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Effects of aging and calorie restriction of Fischer 344 rats on hepatocellular response to proliferative signals
Experimental Gerontology 38 (2003) 431–439 www.elsevier.com/locate/expgero
Effects of aging and calorie restriction of Fischer 344 rats on hepatocellular response to proliferative signals Shizuo Ikeyamaa,*, Gertrude Kokkonena, Jennifer L. Martindalea, Xian-Tao Wanga, Myriam Gorospea, Nikki J. Holbrooka,b a
Cell Stress and Aging Section, Laboratory of Cellular and Molecular Biology, National Institute on Aging, Baltimore, MD 21224, USA b Yale University School of Medicine, Section of Geriatrics, New Haven, CT 06520, USA Received 8 August 2002; received in revised form 8 October 2002; accepted 30 October 2002
Abstract It is well established that the proliferative potential of the liver declines with aging. Epidermal growth factor (EGF)-stimulated DNA synthesis is reduced in hepatocytes from aged rats relative to young rats, and this reduction correlates with diminished activation of the extracellular signal-regulated kinase (ERK) pathway and lower phosphorylation of the EGF receptor on residue Y1173. Calorie restriction (CR) can increase rodent life span and retard many age-associated declines in physiologic function, but its influence on cell proliferation is unknown. Here, we investigated the effects of long-term CR on proliferation of hepatocytes derived from young and aged rats following in vitro stimulation with either low-dose hydrogen peroxide or EGF. CR reduced the proliferative response of hepatocytes derived from young hosts, but long-term CR was associated with enhanced proliferation in aged cells relative to that of ad libitum (AL)-fed animals. ERK activation mirrored the effects of CR on proliferation, in that young CR cells exhibited lower ERK activation than young AL cells, but old CR cells showed higher ERK activation than old AL cells. Finally, a decline in EGF receptor phosphorylation on Y1173, which normally occurs with aging, was absent in cells of old hosts maintained on long-term CR, supporting the view that alterations in this early signaling event underlie the age-related decline in proliferative potential in rat hepatocytes. q 2002 Elsevier Science Inc. All rights reserved. Keywords: Hepatocytes; Aging; Oxidative stress; Proliferation; Epidermal growth factor
1. Introduction A decline in proliferative capacity constitutes an important hallmark of mammalian aging in many cell types (Hornsby, 2001). The liver, and hepatocytes in particular, have proven to be a particularly good model to study the mechanisms associated with this phenomenon. Liver regeneration following partial hepatectomy is impaired in aged animals as evidenced by a delay and reduction in magnitude of DNA synthesis (Moolten and Bucher, 1967; Tsukamoto et al., 1993; Wang et al., 2001).
* Corresponding author. Address: Laboratory of Cellular and Molecular Biology, Gerontology Research Center, Box 12, National Institute on Aging-Intramural Research Program, National Institutes of Health, 5600 Nathan Shock Drive, Baltimore, MD 21224, USA. Tel.: þ 1-410-558-8484; fax: þ 1-410-558-8386. E-mail address: [email protected] (S. Ikeyama).
Likewise, epidermal growth factor (EGF)-stimulated DNA synthesis is markedly lower in primary hepatocytes derived from aged donors compared to similarly treated cells of young donors (Ishigami et al., 1993). We and others have demonstrated that this reflects a decline in the activation of extracellular signal-regulated kinase (ERK), which plays a critical role in transmitting proliferative signals to the nucleus to effect changes in gene expression necessary for cell growth (Liu et al., 1996; Palmer et al., 1999; Hu et al., 1998). The cellular events leading to activation of ERK in response to EGF have been studied extensively (Cobb et al., 1994; Roovers and Assoian, 2000). In brief, binding of EGF to its receptor (EGFR) results in autophosphorylation and activation of the receptor, leading to the recruitment of adaptor and GDP/GTP exchange proteins necessary for activation of membrane-localized Ras. Activated Ras then initiates the phosphorylation cascade resulting in sequential activation of Raf, MEK, and ERK.
0531-5565/03/$ - see front matter q 2002 Elsevier Science Inc. All rights reserved. PII: S 0 5 3 1 - 5 5 6 5 ( 0 2 ) 0 0 2 3 9 - 5
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The defect resulting in reduced ERK activation in aged cells is believed to lie at the level of the EGFR. Neither EGFR number nor affinity of the receptor for its ligand are altered with age (Ishigami et al., 1993). Likewise, total receptor phosphorylation is unchanged with aging (Palmer et al., 1999; Hutter et al., 2000). However, in old cells the EGFR shows reduced ability to interact with the Shc adaptor protein, and this is associated with reduced phosphorylation of a specific EGFR amino acid residue, Tyrosine 1173, that lies within the Shc binding domain (Palmer et al., 1999; Hutter et al., 2000). Like growth factors, oxidants can activate the EGFR in a dose-dependent manner, initiating the signaling cascade leading to subsequent activation of ERK (Knebel et al., 1996; Zanella et al., 1996). Indeed, low concentrations of H2O2 have been shown to be mitogenic for many cell types (Burdon, 1995). At higher concentrations, however, H2O2 becomes toxic and is associated with either inhibition of growth or induction of cell death. In such instances, activation of ERK has been shown to favor cell survival (Guyton et al., 1996; Aikawa et al., 1997; Ikeyama et al., 2001). As in the case of EGF stimulation, aged hepatocytes show reduced activation of ERK in response to toxic concentrations of H2O2, and this is correlated with reduced survival (Ikeyama et al., 2001). Calorie restriction (CR), or the limiting of food intake without deprivation of essential nutrients, can extend life span in a wide variety of species (Masoro, 2001; Hart and Turturro, 1998; Masoro, 1998). In both rats and mice, CR has been shown to prevent or slow the progression of many age-related diseases, and attenuate declines in physiologic function that occur with normal aging (Van Remmen et al., 2001; Meydani, 2001). We have demonstrated that CR can prevent the age-related decline in ERK activation by toxic concentrations of H2O2 (300 – 1000 mM), and have likewise shown that this correlates with improved survival of aged rat hepatocytes (Ikeyama et al., 2001). Studies examining the influence of CR on hepatocyte proliferation with aging have produced variable findings ranging from inhibition to enhancement (Higami et al., 1997; Wolf et al., 1995; Shaddock et al., 1996; Taguchi et al., 2001; Himeno et al., 1992; Cuenca et al., 2001; Rozga, 2002). Therefore, the present study sought to examine the impact of CR on the proliferative capacity of young versus aged hepatocytes using either EGF or low concentrations of H2O2 as proliferative stimuli. We demonstrate that CR alone causes a modest, but significant diminution in the mitogenic response to both EGF and H2O2 in hepatocytes from young rats. However, aged hepatocytes from animals maintained on CR display enhanced proliferative capacity relative to their ad libitum (AL)-fed counterparts. The effect of CR on proliferation is paralleled by similar changes in ERK activity and site-specific phosphorylation of the EGFR.
