Impaired signal transduction in mitogen activated rat splenic lymphocytes during aging

Mechanisms of Ageing and Development 113 (2000) 85 – 99 www.elsevier.com/locate/mechagedev

Impaired signal transduction in mitogen activated rat splenic lymphocytes during aging Min Li, Robin Walter, Claudio Torres, Felipe Sierra * Center for Gerontological Research, MCP Hahnemann Uni6ersity, 2900 Queen Lane, Philadelphia, PA 19129, USA Received 30 June 1999; received in revised form 18 October 1999; accepted 20 October 1999

Abstract Mitogen activated protein kinases (MAPK) are activated by a wide variety of signals leading to cell proliferation and differentiation in different cell types. With aging, there is a marked decrease in proliferation of T-lymphocytes in response to a variety of mitogens. Several age-related changes in the activation of MAPK pathways in T-lymphocytes activated via the T-cell receptor (TCR) have been described in different species. This way, some TCR proximal defects in tyrosine kinase activity have been delineated. In this study, we have used rat splenic lymphocytes to measure the effect of aging on the activation of two MAP kinase families: ERK and JNK. In order to bypass the receptor-proximal age-dependent defects previously described, we used phorbol ester (PMA) and Ca2 + ionophore (A23187) as co-mitogens. Our results demonstrate that splenic lymphocytes from old rats have a disturbance in the activation of the ERK and JNK MAPK signal transduction pathways, that are located downstream of the receptor-proximal events. At least part of the age-related defect leading to decreased ERK activity appears to be located upstream of ERK itself, since activation of MEK is also impaired. On the other hand, the observed defects in MAPK activation do result in decreased activation of downstream events, such as c-Jun phosphorylation. Thus, we conclude that aging of splenic lymphocytes results in a functional decline in signal transduction, and at least some of these defects are located downstream of the

Abbre6iations: CaI, calcium ionophore; Con A, concanavalin A; ERK, extracellular signal regulated protein kinase; IL-2, interleukin-2; IL-2R, IL-2 receptor; JNK, c-Jun N-terminal kinase; MAPK, mitogen activated protein kinases; PMA, phorbol-12-myristate 13-acetate; TCR, T-cell receptor; WCE, whole cell extract. * Corresponding author. Present address: Lankenau Medical Research Center, 100 Lancaster Avenue, Wynnewood, PA 19096, USA. Tel.: + 1-610-6458583; fax: +1-610-6452290. E-mail address: [email protected] (F. Sierra) 0047-6374/00/$ - see front matter © 2000 Published by Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 4 7 - 6 3 7 4 ( 9 9 ) 0 0 0 9 6 - 2

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receptor-proximal events previously described by others. The impaired activity of these two MAP kinase pathways is likely to play a role in the diminished lymphoproliferation observed in old individuals. © 2000 Published by Elsevier Science Ireland Ltd. All rights reserved. Keywords: Aging; Signal transduction; ERK; JNK; Lymphocytes

