SIGNIFICANCE

For cells living in an aerobic environment, exposure to ROS is continuous and unavoidable. Oxidative stress is an important factor in the development of many age-associated degenerative diseases and is believed to contribute to the general decline in physiological function that accompanies normal aging. In mammalian cells, acute oxidant injury triggers the activation of multiple signaling pathways, some of which play an important role in protecting cells against deleterious effects of ROS and promoting survival. We have hypothesized that aging is also associated with a reduced ability to mount these host defenses to oxidant injury, and that this in turn leads to increased vulnerability of aged cells to the insult. Until now, direct experimental support for the above hypothesis has been lacking. The findings described here with hepatocytes derived from young, aged, and calorie-restricted rats provide strong evidence in favor of our hypothesis. We have shown that compared with young cells, old cells do indeed show diminished activation of two prosurvival kinases, ERK and Akt, in response to H₂O₂ treatment, and this is correlated with greater sensitivity to oxidant exposure. That the diminished activities of these prosurvival pathways contribute to the aged cell’s reduced tolerance to oxidative stress was supported by additional experiments in which we modulated their activities in young and aged cells through pharmacologic manipulation or caloric restriction. Inhibition of ERK and Akt activation in young cells rendered them more sensitive to H₂O₂ whereas enhancement of these activities in aged cells reduced their sensitivity to the oxidant and improved their survival. The findings are summarized in Fig. 3 .

View larger version (19K): [in this window] [in a new window]   Figure 3. Schematic diagram illustrating differential activation of ERK and Akt by oxidants in young vs. aged cells, its consequences for survival and modulation of these effects through suppression (via specific pathway inhibitors) or enhancement (calorie restriction) of kinase activities.

The significance of our findings is severalfold. First, although there has been much anecdotal support for the notion that mammalian aging is associated with a reduced ability to cope with environmental stresses, experimental evidence linking the decline in stress tolerance to alterations in specific pathways known to be involved in regulating stress responsiveness at the cellular level has been lacking. Our studies provide such evidence for two important stress response pathways, those leading to activation of ERK and Akt. Second, our studies suggest that regardless of the long-term accumulation of oxidative damage during aging, strategies aimed at boosting these stress response pathways could provide direct benefits to the aged cell and confer greater tolerance to acute oxidative insults. Finally, our studies point to a link between reduced stress tolerance and reduced proliferative capacity, another hallmark of aging. Although ERK and Akt promote survival during oxidant injury, they are best known for their roles in regulating cell proliferation in response to growth factors such as epidermal growth factor (EGF). DNA synthesis after EGF stimulation is also markedly attenuated in aged hepatocytes, and we have demonstrated that this is associated with reduced activation of ERK. As we and others have demonstrated a central role for growth factor receptor signaling pathways in mediating activation of ERK and Akt by oxidants, it is likely that common or overlapping mechanisms contribute to the age-related reductions in proliferative capacity and stress tolerance. Better understanding of these processes could lead to strategies to prevent or delay the onset of age-related declines in proliferative capacity and our ability to withstand acute environmental insults.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0409fje; to cite this article, use FASEB J. (November 14, 2001) 10.1096/fj.01-0409fje

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