2. Materials and methods 2.1. Animals Male Fischer 344 rats, 4 –6 (young) and 24– 26 (old) months of age, were obtained from the National Institute on Aging Contract Colonies at Harlan Sprague Dawley. They were maintained on a 12:12 h light:dark cycle in a controlled environment with water supplied at all times. Ad libitum-fed (AL) animals were provided with unlimited quantities of a regular NIH-31 pelleted diet. For the CR group, CR was initiated at 14 weeks of age, with a 10% reduction in calorie intake. The restriction was increased to 25% at 15 weeks, to a maximum of 40% at 16 weeks. They were maintained on the CR diet, equal to 60% the average daily calorie intake of AL animals, for the remainder of life. These treatment conditions were approved by the Gerontology Research Center Animal Use and Care Committee (Protocol #NJH-058-Ra/Mi) and are in accordance with the National Institutes of Health guidelines for the care and use of laboratory animals. 2.2. Primary hepatocyte culture and treatment Hepatocytes were isolated from male Fischer 344 rats by the collagenase perfusion method of Seglen as described (Seglen, 1976). The isolated cells were seeded onto Biocoat Collagen I cellware (BD Discovery Labware, Bedford, MA) in William’s E medium supplemented with 5% fetal bovine serum, 1 mM dexamethasone, 10 mg/L insulin, 2 mM L glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin for 2 h in 5% CO2 at 37 8C to allow attachment to the dishes. The medium was then replaced with serum-free William’s E medium plus the above supplements, and cells were cultured for an additional 16 h prior to treatment. This procedure results in less than 5% contamination with nonhepatocyte cells. Cultured hepatocytes were treated with various concentrations of H2O2 or 100 ng/ml of EGF (Invitrogen, Carlsbad, CA) for the indicated times. H2O2 was prepared freshly from a concentrated stock solution. 2.3. Immunoprecipitation and western blot analysis For western blot analysis, total cell lysates were prepared using a lysis buffer containing Hepes (20 mM, pH 7.4), EGTA (2 mM), b-glycerophosphate (50 mM), Na3VO4 (1 mM), NaF (5 mM), 1% (v/v) Triton X-100, 10% (v/v) glycerol, dithiothreitol (DTT) (1 mM), PMSF (1 mM), leupeptin (10 mg/ml), aprotinin (10 mg/ml). Cells were centrifuged at 4 8C, 15,000 rpm, for 20 min. Protein (50 – 150 mg) was mixed with 2 £ sample buffer containing 130 mM Tris-Cl, pH 8.0, 20% (v/v) Glycerol, 4.6% (w/v) SDS, 0.02% Bromophenol blue, 2% DTT, and size fractionated by electrophoresis through Tris-Glycine Gel (Invitrogen, Carlsbad, CA). Size-separated proteins were then transferred onto polyvinylidene difluoride membranes
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(Millipore, Bedford, MA) and immunoblot analysis was carried out using the appropriate antibodies. Specific proteins were detected with the enhanced chemiluminescence (ECL) system (Amersham Pharmacia Biotech). Some blots were subjected to multiple probings with different antibodies. In such instances, the blots were stripped between probings using a buffer containing 50 mM TrisHCl, pH 6.8, 2% SDS, and 0.1 M of b-mercaptoethanol. All blots were normalized to total ERK2 or b-actin (Abcam Ltd UK) levels. For immunoprecipitation analysis, cells were treated, then total cell lysates were prepared using lysis buffer, and 500 mg protein incubated with 5 mg of anti-EGFR antibody and 40 ml of protein A-Sepharose for 4 h at 4 8C. Immune complexes were washed four times with lysis buffer and resuspended in 2 £ sample buffer. Immunoblot analysis was carried out using the appropriate antibodies. Specific proteins were detected by western blot analysis. AntiERK2 and EGF-polyclonal antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). PD98059 was purchased from Cell Signaling Technology (Beverly, MA). Anti-phospho-MEK1/2 and the anti-phospho ERK1/2 antibodies, which recognized phosphorylated/active forms of the respective proteins, were from Cell Signaling Technology (Beverly, MA) and Promega (Madison, WI). The anti-phospho-EGF Receptor antibody, specific for residue Y1173, was obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). 2.4. Northern blot analysis For RNA analysis, cells were harvested at various time points following treatment, and total RNA extracted using STAT-60 (Tel-Test B, Friendswood, Tex.) according to the manufacturer’s recommendations. Twenty mg RNA was run on agarose-formaldehyde gels and transferred to a GeneScreen Plus membrane (NEN Life Science Products). Northern blot analysis was performed using either cDNA probes specific for rat c-jun, and MKP-1 or an oligonucleotide complimentary to 18S RNA. 2.5. Assessment of DNA synthesis by monitoring [3H] thymidine and BrdU incorporation [Methyl-3H] thymidine (85 Ci/mmol) was obtained from Amersham (Arlington Heights, IL) and its incorporation into DNA was investigated as follows. Hepatocytes were plated at a density of 0.4 £ 105 cells/well in 12-well plates pre-coated with type I collagen. Twenty-four to 72 h after treatment with either EGF or H2O2, cells were incubated with 10 mCi [3H] thymidine per ml for 2 h, after which they were washed twice with cold-PBS, immersed in 1 ml of cold 10% trichloroacetic acid (TCA) for 10 min and solubilized by incubation in 0.5 ml of 1 N NaOH at 37 8C for 30 min. TCA was added to achieve a final concentration of 15% TCA, and macromolecules were allowed to precipitate
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overnight. After centrifugation, the precipitate was washed with 10% TCA and hydrolyzed in 0.5 ml of 10% TCA at 90 8C for 15 min. Radioactivity in the hot TCA-soluble fraction was measured by liquid scintillation counting. Data was shown as cpm incorporated per mg of protein [measured by the Bradford method (Bio-Rad, Hercules, CA.)] in whole-cell lysates. A cell proliferation ELISA kit (Roche Molecular Biochemicals, IN) that measures BrdU incorporation was also used to monitor DNA synthesis. Briefly, 0.4 £ 105 cells/well in 12-well plates pre-coated with type I collagen were labeled with BrdU for 2 h at 37 8C. After its incorporation into DNA, BrdU was detected by immunoassay. 2.6. Statistical analysis Statistical analysis was performed using a one-way ANOVA. Differences between individual age or treatment groups were evaluated using the unpaired two-tailed Student’s t test.
3. Results 3.1. Effect of aging on proliferation and ERK activity in H2O2 –stimulated hepatocytes Low concentrations of H2O2 have been shown to be mitogenic for many cell types. To determine if this is true for primary rat hepatocytes, and to examine whether host donor age affects the response, hepatocytes derived from young (4 – 6-mo old) and old (24 –26-mo old) male Fischer 344 rats were exposed to varying amounts of H2O2 and 24 h later evaluated for the incorporation of [3H] thymidine. Cells derived from young donors showed a marked elevation in [3H]-thymidine incorporation (. four-fold) in response to both 50 and 100 mM H2O2, but cells from aged donors exhibited a much attenuated response to the mitogen (Fig. 1A). Only at 50 mM H2O2 was a significant increase in [3H]-thymidine incorporation seen in old cells. Consistent with our previous studies, higher concentrations of H2O2 were found to be toxic for the hepatocytes, resulting in [3H] thymidine incorporation below that of control cultures. This toxic effect was more evident in old cells relative to young cells. The mitogenic effects of 50 and 100 mM H2O2, as well as the differential responsiveness of young and old hepatocytes were confirmed using BrdU incorporation (Fig. 1B). Relative amounts of phosphorylated ERK1/2 in young and old hepatocytes treated with H2O2 were examined by western blot analysis as an assessment of kinase activation. In keeping with the known role of ERK in regulating proliferation, ERK1/2 was found to undergo significant activation in response to both 50 and 100 mM H2O2 in young hepatocytes, but this effect was much reduced in aged hepatocytes (Fig. 2). ERK activation was sustained with
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Fig. 1. Age-associated decline in proliferative response of primary cultured rat hepatocytes to H2O2 treatment. Primary hepatocytes derived from young adult (4–6 mo) or aged (24–26 mo) hosts were treated with the indicated concentrations of H2O2 and DNA synthesis was assessed by either [3H] thymidine incorporation (A) or BrdU incorporation (B) 24 h after treatment. Results shown are the means ^ S.E. of three independent experiments. *p , 0.05 comparing H2O2-treated cells with vehicle controls. **p , 0.05 comparing response of young cells to old cells.
higher concentrations of the oxidant in young cells, but underwent further attenuation in old cells. These findings are consistent with our previous studies in which we demonstrated that ERK activation at concentrations between 300 –1000 mM were important for cell survival (Ikeyama et al., 2001). That the activation of ERK by H2O2 is important for the proliferative response is evidenced by the data shown in Fig. 3. Treatment of young hepatocytes with PD98059, which acts to inhibit the activity of the ERK phosphorylating kinase MEK and thereby completely prevents ERK activation (Fig. 3A), effectively blocked BrdU incorporation in response to H2O2 (Fig. 3B). 3.2. Effects of CR on hepatocyte response to EGF and H2O2 CR has been shown to extend lifespan in rats, and prevent the age-related decline in many physiologic functions
Fig. 2. H2O2-triggered ERK activation in hepatocytes from young and aged rats. (A) Representative western blot showing relative amounts of phosphorylated (active) ERK (pERK1/pERK2) in cultures 30 min following treatment with various H2O2 concentrations. Note that total ERK2 protein levels do not differ between age or treatment groups. (B) Quantification of results obtained from five independent experiments comparing amounts of phosphorylated ERK1/2 in H2O2-stimulated cells from young versus aged hosts relative to levels in untreated young cells. Shown are the means ^ S.E. of five independent experiments. *p , 0.05 comparing H2O2-treatment with untreated controls. **p , 0.05 comparing values for young and old cells.