1. Introduction Age-associated decreases in lymphoproliferation in response to mitogens such as concanavalin A (Con A) or phorbol-12-myristate 13-acetate (PMA) plus calcium ionophore (A23187) have long been observed (Kruisbeek, 1976; Kay et al., 1979; Makinodan and Kay, 1980; Holbrook et al., 1989; Goonewardene and Murasko, 1993). The decreased proliferation of lymphocytes correlates with decreased IL-2 production (Miller, 1995; Pahlavani and Richardson, 1996), which is required for T-cell proliferation. A significant decline in IL-2 production after Con A or phytohemagglutinin (PHA) stimulation was found in different rat strains, including Brown Norway and F344 (Bash, 1983; Cheung et al., 1983; Goonewardene and Murasko, 1993). Unlike the consistent observation of decreased IL-2 production in mice and humans, there are some discrepancies in the literature concerning IL-2 activity, as well as IL-2 mRNA levels and IL-2 receptor cell surface expression in lymphocytes from young and old rats stimulated with either Con A or PMA plus CaI. While some authors have observed a decline with age in these parameters (Thoman and Weigle, 1981; Nagel et al., 1988; Ernst et al., 1989; Pahlavani and Richardson, 1996), other have observed no differences (Wu et al., 1986; Holbrook et al., 1989; Murasko and Goonewardene, 1990). This suggests that some factors other than decreased expression of IL-2 and IL-2 receptors might also account for the diminished proliferative capacity of aged rat lymphocytes following mitogen induction. Considerable progress has been achieved in understanding the mechanisms by which mitogens stimulate gene transcription, IL-2 production and eventually cell proliferation. Several signaling pathways, including ERK and JNK pathways, PKC and Ca2 + /calcineurin, are activated upon T-cell receptor ligation (Crabtree and Clipstone, 1994). Specifically, both the ERK and JNK pathways are known to be critical for lymphocyte proliferation (DeSilva et al., 1998; Lafont et al., 1998; Swat et al., 1998). The limited studies available suggest that age-related disturbances in signal transduction pathways might explain the diminished IL-2 gene expression. Thus, it has been shown that tyrosine phosphorylation of several proteins is affected during aging in purified T-lymphocytes (Ghosh and Miller, 1995; Utsuyama et al., 1997; Whisler et al., 1997). Consistent with these results, a significant age-related reduction of ERK activation was observed in mouse splenic T-cells stimulated via the TCR using anti-CD3 antibodies (Gorgas et al., 1997), in Con A induced splenic T-lymphocytes from rats (Pahlavani et al., 1998) and in peripheral blood T-cells from humans (Whisler et al., 1996). Similarly, an age-related decline in JNK

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activation in response to anti-CD3 plus PMA was observed in human peripheral T-lymphocytes (Liu et al., 1997). These studies suggest some age-related impairment in the ERK/JNK MAPK pathways via the TCR/CD3 complex. Signaling via the TCR results in a variety of biochemical signaling events that include a rise in intracellular free calcium and activation of protein kinase C. These two signals can also be generated by CaI and by activators of protein kinase C, such as PMA (Nau et al., 1988). This combination provides a unique opportunity to circumvent the early events in TCR activation, which are known to be defective during aging. Together, these compounds act as effective co-mitogens (Larsen, 1990). Most studies have indicated that proliferation is significantly impaired (30– 50%) in aged lymphocytes stimulated with either Con A or PMA plus A23187 in different species (Thoman and Weigle, 1988; Holbrook et al., 1989; Negoro and Hara, 1992; Utsuyama et al., 1997). However, the difference in responsiveness to PMA plus CaI between young and old T-cells is less pronounced than that observed in response to receptor-dependent stimuli. This has led some authors to suggest that the components of the activation pathways that lie downstream from the generation of calcium and diacylglycerol (DAG) may be relatively unimpaired by aging (Miller, 1986, 1995). Nevertheless, there appears to be a significant portion of the age-related decrease in responsiveness that still can not be overcome by bypassing the early events through the use of PMA plus Ca2 + as inducers. Therefore, it seems that, besides the major blockade in TCR-dependent signal transduction located just after the TCR, an additional blockade might also be present at sites lying downstream of these events. To further understand the mechanisms underlying these phenomena, we have studied the activation of the MAPK signaling cascades in total splenocytes isolated from F344 rats. Lymphocytes from young, middle-aged and old rats were stimulated for 15 min with two different concentrations of PMA plus A23187. The low concentration of inducers has been reported to induce optimal splenic lymphocyte proliferation with decreased proliferative capacity in lymphocytes from old animals (Holbrook et al., 1989). The high concentration of PMA plus A23187 has also been used in the literature, for studies designed to investigate the activation of different signaling cascades in Jurkat cells (Su et al., 1994). Our data indicate the presence of an age-related blockade in MAPK signal transduction, located downstream of Ras activation, which results in decreased phosphorylation of key transcription factors required for further downstream events.

2. Materials and methods

2.1. Animals Young (6 months), middle-aged (15 months) and old (24 months) Fisher F344 male, ad libitum fed rats were purchased from the Natonal Institutes on Aging (NIA). Upon arrival, they were housed in our specific pathogen free facility for 2 weeks before use. The mean lifespan of male F344 rats is  24 months. Rats with

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tumors or with spleen or other organ enlargements were excluded from the studies. In addition, serum levels of the acute phase protein, a1-acid glycoprotein (AGP) were determined as a measure of inflammatory processes (data not shown). Rats that exhibited a high level of expression of AGP were eliminated from the study.

2.2. Spleen lymphocyte isolation Animals were decapitated, and spleens were removed and homogenized in a Tenbroeck tissue grinder (Fisher) in RPMI 1640 (Whittaker Bioproducts). The cell suspension was centrifuged at 400×g for 10 min at 4°C, the pellets were resuspended in 5 ml RPMI and were overlaid on Ficoll (1-Step™ 1.077/265 Animal; Accurate Chemical & Scientific) and centrifuged at 800× g for 20 min at room temperature. Splenocytes were carefully collected from the interphase, washed and resuspended in RPMI.

2.3. Cell stimulation After two washes in RPMI, splenocytes from individual rats were resuspended at 5× 106 cells/ml in RPMI medium containing 10% fetal calf serum, 2 mM glutamine, 100 U/ml penicillin and 100 mg/ml streptomycin. Aliquots of 107 cells from each age group were placed in 35-mm tissue culture plates and kept at 37°C, 5% CO2. Cells were stimulated with low concentration of PMA plus A23187 (10 ng/ml and 0.05 mg/ml, respectively) or high concentration of PMA plus A23187 (50 ng/ml and 1 mg/ml, respectively) for 15 min. Uninduced splenocytes were used as control.

2.4. Whole cell extract preparation To prepare whole cell extracts (WCE), the cells were pelleted at 16 000× g for 10 s, and resuspended in lysis buffer (25 mM Hepes, pH 7.7, 0.3 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.1% Triton X-100, 2 mM sodium pyrophosphate, 0.5 mM DTT, 20 mM b-glycerophosphate, 5 mg/ml leupeptin, 10 mg/ml aprotinin, 100 mg/ml PMSF, and 0.1 mM sodium orthovanadate (modified from (Su et al., 1994). After shaking the cell suspension at 4°C for 30 min, the extract was clarified by centrifugation at 12 000× g for 10 min. The supernatants (WCE) were collected and protein concentration was measured by the Bradford method as commercialized by BioRad.

2.5. Western blot Aliquots (35 mg) of WCE were used for Western blot analysis. Samples were denatured by boiling in Laemmli SDS loading buffer, reduced with 5% b-mercaptoethanol, and fractionated by SDS–PAGE using 10% mini gels. Proteins were then electrophoretically transferred to nitrocellulose filters (BA 85, Schleicher and Schuell) for 60 min at 100 V in a BioRad mini electrotransfer apparatus. The blots were reversibly stained with Ponceau S (0.05% Ponceau S in 3% TCA) for 1 min,

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followed by two washes with ddH2O. After photography, the membranes were blocked with BSA buffer (1% BSA, 10 mM Tris, pH 7.5, 100 mM NaCl, 0.1% Tween 20) for 1 h at room temperature, and probed with horseradish peroxidase conjugated anti-phosphotyrosine monoclonal antibody (PY-20, Transduction Laboratories) for 1 h at room temperature. After washing three times for 15 min in TNT buffer (10 mM Tris, pH 7.5, 100 mM NaCl, 0.1% Tween 20), detection was performed using the enhanced chemiluminescence (ECL) system from Amersham, according to the instructions supplied by the manufacturer. As a control for loading, the membranes were probed with anti-actin antibody (ICN). Western blot analysis with the anti-active ERK antibody was performed exactly as suggested by the manufacturer (Promega), while the analysis with anti-phospho JNK, anti-phospho MEK1/2, and anti-phospho c-Jun were done according to the protocol provided by New England Biolabs.

2.6. Data analysis Films were scanned and quantitated using the ImageQuaNT image quantitation software. The activities of ERK, JNK and MEK1/2 are expressed as fold induction relative to their uninduced cells of each age. The values represent the mean9 S.D. from three independent experiments. Statistical comparison of the values relative to young animals was performed by a two-tailed, paired Student’s t-test.