(Masoro, 2001; Hart and Turturro, 1998; Masoro, 1998; Van Remmen et al., 2001; Meydani, 2001). To determine the impact of CR on proliferative capacity of hepatocytes, we compared the responsiveness of hepatocytes derived from young and old rats maintained on either AL or CR diets to treatment with either 50 or 100 mM H2O2 or 100 ng/ ml EGF. As shown in Fig. 4, BrdU incorporation was higher with EGF treatment than with H2O2. CR itself resulted in a reduced response to the proliferative signals, as evidenced by the lower BrdU incorporation in cells derived from young CR rats relative to young AL animals. Interestingly, BrdU incorporation did not differ significantly between cells of young CR and old CR animals, but both were markedly different (higher) from that seen in cells from old AL rats. Examination of ERK activation in these same animals produced results that mirrored those for BrdU incorporation (Fig. 5). Cells from young CR animals showed lower ERK activation compared to cells of young AL animals, but young and old CR cells did not differ in levels of ERK activation, both showing significantly higher levels than seen in old AL cells.
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Fig. 4. Effect of CR on proliferation of young and aged hepatocytes treated with EGF or H2O2. Hepatocytes derived from young and old AL-fed and CR rats were treated with either EGF or H2O2. DNA synthesis was determined based on BrdU incorporation as described for Fig. 1. Results shown are the means ^ S.E. of three independent experiments. *p , 0.05 comparing young AL and young CR cells. **p , 0.05 comparing young AL and old AL cells. ***p , 0.05 comparing old CR and old AL cells. Note that young CR and old CR do not differ.
Fig. 3. Effect of the MEK inhibitor PD98059 on DNA synthesis in young and old hepatocytes. Cells were pretreated with PD98059 for 30 min prior to the addition of H2O2. (A) ERK phosphorylation was monitored by western blot analysis using a phospho-specific antibody. (B) DNA synthesis was measured as described in Fig. 1 based on BrdU incorporation. Results shown are the means ^ S.E. of three independent experiments. *p , 0.05 comparing BrdU incorporation of H2O2-treated cells relative to untreated or PD98059 þ H2O2-treated cells.
3.3. c-jun and MAP kinase phosphatase 1 (MKP-1) mRNA expression in response to EGF and H2O2 c-jun and MKP-1 are immediate-early genes whose induction by growth factors is at least partially dependent on ERK (Liu et al., 1996). Since old hepatocytes showed reduced ERK activation in response to EGF treatment relative to young hepatocytes, we compared c-jun and MKP-1 mRNA expression in EGF-treated hepatocytes derived from young and old AL or CR rats (Fig. 6). With
H2O2, only the young AL cells showed significant induction of either gene (not shown). EGF treatment, on the other hand, led to marked induction of both genes, the magnitude of which reflected that seen for ERK phosphorylation. That is, levels of c-jun and MKP-1 mRNAs were up regulated to a greater extent in young AL hepatocytes relative to old AL hepatocytes. Young CR hepatocytes showed a lesser induction of c-jun and MKP-1 compared to that seen in young AL cells, but old CR hepatocytes, displayed a more pronounced induction of c-jun and MKP-1 mRNAs than old AL hepatocytes. 3.4. Effect of aging on EGFR phosphorylation by EGF and H2O2 Previous studies had demonstrated that while total EGFR phosphorylation in response to EGF does not differ between young and old hepatocytes, aged cells exhibit reduced phosphorylation of a specific EGFR residue, Tyrosine 1173, in response to EGF treatment (Palmer et al., 1999). Since CR enhances ERK activation and improves the proliferative response of aged hepatocytes, we examined whether CR also influences phosphorylation of Tyrosine 1173 in aged cells. Consistent with a previous report (Palmer et al., 1999), total receptor phosphorylation did not differ between old and young cells (Fig. 7A), but aged cells showed a selective
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Fig. 7. Western blot analysis of total and Tyrosine 1173 specific phosphorylation of the EGFR in EGF- and H2O2-treated young and old hepatocytes. (A) Cells were treated with 100 ng/ml of EGF for 10 min, and whole-cell extracts were immunoprecipitated with EGFR antibody and immunoblotted with a general anti-phosphotyrosine antibody (B) Following treatment with EGF or H2O2 for 10 min, whole-cell extracts were immunoblotted with an antibody that specifically recognizes the phosphotyrosine at Y1173 on the EGFR, and with phospho-MEK 1/2 antibody. bactin signals are shown to reflect differences in loading and transfer among samples. The results are representative of three independent experiments.
Fig. 5. Effect of CR on ERK activation in young and aged hepatocytes treated with EGF or H2O2. Hepatocytes derived from young and old AL-fed and CR rats were treated with either 100 ng/ml EGF or 100 mM H2O2. ERK phosphorylation was monitored by western blot analysis using a phosphospecific antibody. Samples were harvested 30 min after stimulation. (A) Representative blots; ERK2 signals are shown to reflect evenness in loading and transfer among samples. (B) Summary of results obtained in five independent experiments. Shown are the means ^ S.E. *p , 0.05 comparing young AL and young CR cells. **p , 0.05 comparing young AL and old AL cells. ***p , 0.05 comparing old CR and old AL cells. Note that young CR and old CR do not differ.
reduction in phosphorylation of Tyrosine 1173 compared to young cells in response to EGF and H2O2 (Fig. 7B, upper panel). However, this effect was largely absent in cells from aged CR rats, supporting the notion that the defect leading to
Fig. 6. c-jun and MKP-1 expression in EGF-stimulated hepatocytes from young and aged AL- or CR-fed hosts. Representative northern blot showing c-jun and MKP-1 induction in EGF-stimulated hepatocytes for the times indicated. The 18S signals are shown as a control for loading, transfer and integrity of the various RNA samples.
reduced ERK activation in response to proliferative signals in old cells lies at this level. Indeed, MEK phosphorylation, which lies intermediate to EGFR and ERK in the signaling cascade, was similarly affected by age and CR (Fig. 7B, lower panel).