3. Results

3.1. Protein tyrosine phosphorylation in response to mitogen stimulation is impaired during aging in rat splenocytes It has been established that the rate and extent of total protein tyrosine phosphorylation (P-Tyr) is impaired as a function of age in T-lymphocytes activated via the TCR in mouse, human and rat (Shi and Miller, 1992; Ghosh and Miller, 1995; Whisler et al., 1997; Pahlavani et al., 1998). We wanted to investigate if this age-related defect was apparent for specific proteins in rat splenocytes activated by PMA plus A23187. Because of our interest in signaling molecules, we focused on the 40 – 50-kDa molecular weight range. The results presented in Fig. 1A indicate that, in this range, protein tyrosine (Tyr) phosphorylation also declines with age after PMA plus A23187 induction of rat splenocytes. A decline in Tyr phosphorylation of proteins in this molecular weight range is already observed in middle-aged animals, and this decrease becomes more dramatic in splenocytes isolated from old rats. Therefore, we conclude that tyrosine phosphorylation in the 40– 50-kDa range in response to PMA plus A23187 is impaired during aging in rats. The same membranes were stripped and reprobed with anti-actin antibody as an internal control (Fig. 1B). The figure shown is representative of three independent experiments that yielded consistent results.

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3.2. Signal transduction is impaired in splenocytes from old rats induced with PMA/A23187 3.2.1. ERK Since ERK activation is dependent upon tyrosine and threonine phosphorylation (Charest et al., 1993; Sugiura et al., 1997), we measured the phosphorylation of ERK proteins after PMA plus A23187 induction in rat splenocytes. For this, the same membranes used in Fig. 1 were stripped and reprobed with an anti-active ERK antibody that recognizes dually phosphorylated (tyrosine and threonine) ERK1 (44 kDa) and ERK2 (42 kDa), but does not significantly recognize the corresponding inactive form of the enzymes. The results shown in Fig. 2A indicate that both ERK activities (ERK1 and ERK2) are induced in response to PMA plus A23187 in splenocytes isolated from rats of all age groups. However, phosphorylation of both ERK1 and ERK2 is slightly but reproducibly diminished in both middle-aged and old animals. The same membranes were stripped and reprobed with an anti-ERK2 antibody (Transduction Labs). The basal and induced ERK2 protein levels did not change (Fig. 2A). As before, the data shown in Fig. 2A are a representative of three independent experiments. Quantitation of this data (Fig. 2B) indicates that the fold induction relative to the basal levels of both ERK1 and ERK2 activities is significantly diminished in both the middle and old age groups, as compared to their younger counterparts (P B 0.05). Thus, the results indicate that, in response to PMA and CaI, the capacity of ERK activation is diminished with age.

Fig. 1. Protein tyrosine phosphorylation in response to mitogen induction is impaired in rat splenocytes during aging. Splenocytes from three individual animals of each age group, young (6 months), middle-aged (15 months) and old (24 months) rats were stimulated with different concentration of PMA/A23187 for 15 min. WCE were prepared, aliquots (35 mg) were resolved on 10% SDS – PAGE gels and Western analysis was performed. Lane 1: untreated; lane 2: PMA (10 ng/ml) plus A23187 (0.05 mg/ml); lane 3: PMA (50 ng/ml) plus A23187 (1 mg/ml). Panel A: Tyrosine phosphorylated proteins were detected using anti-phospho-tyrosine antibody (P-Tyr, PY 20, Transduction Laboratories). Panel B: Anti-actin antibody (ICN) was used to reprobe the same membrane as an internal control. The results shown are representative of three independent experiments.

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Fig. 2. ERK phosphorylation is impaired in response to PMA/A23187 in old rat splenocytes. Panel A: The same membrane as in Fig. 1 was stripped and re-probed with either anti-active ERK antibody (top) or anti-ERK2 antibody (bottom). Panel B: Quantitation of three independent experiments similar to the one shown in panel A. Phosphorylated ERK1 or ERK2 (P-ERK1 or P-ERK2) is represented as fold induction relative to uninduced cells in each age group. The values represent the mean 9 S.D. Stars represent values that display a statistically significant difference relative to young rats (P B0.05).

3.2.2. JNK Since JNK is also an important constituent of the MAPK signaling cascade, we have studied the effect of age on JNK activation. Fig. 3A shows a representative Western blot using an anti-phospho JNK antibody. As in the case of ERK, this antibody detects dually phosphorylated JNK1 (46 kDa) and JNK2 (54 kDa), but does not cross-react with the corresponding phosphorylated form of either ERK or p38 MAPK. The results indicate that JNK is activated only by high, but not by low concentrations of PMA plus A23187. Interestingly, the basal level of JNK activity is relatively high in splenocytes from old rats, as compared to cells from young or middle-aged animals. In addition, splenocytes from old animals display only a weak induction of JNK activity in response to the same concentration of inducers that is

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effective in cells from younger animals. The induced level of JNK activity in old animals is still considerably lower than that observed in younger rats. Further reprobing of the same membranes with an anti-JNK1 antibody that does not distinguish between phosphorylated and dephosphorylated forms of the enzyme

Fig. 3. JNK phosphorylation is impaired in response to PMA/A23187 in old rat splenocytes. Panel A: The same membrane in Fig. 1 was stripped and reprobed with anti-phospho JNK antibody (top) or anti-JNK1 antibody (bottom). Panel B: Quantitation of three independent experiments similar to the one shown in panel A. Phosphorylated JNK1 or JNK2 (P-JNK1 or P-JNK-2) is represented either as arbitrary units (left panels) or as fold induction relative to uninduced cells in each age group (right panels). The values represent the mean 9 S.D. Stars represent values that display a statistically significant difference relative to young rats (P B0.05).

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Fig. 4. MEK1/2 phosphorylation is impaired in response to PMA/A23187 in old rat splenocytes. Panel A: The same membranes as in Fig. 1 above were stripped and reprobed with anti-phospho-MEK1/2 antibody (ser217/221). Panel B: Quantitation of three independent experiments similar to the one shown in panel A. Phosphorylated MEK1/2 (P-MEK1/2) is represented as fold induction relative to uninduced cells in each age group. The values represent the mean 9 S.D. Stars represent values that display a statistically significant difference in fold induction relative to young animals (PB 0.05).

indicates that the steady state level of JNK1 does not change as a function of age (Fig. 3A). Quantitation of the data obtained from three independent experiments is shown in Fig. 3B. It indicates that the absolute activities of JNK1 and JNK2 are statistically diminished in cells from old animals, but not in those from middle-aged ones (P B 0.05). Furthermore, the fold induction of JNK2, but not JNK1, relative to their basal activities is also diminished in splenocytes from old rats as compared to younger counterparts (P B0.05).

3.3. The age-related defect in ERK acti6ation is paralleled, at least in part, by decreased acti6ation of MEK Signaling upstream of ERK involves Ras, Raf-1 and MEK. To date, ERK1/ ERK2 are the only known substrates for MEK1/2 (Crews et al., 1992; Franklin et al., 1994b; Zheng and Guan, 1994), while JNK1/JNK2 are the only known substrates for SEK1 (also known as MKK4 or JNKK) (Davis, 1994; Sanchez et al., 1994; Yan et al., 1994). MEK activity is increased upon stimulation with either anti-CD3, PMA or calcium influx (Franklin et al., 1994b; Rosen et al., 1994), and we measured MEK activity to determine whether there are any defects upstream of ERK upon induction with PMA plus CaI. Fig. 4A shows a representative Western blot using anti-phospho MEK1/2 antibody (New England Biolabs). This antibody selectively recognizes active MEK only when it is phosphorylated at Ser217/221, and does not cross-react with other related family members such as MKK4, MKK3 or MKK6. Hence, it is an excellent marker of MEK1/2 activity. The results indicate

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that MEK is activated to a similar extent with both concentrations of PMA plus A23187, and this activation declines considerably in the old. Quantitation of three independent experiments (Fig. 4B) indicates that the decrease in MEK activation with age is statistically significant (PB 0.05). Thus, we conclude that MEK1/2 activation is also affected by the age of the splenocyte donor, even if the TCR is bypassed. This in turn might affect ERK activation during aging.