4. Discussion While it is well established that aging is associated with reduced proliferative potential of hepatocytes, the effect of CR on this process has been less clear. As we had previously demonstrated that aging is associated with a decline in the proliferative response of primary cultured hepatocytes to EGF stimulation (Liu et al., 1996), we investigated here the ability of CR to influence this response. The main findings of our studies are as follows. First, CR reduces proliferation of in vitro cultured hepatocytes from young hosts, but despite this, increases the proliferative capacity of old cells relative to that seen in cells from old AL animals. Second, the effects of aging and/or CR on the proliferative response to EGF stimulation are reflected by the relative degree of ERK activation and site-specific phosphorylation of EGFR residue Tyrosine 1173. The finding that CR reduces proliferation of young hepatocytes is consistent with several previous studies demonstrating lower basal rates of DNA synthesis in hepatocytes of either mice or rats placed on CR relative to those of AL fed counterparts (Higami et al., 1997; Wolf et al., 1995). In addition, two other studies reported that CR
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decreases the proliferative capacity of hepatocytes isolated from young animals subjected to partial hepatectomy (Shaddock et al., 1996; Taguchi et al., 2001). However, another group reported that in both young adult mice and rats, CR enhanced DNA synthesis during liver regeneration in vivo (Himeno et al., 1992; Cuenca et al., 2001). The reason for the differences between these findings is unclear, but could reflect differences in the model systems employed; for example, analysis of proliferation in vivo rather than in vitro, or the nature of the stimulus. Indeed, the response to partial hepatectomy in vivo is much more complex than in vitro stimulation with EGF and, in addition to inducing proliferation, leads to activation of other host stress response mechanisms that could contribute to the effects observed (Rozga, 2002; Fausto, 2001). Whether or not CR can prevent an age-related loss in proliferative capacity is also unclear. Studies examining the proliferative potential of aortic smooth muscle cells and skin fibroblasts during aging observed little or no effect of CR (Pignolo et al., 1992; Volicer et al., 1983). On the other hand, CR has been reported to inhibit an age-related decline in the responsiveness of T cells to mitogenic stimulation (Pahlavani and Vargas, 2000). Lu et al. (2002) reported that 24 month-old rats maintained on CR showed reduced cell proliferation in the liver of unstimulated animals. Shaddock et al. (1996) and Chou et al. (1995), on the other hand, found that although CR reduced the proliferative capacity of hepatocytes derived from young animals subjected to partial hepatectomy (PH) relative to AL fed young hosts, it increased proliferation in hepatocytes from old PH animals. These investigators suggested that the decrease in proliferation in young animals serves to preserve the proliferative capacity of hepatocytes over the long term, permitting aged animals to respond more efficiently with induced compensatory cellular replication in old age. Our own studies are consistent with this view. Pendergrass et al. (1995) and Wolf et al. (1995) similarly demonstrated that CR preserves proliferative capacity in a variety of other cell types and tissues of mice and likewise hypothesized that this may enable CR animals to better respond to proliferative stresses in old age. The notion that inhibiting proliferation early in life could be an important factor in the longevity-enhancing effects of CR is also consistent with features of several longevity mutants of mice. For example, the Ames dwarf and Snell dwarf mice, and mice lacking functional growth hormone receptors, all exhibit growth retardation and increased life span (Brown-Borg et al., 1996; Flurkey et al., 2001; Miller, 2001). It will be interesting to examine the proliferative potential of hepatocytes and other cells/tissues in these animals relative to their wild type counterparts at various stages of life. Our studies have demonstrated a close relationship between the degree of ERK activation and proliferation in hepatocytes. We observed this correlation with respect to both aging and CR treatment and also with two different
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proliferative stimuli, EGF and low concentrations of H2O2. We have reported a similar relationship between ERK activation and cell responsiveness to oxidative stress in hepatocytes (Ikeyama et al., 2001). That is, we demonstrated an age-related decline in ERK activation in response to high H2O2 treatment that was correlated with reduced survival of aged cells (Ikeyama et al., 2001). CR prevented the age-related decline in ERK activation and improved survival of aged cells. One major difference between our previous studies on oxidative stress with high H2O2 concentration and our current study examining proliferation with low H2O2 concentrations or EGF, is that CR did not affect the magnitude of ERK activation in response to high concentrations of H2O2, as ERK activation was equally robust in young CR and young AL animals treated with 300 – 600 mM H2O2 (Ikeyama et al., 2001). Likewise, CR did not reduce the sensitivity of young animals to H2O2. While the pathway leading to ERK activation following oxidant injury is known to overlap largely with that of growth factors, and is mediated at least partly through the EGFR (reviewed in Martindale and Holbrook, 2002; Holbrook and Ikeyama, 2002), clearly there is some distinction between the two signals. We and others have provided evidence that the defect resulting in reduced ERK activation in response to EGF in aged cells is believed to lie at the level of the EGFR and its ability to interact with the adaptor protein Shc (Palmer et al., 1999; Hutter et al., 2000). This was associated with reduced phosphorylation of the EGFR Tyrosine residue 1173, which lies within the Shc binding domain (Hutter et al., 2000). As shown now, the ability of CR to prevent the age-related decline in Tyrosine 1173 phosphorylation, further supports its importance in regulating the responsiveness to EGF. Why phosphorylation declines with age, and how CR alters this effect remains to be determined. The importance of cell membrane structure and fluidity in regulating signal transduction has become increasingly evident over recent years, and it has been suggested that aging is associated with changes in cell membrane composition and fluidity (Spiteller, 2002; Alvarez et al., 2001; Park et al., 2001). Such changes could influence the stoichiometry of protein interactions important in influencing phosphorylation of EGFR at specific sites such as at Tyrosine 1173. By preventing such changes in membrane structure composition, CR could act to preserve signal transduction processes. This idea is consistent with the finding that diverse signal transduction processes emanating from the cell membrane-localized receptors likewise display alterations with aging (Pahlavani, 1998; Podolin et al., 2001; Elias and Ghadially, 2002; Roka et al., 2000). Finally, it is important to consider the possibility that CR could influence the proliferative capacity of old cells not through preventing a loss of certain function with age, but rather through some direct effect(s) of CR that modulates/potentiates the response of old cells. An acute effect of CR on membrane composition, fluidity of both offers one such
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possibility. We are currently investigating the ability of short-term CR to influence proliferation on old cells to address such possibility.
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Ishigami, A., Reed, T.D., Roth, G.S., 1993. Effect of aging on EGF stimulated DNA synthesis and EGF receptor levels in primary cultured rat hepatocytes. Biochem. Biophys. Res. Commun. 196, 181–186. Knebel, A., Rahmsdorf, H.J., Ullrich, A., Herrlich, P., 1996. Dephosphorylation of receptor tyrosine kinases as target of regulation by radiation, oxidants or alkylating agents. EMBO J. 15, 5314–5325. Liu, Y., Guyton, K.Z., Gorospe, M., Xu, Q., Kokkonen, G.C., Mock, Y.D., Roth, G.S., Holbrook, N.J., 1996. Age-related decline in mitogenactivated protein kinase activity in epidermal growth factor-stimulated rat hepatocytes. J. Biol. Chem. 271, 3604–3607. Lu, M.H., Warbritton, A., Tang, N., Bucci, T.J., 2002. Dietary restriction alters cell proliferation in rats: An immunohistochemical study by labeling proliferating cell nuclear antigen. Mech. Ageing Dev. 123, 391 –400. Martindale, J., Holbrook, N.J., 2002. Cellular response to oxidative stress: Signaling for suicide and survival. J. Cell Physiol. 192, 1–15. Masoro, E.J., 1998. Hormesis and the antiaging action of dietary restriction. Exp. Gerontol. 33, 61–66. Masoro, E.J., 2001. An experimental approach to the study of the biology of aging. In: Masoro, E.J., Austad, S.N. (Eds.), Handbook of the Biology of Aging, Fifth ed., Academic Press, San Diego, pp. 396–420. Meydani, M., 2001. Nutrition interventions in aging and age-associated disease. Ann. N.Y. Acad. Sci. 928, 226 –235. Miller, R.A., 2001. Genetics of increased longevity and retarded aging in mice. In: Masoro, E.J., Austad, S.N. (Eds.), Handbook of the Biology of Aging, Academic Press, San Diego, pp. 369–395. Moolten, F.L., Bucher, N.L., 1967. Regeneration of rat liver: transfer of humoral agent by cross circulation. Science 158, 272–274. Pahlavani, M.A., 1998. T cell signaling: Effect of age. Frontiers in Biosci. 3, 1120–1133. Pahlavani, M.A., Vargas, D.M., 2000. Influence of aging and caloric restriction on activation of Ras/MAPK, calcineurin and CaMK-IV activities in rat T cells. Proc. Soc. Exp. Biol. Med. 223, 163–169. Palmer, H.J., Tuzon, C.T., Paulson, K.E., 1999. Age-dependent decline in mitogenic stimulation of hepatocytes. Reduced association between Shc and the epidermal growth factor receptor is coupled to decreased activation of Raf and extracellular signal-regulated kinases. J. Biol. Chem. 274, 11424– 11430. Park, W.Y., Cho, K.A., Park, J.S., Kim, D.I., Park, S.C., 2001. Attenuation of EGF signaling in senescent cells by caveolin. Ann. N.Y. Acad. Sci. 928, 79–84. Pendergrass, W., Li, Y., Jiang, D., Fei, R.G., Wolf, N.S., 1995. Caloric restriction: Conservation of cellular replicative capacity in vitro accompanies life-span extension in mice. Exp. Cell Res. 217, 309 –316. Pignolo, R.J., Masoro, E.J., Nichols, W.W., Bradt, C.I., Cristofalo, V.J., 1992. Skin fibroblasts from aged Fischer 344 rats undergo similar changes in replicative life span but not immortalization with caloric restriction of donors. Exp. Cell Res. 201, 16–22. Podolin, D.A., Wills, B.K., Wood, I.O., Lopez, M., Mazzeo, R.S., Roth, D.A., 2001. Attenuation of age-related declines in glucagon-mediated signal transduction in rat liver by exercise training. Am. J. Physiol. Endocrinol. Metab. 281, E516–E523. Roka, F., Freissmuth, M., Nanoff, C., 2000. G protein-dependent signaling and ageing. Exp. Gerontol. 35, 133–143. Roovers, K., Assoian, R.K., 2000. Integrating the MAP kinase signal into the G1 phase cell cycle machinery. Bioessays 22, 818 –826. Rozga, J., 2002. Hepatocyte proliferation in health and in liver failure. Med. Sci. Monit. 8, RA32–38. Seglen, P.O., 1976. Preparation of isolated rat liver cells. Methods Cell Biol. 13, 29 –83. Shaddock, J.G., Chou, M.W., Casciano, D.A., 1996. Effects of age and caloric restriction on cell proliferation in hepatocytes cultures from control and hepatectomized Fischer 344 rats. Mutagenesis 11, 281 –284. Spiteller, G., 2002. Are changes of the cell membrane structure causally involved in the aging process? Ann. N.Y. Acad. Sci. 959, 30–44.