3.4. Acti6ation of transcription factors in response to PMA/A23187 is impaired in splenocytes from old rats Signaling downstream of ERK and JNK results in the activation of several transcription factors, including Elk-1, c-myc, c-Fos, c-Jun and ATF-2 (Davis, 1995). Some of these have been shown to play a crucial role in the regulation of downstream genes such as IL-2. Therefore, it was of primary interest to determine whether activation of these factors is diminished during aging as a result of the decreased activation of ERK and JNK. Since we have observed that the age-related defect in JNK activation is more pronounced than that of ERK, we concentrated on a transcription factor activated by this pathway, namely c-Jun. The results shown in Fig. 5A indicate that c-Jun can be phosphorylated in Ser 63 and 73 only by high concentrations of PMA plus A23187, but not by low concentrations of these inducers. Its activation, however, was only barely detectable in splenocytes isolated from old animals (PB 0.05, Fig. 5B). The pattern looks similar to the one observed for JNK activation, which suggests that phosphoryla-

Fig. 5. c-Jun phosphorylation is impaired in response to PMA/A23187 in old rat splenocytes. Panel A: The same membranes were stripped and reprobed with anti-phospho-c-Jun antibody. Panel B: Quantitation of three independent experiments similar to the one shown in panel A. Phosphorylated c-Jun is represented as fold induction relative to uninduced cells in each age group. The values represent the mean9 S.D. Stars represent values that display a statistically significant difference relative to young animals (P B0.05).

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tion of c-Jun is affected in the old animals as a result of the upstream defect of JNK activation.

4. Discussion In this study, we have analyzed the effect of age on both ERK and JNK activation in unfractionated total rat spleen lymphocytes. Bypassing the early events in either TCR- or B-cell receptor (BCR)-directed signal transduction, PMA plus A23187 can substitute for receptor ligation as a primary signal in cell activation (Nau et al., 1988; Kanno and Siebenlist, 1996; Rudd, 1996). ERK activity can be induced by either high or low concentrations of PMA plus A23187, and the induced activities for each age group are comparable with either concentration, indicating that the low concentration of PMA/CaI can induce full activation of ERK. In fact, at least in Jurkat cells, PMA alone can cause full activation of ERK, with no synergistic effect by A23187. However, CaI does have a synergistic effect with PMA on JNK activation, as only the combination of both effectors leads to full activation of JNK (Su et al., 1994 and our own data). IL-2 synthesis requires co-stimulation with PMA plus CaI (Mohagheghpour et al., 1995), while PMA alone can only induce cell surface expression of IL-2R molecules by T-cells, but not IL-2 synthesis. For these reasons, we used both as comitogens, so that the data for ERK and JNK activation could be directly comparable. Our results suggest that splenic lymphocytes from old rats are afflicted by multiple defects within the MAP kinase pathways, some of which are located downstream of those previously described in the literature. In the case of ERK, we have further investigated the activation of the upstream kinases, MEK1 and MEK2, and found them to be impaired as well. While the decrease in MEK activation was more dramatic, and observed later in life, we conclude that, at least in the old animals, part of the diminished activation of ERK can be ascribed to decreased signaling from its upstream kinase. Medrano et al. have reported that senescent and terminally differentiated human melanocytes are unable to phosphorylate tyrosine residues in ERK2 in response to PMA induction, in spite of presenting normal amounts of ERK2 protein (Medrano et al., 1994). In addition, in their experiments, ERK2 did not show the nuclear accumulation observed in proliferating melanocytes after PMA activation and remained localized in the perinuclear area It is likely that a similar situation might apply to aged splenocytes in our study. Although PMA plus A23187 induce ERK1 and ERK2 activities in rat splenocytes even when used at a low concentration, this treatment fails to induce either JNK1 or JNK2 activation. Thus, JNK isozymes were activated in a dose-dependent manner by these inducers. The ability of splenic lymphocytes to phosphorylate JNK is strongly diminished with age. The results suggest that an age-related defect exists in this pathway, even if the TCR is not utilized. Our results also indicate that splenocytes from old rats have a higher level of basal JNK phosphorylation, as compared to either young or middle-aged animals. It is possible that a disturbance in JNK subcellular location with age might prevent dephosphorylation by specific