S. Ikeyama et al. / Experimental Gerontology 38 (2003) 431–439 Taguchi, T., Fukuda, M., Ohashi, M., 2001. Differences in DNA synthesis in vitro using isolated nuclei from regenerating livers of young and aged rats. Mech. Ageing Dev. 122, 141–155. Tsukamoto, I., Nakata, R., Kojo, S., 1993. Effect of ageing on rat liver regeneration after partial hepatectomy. Biochem. Mol. Biol. Int. 30, 773–778. Van Remmen, H., Guo, Z., Richardson, A., 2001. The anti-ageing action of dietary restriction. Novartis Found. Symp. 235, 221–230. Volicer, L., West, C.D., Greene, L., 1983. Beta adrenergic receptor sensitivity in cultured vascular smooth muscle cells: Effect of age and of dietary restriction. Mech. Ageing Dev. 21, 283 –293.
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Effects of aging and calorie restriction of Fischer 344 rats on hepatocellular response to proliferative signals Shizuo Ikeyamaa,*, Gertrude Kokkonena, Jennifer L. Martindalea, Xian-Tao Wanga, Myriam Gorospea, Nikki J. Holbrooka,b a
Cell Stress and Aging Section, Laboratory of Cellular and Molecular Biology, National Institute on Aging, Baltimore, MD 21224, USA b Yale University School of Medicine, Section of Geriatrics, New Haven, CT 06520, USA Received 8 August 2002; received in revised form 8 October 2002; accepted 30 October 2002
Abstract It is well established that the proliferative potential of the liver declines with aging. Epidermal growth factor (EGF)-stimulated DNA synthesis is reduced in hepatocytes from aged rats relative to young rats, and this reduction correlates with diminished activation of the extracellular signal-regulated kinase (ERK) pathway and lower phosphorylation of the EGF receptor on residue Y1173. Calorie restriction (CR) can increase rodent life span and retard many age-associated declines in physiologic function, but its influence on cell proliferation is unknown. Here, we investigated the effects of long-term CR on proliferation of hepatocytes derived from young and aged rats following in vitro stimulation with either low-dose hydrogen peroxide or EGF. CR reduced the proliferative response of hepatocytes derived from young hosts, but long-term CR was associated with enhanced proliferation in aged cells relative to that of ad libitum (AL)-fed animals. ERK activation mirrored the effects of CR on proliferation, in that young CR cells exhibited lower ERK activation than young AL cells, but old CR cells showed higher ERK activation than old AL cells. Finally, a decline in EGF receptor phosphorylation on Y1173, which normally occurs with aging, was absent in cells of old hosts maintained on long-term CR, supporting the view that alterations in this early signaling event underlie the age-related decline in proliferative potential in rat hepatocytes. q 2002 Elsevier Science Inc. All rights reserved. Keywords: Hepatocytes; Aging; Oxidative stress; Proliferation; Epidermal growth factor
1. Introduction A decline in proliferative capacity constitutes an important hallmark of mammalian aging in many cell types (Hornsby, 2001). The liver, and hepatocytes in particular, have proven to be a particularly good model to study the mechanisms associated with this phenomenon. Liver regeneration following partial hepatectomy is impaired in aged animals as evidenced by a delay and reduction in magnitude of DNA synthesis (Moolten and Bucher, 1967; Tsukamoto et al., 1993; Wang et al., 2001).
* Corresponding author. Address: Laboratory of Cellular and Molecular Biology, Gerontology Research Center, Box 12, National Institute on Aging-Intramural Research Program, National Institutes of Health, 5600 Nathan Shock Drive, Baltimore, MD 21224, USA. Tel.: þ 1-410-558-8484; fax: þ 1-410-558-8386. E-mail address: [email protected] (S. Ikeyama).
Likewise, epidermal growth factor (EGF)-stimulated DNA synthesis is markedly lower in primary hepatocytes derived from aged donors compared to similarly treated cells of young donors (Ishigami et al., 1993). We and others have demonstrated that this reflects a decline in the activation of extracellular signal-regulated kinase (ERK), which plays a critical role in transmitting proliferative signals to the nucleus to effect changes in gene expression necessary for cell growth (Liu et al., 1996; Palmer et al., 1999; Hu et al., 1998). The cellular events leading to activation of ERK in response to EGF have been studied extensively (Cobb et al., 1994; Roovers and Assoian, 2000). In brief, binding of EGF to its receptor (EGFR) results in autophosphorylation and activation of the receptor, leading to the recruitment of adaptor and GDP/GTP exchange proteins necessary for activation of membrane-localized Ras. Activated Ras then initiates the phosphorylation cascade resulting in sequential activation of Raf, MEK, and ERK.
0531-5565/03/$ - see front matter q 2002 Elsevier Science Inc. All rights reserved. PII: S 0 5 3 1 - 5 5 6 5 ( 0 2 ) 0 0 2 3 9 - 5
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The defect resulting in reduced ERK activation in aged cells is believed to lie at the level of the EGFR. Neither EGFR number nor affinity of the receptor for its ligand are altered with age (Ishigami et al., 1993). Likewise, total receptor phosphorylation is unchanged with aging (Palmer et al., 1999; Hutter et al., 2000). However, in old cells the EGFR shows reduced ability to interact with the Shc adaptor protein, and this is associated with reduced phosphorylation of a specific EGFR amino acid residue, Tyrosine 1173, that lies within the Shc binding domain (Palmer et al., 1999; Hutter et al., 2000). Like growth factors, oxidants can activate the EGFR in a dose-dependent manner, initiating the signaling cascade leading to subsequent activation of ERK (Knebel et al., 1996; Zanella et al., 1996). Indeed, low concentrations of H2O2 have been shown to be mitogenic for many cell types (Burdon, 1995). At higher concentrations, however, H2O2 becomes toxic and is associated with either inhibition of growth or induction of cell death. In such instances, activation of ERK has been shown to favor cell survival (Guyton et al., 1996; Aikawa et al., 1997; Ikeyama et al., 2001). As in the case of EGF stimulation, aged hepatocytes show reduced activation of ERK in response to toxic concentrations of H2O2, and this is correlated with reduced survival (Ikeyama et al., 2001). Calorie restriction (CR), or the limiting of food intake without deprivation of essential nutrients, can extend life span in a wide variety of species (Masoro, 2001; Hart and Turturro, 1998; Masoro, 1998). In both rats and mice, CR has been shown to prevent or slow the progression of many age-related diseases, and attenuate declines in physiologic function that occur with normal aging (Van Remmen et al., 2001; Meydani, 2001). We have demonstrated that CR can prevent the age-related decline in ERK activation by toxic concentrations of H2O2 (300 – 1000 mM), and have likewise shown that this correlates with improved survival of aged rat hepatocytes (Ikeyama et al., 2001). Studies examining the influence of CR on hepatocyte proliferation with aging have produced variable findings ranging from inhibition to enhancement (Higami et al., 1997; Wolf et al., 1995; Shaddock et al., 1996; Taguchi et al., 2001; Himeno et al., 1992; Cuenca et al., 2001; Rozga, 2002). Therefore, the present study sought to examine the impact of CR on the proliferative capacity of young versus aged hepatocytes using either EGF or low concentrations of H2O2 as proliferative stimuli. We demonstrate that CR alone causes a modest, but significant diminution in the mitogenic response to both EGF and H2O2 in hepatocytes from young rats. However, aged hepatocytes from animals maintained on CR display enhanced proliferative capacity relative to their ad libitum (AL)-fed counterparts. The effect of CR on proliferation is paralleled by similar changes in ERK activity and site-specific phosphorylation of the EGFR.
2. Materials and methods 2.1. Animals Male Fischer 344 rats, 4 –6 (young) and 24– 26 (old) months of age, were obtained from the National Institute on Aging Contract Colonies at Harlan Sprague Dawley. They were maintained on a 12:12 h light:dark cycle in a controlled environment with water supplied at all times. Ad libitum-fed (AL) animals were provided with unlimited quantities of a regular NIH-31 pelleted diet. For the CR group, CR was initiated at 14 weeks of age, with a 10% reduction in calorie intake. The restriction was increased to 25% at 15 weeks, to a maximum of 40% at 16 weeks. They were maintained on the CR diet, equal to 60% the average daily calorie intake of AL animals, for the remainder of life. These treatment conditions were approved by the Gerontology Research Center Animal Use and Care Committee (Protocol #NJH-058-Ra/Mi) and are in accordance with the National Institutes of Health guidelines for the care and use of laboratory animals. 2.2. Primary hepatocyte culture and treatment Hepatocytes were isolated from male Fischer 344 rats by the collagenase perfusion method of Seglen as described (Seglen, 1976). The isolated cells were seeded onto Biocoat Collagen I cellware (BD Discovery Labware, Bedford, MA) in William’s E medium supplemented with 5% fetal bovine serum, 1 mM dexamethasone, 10 mg/L insulin, 2 mM L glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin for 2 h in 5% CO2 at 37 8C to allow attachment to the dishes. The medium was then replaced with serum-free William’s E medium plus the above supplements, and cells were cultured for an additional 16 h prior to treatment. This procedure results in less than 5% contamination with nonhepatocyte cells. Cultured hepatocytes were treated with various concentrations of H2O2 or 100 ng/ml of EGF (Invitrogen, Carlsbad, CA) for the indicated times. H2O2 was prepared freshly from a concentrated stock solution. 2.3. Immunoprecipitation and western blot analysis For western blot analysis, total cell lysates were prepared using a lysis buffer containing Hepes (20 mM, pH 7.4), EGTA (2 mM), b-glycerophosphate (50 mM), Na3VO4 (1 mM), NaF (5 mM), 1% (v/v) Triton X-100, 10% (v/v) glycerol, dithiothreitol (DTT) (1 mM), PMSF (1 mM), leupeptin (10 mg/ml), aprotinin (10 mg/ml). Cells were centrifuged at 4 8C, 15,000 rpm, for 20 min. Protein (50 – 150 mg) was mixed with 2 £ sample buffer containing 130 mM Tris-Cl, pH 8.0, 20% (v/v) Glycerol, 4.6% (w/v) SDS, 0.02% Bromophenol blue, 2% DTT, and size fractionated by electrophoresis through Tris-Glycine Gel (Invitrogen, Carlsbad, CA). Size-separated proteins were then transferred onto polyvinylidene difluoride membranes
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(Millipore, Bedford, MA) and immunoblot analysis was carried out using the appropriate antibodies. Specific proteins were detected with the enhanced chemiluminescence (ECL) system (Amersham Pharmacia Biotech). Some blots were subjected to multiple probings with different antibodies. In such instances, the blots were stripped between probings using a buffer containing 50 mM TrisHCl, pH 6.8, 2% SDS, and 0.1 M of b-mercaptoethanol. All blots were normalized to total ERK2 or b-actin (Abcam Ltd UK) levels. For immunoprecipitation analysis, cells were treated, then total cell lysates were prepared using lysis buffer, and 500 mg protein incubated with 5 mg of anti-EGFR antibody and 40 ml of protein A-Sepharose for 4 h at 4 8C. Immune complexes were washed four times with lysis buffer and resuspended in 2 £ sample buffer. Immunoblot analysis was carried out using the appropriate antibodies. Specific proteins were detected by western blot analysis. AntiERK2 and EGF-polyclonal antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). PD98059 was purchased from Cell Signaling Technology (Beverly, MA). Anti-phospho-MEK1/2 and the anti-phospho ERK1/2 antibodies, which recognized phosphorylated/active forms of the respective proteins, were from Cell Signaling Technology (Beverly, MA) and Promega (Madison, WI). The anti-phospho-EGF Receptor antibody, specific for residue Y1173, was obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). 2.4. Northern blot analysis For RNA analysis, cells were harvested at various time points following treatment, and total RNA extracted using STAT-60 (Tel-Test B, Friendswood, Tex.) according to the manufacturer’s recommendations. Twenty mg RNA was run on agarose-formaldehyde gels and transferred to a GeneScreen Plus membrane (NEN Life Science Products). Northern blot analysis was performed using either cDNA probes specific for rat c-jun, and MKP-1 or an oligonucleotide complimentary to 18S RNA. 2.5. Assessment of DNA synthesis by monitoring [3H] thymidine and BrdU incorporation [Methyl-3H] thymidine (85 Ci/mmol) was obtained from Amersham (Arlington Heights, IL) and its incorporation into DNA was investigated as follows. Hepatocytes were plated at a density of 0.4 £ 105 cells/well in 12-well plates pre-coated with type I collagen. Twenty-four to 72 h after treatment with either EGF or H2O2, cells were incubated with 10 mCi [3H] thymidine per ml for 2 h, after which they were washed twice with cold-PBS, immersed in 1 ml of cold 10% trichloroacetic acid (TCA) for 10 min and solubilized by incubation in 0.5 ml of 1 N NaOH at 37 8C for 30 min. TCA was added to achieve a final concentration of 15% TCA, and macromolecules were allowed to precipitate
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overnight. After centrifugation, the precipitate was washed with 10% TCA and hydrolyzed in 0.5 ml of 10% TCA at 90 8C for 15 min. Radioactivity in the hot TCA-soluble fraction was measured by liquid scintillation counting. Data was shown as cpm incorporated per mg of protein [measured by the Bradford method (Bio-Rad, Hercules, CA.)] in whole-cell lysates. A cell proliferation ELISA kit (Roche Molecular Biochemicals, IN) that measures BrdU incorporation was also used to monitor DNA synthesis. Briefly, 0.4 £ 105 cells/well in 12-well plates pre-coated with type I collagen were labeled with BrdU for 2 h at 37 8C. After its incorporation into DNA, BrdU was detected by immunoassay. 2.6. Statistical analysis Statistical analysis was performed using a one-way ANOVA. Differences between individual age or treatment groups were evaluated using the unpaired two-tailed Student’s t test.