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phosphatases, which would lead to a gradual accumulation of phosphorylated JNK in the cells. However, we observed no evidence of activation of downstream transcription factors in resting splenocytes from old animals (Fig. 5B, and data not shown). This could be due to lack of activity of the phosphorylated kinases in vivo, possibly due to anomalous subcellular distribution. In principle, splenocytes from old rats could have a disturbance at the level of expression of a JNK-specific MKP. A recent study in hepatocytes has provided evidence that the levels of MKP-1 mRNA are increased in EGF-stimulated hepatocytes from old rats compared to those from young animals (Liu et al., 1996). Thus, it is reasonable to assume that an alteration in the balance between MAPK and MKP activities might contribute to the age-related defects in MAPK activities we have observed in lymphocytes. Consistent with the observed decrease in JNK activation, our results confirm that phosphorylation of c-Jun is also impaired upon PMA plus A23187 induction in rat splenocytes. JNK binds tightly and specifically to the N-terminal region of c-Jun and phosphorylates it at Ser63 and 73 (Derijard et al., 1994; Kyriakis et al., 1994). The phospho c-Jun (Ser63) antibody we have used reacts with phosphorylated c-Jun, but does not cross-react with the corresponding phosphorylated forms of JunD or JunB. In this study, we have used a mixed lymphocyte population, consisting primarily of B-cells, T-cells, and a small amount of macrophages, NK cells, etc. The interactions between B-cells and T-cells are complex and bidirectional, as B-cells present antigen to T-cells, and also receive signals from T-cells for division and differentiation. Such unfractionated splenic lymphocyte populations have therefore been used by many investigators, since they represent a situation that is closer to the in vivo condition, in which different cell types coexist and interact with each other. However, an obvious problem arises when these cells are used in conjunction with relatively non-specific inducers such as PMA plus A23187, which not only activate T-cells but also other cell populations, including B-cells. Both MAPK family members under study, ERK and JNK, are known to be activated in B-cells (Franklin et al., 1994a; Weiss and Littman, 1994and our own data). Thus, we can not infer from our studies which cell population plays a critical role in the decrease in MAPK activation with age. Indeed, it is very likely that a defect in the rate and/or extent of activation of these pathways represents a combined defect of activation of both T- and B-cells in our model system. In summary, our studies have revealed the presence of novel defects in both the ERK and JNK MAPK pathways of signal transduction, even when the receptor proximal defects are bypassed by PMA plus CaI. While we did not measure proliferative responses in parallel, it is well established that proliferation is significantly impaired (30 – 50%) in aged lymphocytes stimulated with either Con A or PMA plus A23187 in several species (Thoman and Weigle, 1988; Holbrook et al., 1989; Negoro and Hara, 1992; Utsuyama et al., 1997). The defects we have observed can be attributed, at least partially, to the decreased activation of upstream signaling molecules, such as MEK or JNKK. Indeed, we have identified age-associated defects in MEK activation. Since both MEK and JNKK are located

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at the same levels in their corresponding signaling cascades, we predict that there might be some age-related defects in JNKK activation as well. Possible mechanisms to explain our results include age-related changes in MAPK phosphatase activities, disturbances in signal molecule subcellular location, and changes in the subpopulation of lymphocytes during aging. Our data complement other investigators’ findings, and further indicate that the age-related defects in lymphocyte activation are not limited to TCR proximal events, but extend to multiple steps in the signaling cascades. The outcome of signal transduction and all the related biochemical changes in response to lymphocyte activation is the induction of cell proliferation and acquisition of immune functions. It is clear that proliferation and differentiation of both T- and B-cells is critical to effective humoral and cell-mediated immune responsiveness to pathogens, and that the proliferative response is diminished during aging. The observed decline in signaling might contribute significantly to the impairment of immune functions during aging.

Acknowledgements This work was supported by grants AG14232 and AG00378 from the NIH, and by a pilot project grant from the Nathan Shock Center of Excellence in Aging Research, AG 13282-05. We are gratefully indebted to Drs Vincent J. Cristofalo and Donna M. Murasko for their unwavering support and to Dr. Mary K Francis for critical reading of the manuscript. We also thank the Murasko lab for their invaluable help in setting the splenocyte model system.

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