3. Results 3.1. Effect of aging on proliferation and ERK activity in H2O2 –stimulated hepatocytes Low concentrations of H2O2 have been shown to be mitogenic for many cell types. To determine if this is true for primary rat hepatocytes, and to examine whether host donor age affects the response, hepatocytes derived from young (4 – 6-mo old) and old (24 –26-mo old) male Fischer 344 rats were exposed to varying amounts of H2O2 and 24 h later evaluated for the incorporation of [3H] thymidine. Cells derived from young donors showed a marked elevation in [3H]-thymidine incorporation (. four-fold) in response to both 50 and 100 mM H2O2, but cells from aged donors exhibited a much attenuated response to the mitogen (Fig. 1A). Only at 50 mM H2O2 was a significant increase in [3H]-thymidine incorporation seen in old cells. Consistent with our previous studies, higher concentrations of H2O2 were found to be toxic for the hepatocytes, resulting in [3H] thymidine incorporation below that of control cultures. This toxic effect was more evident in old cells relative to young cells. The mitogenic effects of 50 and 100 mM H2O2, as well as the differential responsiveness of young and old hepatocytes were confirmed using BrdU incorporation (Fig. 1B). Relative amounts of phosphorylated ERK1/2 in young and old hepatocytes treated with H2O2 were examined by western blot analysis as an assessment of kinase activation. In keeping with the known role of ERK in regulating proliferation, ERK1/2 was found to undergo significant activation in response to both 50 and 100 mM H2O2 in young hepatocytes, but this effect was much reduced in aged hepatocytes (Fig. 2). ERK activation was sustained with
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Fig. 1. Age-associated decline in proliferative response of primary cultured rat hepatocytes to H2O2 treatment. Primary hepatocytes derived from young adult (4–6 mo) or aged (24–26 mo) hosts were treated with the indicated concentrations of H2O2 and DNA synthesis was assessed by either [3H] thymidine incorporation (A) or BrdU incorporation (B) 24 h after treatment. Results shown are the means ^ S.E. of three independent experiments. *p , 0.05 comparing H2O2-treated cells with vehicle controls. **p , 0.05 comparing response of young cells to old cells.
higher concentrations of the oxidant in young cells, but underwent further attenuation in old cells. These findings are consistent with our previous studies in which we demonstrated that ERK activation at concentrations between 300 –1000 mM were important for cell survival (Ikeyama et al., 2001). That the activation of ERK by H2O2 is important for the proliferative response is evidenced by the data shown in Fig. 3. Treatment of young hepatocytes with PD98059, which acts to inhibit the activity of the ERK phosphorylating kinase MEK and thereby completely prevents ERK activation (Fig. 3A), effectively blocked BrdU incorporation in response to H2O2 (Fig. 3B). 3.2. Effects of CR on hepatocyte response to EGF and H2O2 CR has been shown to extend lifespan in rats, and prevent the age-related decline in many physiologic functions
Fig. 2. H2O2-triggered ERK activation in hepatocytes from young and aged rats. (A) Representative western blot showing relative amounts of phosphorylated (active) ERK (pERK1/pERK2) in cultures 30 min following treatment with various H2O2 concentrations. Note that total ERK2 protein levels do not differ between age or treatment groups. (B) Quantification of results obtained from five independent experiments comparing amounts of phosphorylated ERK1/2 in H2O2-stimulated cells from young versus aged hosts relative to levels in untreated young cells. Shown are the means ^ S.E. of five independent experiments. *p , 0.05 comparing H2O2-treatment with untreated controls. **p , 0.05 comparing values for young and old cells.
(Masoro, 2001; Hart and Turturro, 1998; Masoro, 1998; Van Remmen et al., 2001; Meydani, 2001). To determine the impact of CR on proliferative capacity of hepatocytes, we compared the responsiveness of hepatocytes derived from young and old rats maintained on either AL or CR diets to treatment with either 50 or 100 mM H2O2 or 100 ng/ ml EGF. As shown in Fig. 4, BrdU incorporation was higher with EGF treatment than with H2O2. CR itself resulted in a reduced response to the proliferative signals, as evidenced by the lower BrdU incorporation in cells derived from young CR rats relative to young AL animals. Interestingly, BrdU incorporation did not differ significantly between cells of young CR and old CR animals, but both were markedly different (higher) from that seen in cells from old AL rats. Examination of ERK activation in these same animals produced results that mirrored those for BrdU incorporation (Fig. 5). Cells from young CR animals showed lower ERK activation compared to cells of young AL animals, but young and old CR cells did not differ in levels of ERK activation, both showing significantly higher levels than seen in old AL cells.
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Fig. 4. Effect of CR on proliferation of young and aged hepatocytes treated with EGF or H2O2. Hepatocytes derived from young and old AL-fed and CR rats were treated with either EGF or H2O2. DNA synthesis was determined based on BrdU incorporation as described for Fig. 1. Results shown are the means ^ S.E. of three independent experiments. *p , 0.05 comparing young AL and young CR cells. **p , 0.05 comparing young AL and old AL cells. ***p , 0.05 comparing old CR and old AL cells. Note that young CR and old CR do not differ.
Fig. 3. Effect of the MEK inhibitor PD98059 on DNA synthesis in young and old hepatocytes. Cells were pretreated with PD98059 for 30 min prior to the addition of H2O2. (A) ERK phosphorylation was monitored by western blot analysis using a phospho-specific antibody. (B) DNA synthesis was measured as described in Fig. 1 based on BrdU incorporation. Results shown are the means ^ S.E. of three independent experiments. *p , 0.05 comparing BrdU incorporation of H2O2-treated cells relative to untreated or PD98059 þ H2O2-treated cells.
3.3. c-jun and MAP kinase phosphatase 1 (MKP-1) mRNA expression in response to EGF and H2O2 c-jun and MKP-1 are immediate-early genes whose induction by growth factors is at least partially dependent on ERK (Liu et al., 1996). Since old hepatocytes showed reduced ERK activation in response to EGF treatment relative to young hepatocytes, we compared c-jun and MKP-1 mRNA expression in EGF-treated hepatocytes derived from young and old AL or CR rats (Fig. 6). With
H2O2, only the young AL cells showed significant induction of either gene (not shown). EGF treatment, on the other hand, led to marked induction of both genes, the magnitude of which reflected that seen for ERK phosphorylation. That is, levels of c-jun and MKP-1 mRNAs were up regulated to a greater extent in young AL hepatocytes relative to old AL hepatocytes. Young CR hepatocytes showed a lesser induction of c-jun and MKP-1 compared to that seen in young AL cells, but old CR hepatocytes, displayed a more pronounced induction of c-jun and MKP-1 mRNAs than old AL hepatocytes. 3.4. Effect of aging on EGFR phosphorylation by EGF and H2O2 Previous studies had demonstrated that while total EGFR phosphorylation in response to EGF does not differ between young and old hepatocytes, aged cells exhibit reduced phosphorylation of a specific EGFR residue, Tyrosine 1173, in response to EGF treatment (Palmer et al., 1999). Since CR enhances ERK activation and improves the proliferative response of aged hepatocytes, we examined whether CR also influences phosphorylation of Tyrosine 1173 in aged cells. Consistent with a previous report (Palmer et al., 1999), total receptor phosphorylation did not differ between old and young cells (Fig. 7A), but aged cells showed a selective
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Fig. 7. Western blot analysis of total and Tyrosine 1173 specific phosphorylation of the EGFR in EGF- and H2O2-treated young and old hepatocytes. (A) Cells were treated with 100 ng/ml of EGF for 10 min, and whole-cell extracts were immunoprecipitated with EGFR antibody and immunoblotted with a general anti-phosphotyrosine antibody (B) Following treatment with EGF or H2O2 for 10 min, whole-cell extracts were immunoblotted with an antibody that specifically recognizes the phosphotyrosine at Y1173 on the EGFR, and with phospho-MEK 1/2 antibody. bactin signals are shown to reflect differences in loading and transfer among samples. The results are representative of three independent experiments.
Fig. 5. Effect of CR on ERK activation in young and aged hepatocytes treated with EGF or H2O2. Hepatocytes derived from young and old AL-fed and CR rats were treated with either 100 ng/ml EGF or 100 mM H2O2. ERK phosphorylation was monitored by western blot analysis using a phosphospecific antibody. Samples were harvested 30 min after stimulation. (A) Representative blots; ERK2 signals are shown to reflect evenness in loading and transfer among samples. (B) Summary of results obtained in five independent experiments. Shown are the means ^ S.E. *p , 0.05 comparing young AL and young CR cells. **p , 0.05 comparing young AL and old AL cells. ***p , 0.05 comparing old CR and old AL cells. Note that young CR and old CR do not differ.
reduction in phosphorylation of Tyrosine 1173 compared to young cells in response to EGF and H2O2 (Fig. 7B, upper panel). However, this effect was largely absent in cells from aged CR rats, supporting the notion that the defect leading to
Fig. 6. c-jun and MKP-1 expression in EGF-stimulated hepatocytes from young and aged AL- or CR-fed hosts. Representative northern blot showing c-jun and MKP-1 induction in EGF-stimulated hepatocytes for the times indicated. The 18S signals are shown as a control for loading, transfer and integrity of the various RNA samples.
reduced ERK activation in response to proliferative signals in old cells lies at this level. Indeed, MEK phosphorylation, which lies intermediate to EGFR and ERK in the signaling cascade, was similarly affected by age and CR (Fig. 7B, lower panel).
4. Discussion While it is well established that aging is associated with reduced proliferative potential of hepatocytes, the effect of CR on this process has been less clear. As we had previously demonstrated that aging is associated with a decline in the proliferative response of primary cultured hepatocytes to EGF stimulation (Liu et al., 1996), we investigated here the ability of CR to influence this response. The main findings of our studies are as follows. First, CR reduces proliferation of in vitro cultured hepatocytes from young hosts, but despite this, increases the proliferative capacity of old cells relative to that seen in cells from old AL animals. Second, the effects of aging and/or CR on the proliferative response to EGF stimulation are reflected by the relative degree of ERK activation and site-specific phosphorylation of EGFR residue Tyrosine 1173. The finding that CR reduces proliferation of young hepatocytes is consistent with several previous studies demonstrating lower basal rates of DNA synthesis in hepatocytes of either mice or rats placed on CR relative to those of AL fed counterparts (Higami et al., 1997; Wolf et al., 1995). In addition, two other studies reported that CR
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decreases the proliferative capacity of hepatocytes isolated from young animals subjected to partial hepatectomy (Shaddock et al., 1996; Taguchi et al., 2001). However, another group reported that in both young adult mice and rats, CR enhanced DNA synthesis during liver regeneration in vivo (Himeno et al., 1992; Cuenca et al., 2001). The reason for the differences between these findings is unclear, but could reflect differences in the model systems employed; for example, analysis of proliferation in vivo rather than in vitro, or the nature of the stimulus. Indeed, the response to partial hepatectomy in vivo is much more complex than in vitro stimulation with EGF and, in addition to inducing proliferation, leads to activation of other host stress response mechanisms that could contribute to the effects observed (Rozga, 2002; Fausto, 2001). Whether or not CR can prevent an age-related loss in proliferative capacity is also unclear. Studies examining the proliferative potential of aortic smooth muscle cells and skin fibroblasts during aging observed little or no effect of CR (Pignolo et al., 1992; Volicer et al., 1983). On the other hand, CR has been reported to inhibit an age-related decline in the responsiveness of T cells to mitogenic stimulation (Pahlavani and Vargas, 2000). Lu et al. (2002) reported that 24 month-old rats maintained on CR showed reduced cell proliferation in the liver of unstimulated animals. Shaddock et al. (1996) and Chou et al. (1995), on the other hand, found that although CR reduced the proliferative capacity of hepatocytes derived from young animals subjected to partial hepatectomy (PH) relative to AL fed young hosts, it increased proliferation in hepatocytes from old PH animals. These investigators suggested that the decrease in proliferation in young animals serves to preserve the proliferative capacity of hepatocytes over the long term, permitting aged animals to respond more efficiently with induced compensatory cellular replication in old age. Our own studies are consistent with this view. Pendergrass et al. (1995) and Wolf et al. (1995) similarly demonstrated that CR preserves proliferative capacity in a variety of other cell types and tissues of mice and likewise hypothesized that this may enable CR animals to better respond to proliferative stresses in old age. The notion that inhibiting proliferation early in life could be an important factor in the longevity-enhancing effects of CR is also consistent with features of several longevity mutants of mice. For example, the Ames dwarf and Snell dwarf mice, and mice lacking functional growth hormone receptors, all exhibit growth retardation and increased life span (Brown-Borg et al., 1996; Flurkey et al., 2001; Miller, 2001). It will be interesting to examine the proliferative potential of hepatocytes and other cells/tissues in these animals relative to their wild type counterparts at various stages of life. Our studies have demonstrated a close relationship between the degree of ERK activation and proliferation in hepatocytes. We observed this correlation with respect to both aging and CR treatment and also with two different
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proliferative stimuli, EGF and low concentrations of H2O2. We have reported a similar relationship between ERK activation and cell responsiveness to oxidative stress in hepatocytes (Ikeyama et al., 2001). That is, we demonstrated an age-related decline in ERK activation in response to high H2O2 treatment that was correlated with reduced survival of aged cells (Ikeyama et al., 2001). CR prevented the age-related decline in ERK activation and improved survival of aged cells. One major difference between our previous studies on oxidative stress with high H2O2 concentration and our current study examining proliferation with low H2O2 concentrations or EGF, is that CR did not affect the magnitude of ERK activation in response to high concentrations of H2O2, as ERK activation was equally robust in young CR and young AL animals treated with 300 – 600 mM H2O2 (Ikeyama et al., 2001). Likewise, CR did not reduce the sensitivity of young animals to H2O2. While the pathway leading to ERK activation following oxidant injury is known to overlap largely with that of growth factors, and is mediated at least partly through the EGFR (reviewed in Martindale and Holbrook, 2002; Holbrook and Ikeyama, 2002), clearly there is some distinction between the two signals. We and others have provided evidence that the defect resulting in reduced ERK activation in response to EGF in aged cells is believed to lie at the level of the EGFR and its ability to interact with the adaptor protein Shc (Palmer et al., 1999; Hutter et al., 2000). This was associated with reduced phosphorylation of the EGFR Tyrosine residue 1173, which lies within the Shc binding domain (Hutter et al., 2000). As shown now, the ability of CR to prevent the age-related decline in Tyrosine 1173 phosphorylation, further supports its importance in regulating the responsiveness to EGF. Why phosphorylation declines with age, and how CR alters this effect remains to be determined. The importance of cell membrane structure and fluidity in regulating signal transduction has become increasingly evident over recent years, and it has been suggested that aging is associated with changes in cell membrane composition and fluidity (Spiteller, 2002; Alvarez et al., 2001; Park et al., 2001). Such changes could influence the stoichiometry of protein interactions important in influencing phosphorylation of EGFR at specific sites such as at Tyrosine 1173. By preventing such changes in membrane structure composition, CR could act to preserve signal transduction processes. This idea is consistent with the finding that diverse signal transduction processes emanating from the cell membrane-localized receptors likewise display alterations with aging (Pahlavani, 1998; Podolin et al., 2001; Elias and Ghadially, 2002; Roka et al., 2000). Finally, it is important to consider the possibility that CR could influence the proliferative capacity of old cells not through preventing a loss of certain function with age, but rather through some direct effect(s) of CR that modulates/potentiates the response of old cells. An acute effect of CR on membrane composition, fluidity of both offers one such
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possibility. We are currently investigating the ability of short-term CR to influence proliferation on old cells to address such possibility.
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