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Prevention of Accelerated Cell Aging in Werner Syndrome Using a p38 Mitogen-Activated Protein Kinase Inhibitor

Department of Pathology, School of Medicine, Cardiff University, Wales, United Kingdom.

Address correspondence to David Kipling, D.Phil, Department of Pathology, School of Medicine, Cardiff University, Heath Park, Cardiff CF14 4XN, United Kingdom. E-mail: kiplingd{at}cardiff.ac.uk

	Abstract

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We investigated the role of p38 mitogen-activated protein kinase (MAPK) signalling in the accelerated aging of Werner Syndrome (WS) fibroblasts by use of SB203580, a cytokine-suppressive anti-inflammatory drug that targets p38 activity. SB203580 treatment reverts the aged morphology of young WS fibroblasts to that seen in young normal fibroblasts. In addition, SB203580 increases the life span and growth rate of WS fibroblasts to within the normal range. In young WS cells, p38 is activated coincident with an up-regulation of p21^WAF1, and a reduction in the levels of both activated p38 and p21^WAF1 are seen following treatment with SB203580. As these effects are not seen in young normal cells, our data suggest that the abbreviated replicative life span of WS cells is due to a stress-induced, p38-mediated growth arrest that is independent of telomere erosion. With some p38 inhibitors already in clinical trials, our data suggest a potential route to drug intervention in a premature aging syndrome.

The gene mutated in WS (WRN) encodes a RecQ helicase (4) that interacts with many proteins involved in DNA replication, recombination, and repair (5). Analyses of chromosomes from WS cells show that they have increased numbers of chromosomal aberrations, including deletions, and WS has been classified as a genome instability syndrome (6). However, many but not necessarily all aspects of WS appear to be related to accelerated cell aging. Cultured cells from normal individuals divide only a limited number of times before they enter a state of viable growth arrest termed "cellular senescence" (also known as M1). Senescence has been postulated to contribute to normal human aging (1,2) and is accelerated in WS, with fibroblasts from WS patients having a dramatically reduced cellular replicative life span (7–9). Thus WS cells behave like fibroblasts established from elderly individuals. However, several characteristics of WS cells, such as very slow growth rates, an elongated cell cycle, and a morphology resembling aged normal fibroblasts even at low passages (10–13), suggest that WS is not simply accelerated normal cell aging. These features are reminiscent of cells grown under conditions of stress, and many young WS cells resemble fibroblasts that have undergone oncogenic ras- or arsenite-induced premature senescence (14–17). Premature senescence induced by ras can result from activation of the stress-associated p38 mitogen-activated protein kinase (MAPK) via the activating map kinase kinase 6 (MKK6) (16,17), and the use of the p38 selective inhibitor SB203580 prevents ras-induced senescence in human BJ fibroblasts (17). Activation of p38 leads to the stabilization of the cyclin-dependent kinase inhibitor p21^WAF1 and subsequent cell-cycle arrest (17–19). The similarities between young WS cells and normal cells that have undergone ras-induced premature senescence raise the possibility that the p38 pathway may play a role in the premature senescence seen in WS cells.

The genome instability seen in WS, together with the frequent replication fork stalling seen in WS cells (13), provides a plausible trigger for replication stress in WS cells and a possible involvement for p38 signalling in inducing the shortened replicative life span. We therefore tested the role of p38-transduced stress signalling in WS using SB203580, a cytokine-suppressive anti-inflammatory drug whose major inhibitory target is p38 (20), and primary fibroblasts derived from dermal biopsies of three separate WS patients. Our data revealed an unexpected reversal of the aging phenotype of drug-treated WS fibroblasts, which showed a much-increased replicative life span and growth rate and a morphology that resembled that of young normal cells. If the shortened in vitro life span seen in untreated WS cells does contribute to the accelerated aging seen in WS individuals, this observation opens the route to possible drug intervention in a premature aging disease.

	METHODS

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Cells and Cell Culture
WS fibroblast cells derived from biopsies of human tissue (AG03141, AG05229, and AG12795) were obtained from the Coriell Cell Repository (Camden, NJ), MRC5 cells were obtained from the European Collection of Cell Cultures (Salisbury, U.K.), and WI38 cells were obtained from the American Type Culture Collection (Manassas, VA). All these are primary cells. The telomerized AG03141 cells were produced by infecting primary AG03141 cells with a retrovirus construct containing the catalytic subunit of human telomerase (21). Immortal clones were selected, and AG03141.hTERT clone 8 was used in this study, as it is representative of the various immortal clones for growth rates (21). Cells were grown in Dulbecco's modified Eagle medium as previously described (22). Population doublings (PD) were calculated according to the formula: PD = log(N_t/N_o)/log2, where N_t is number of cells counted and N_o is number of cells seeded.

For drug treatments, the culture medium was supplemented with 10 µM SB203580 (Tocris Chemical Co., Bristol, U.K.) dissolved in dimethyl sulfoxide (DMSO), with the medium being replaced daily. For controls, an equivalent volume of the drug solvent (DMSO) was added to the medium. We used SB203580 at 10 µM, as this concentration is used routinely for studying the effects of SB203580 on p38 activity in cell biological systems (16,19,20).

DNA Synthesis Assays
DNA synthesis was assayed by labelling cells in the presence of 10 µM bromodeoxyuridine (BrdU) for 1 hour, following which BrdU incorporation was detected as previously described by immunoperoxidase (22). The rate of exit of cells from the cell cycle was calculated according to (9).

Telomere Length Analysis
DNA extraction and the determination of XpYp telomere lengths by single telomere length analysis (STELA) were as previously described (23).

Immunoblot Analysis
Protein samples were prepared in lysis buffer containing the phosphatase inhibitors NaF and Na₃VO₄, separated on 12% sodiumdodecyl sulfate–polyacrylamide electrophoresis gels, and electroblotted to Immobilon-P polyvinylidene difluoride membrane (Millipore, Watford, U.K.); antibodies were applied as described previously (22). The antibodies used were: mouse monoclonal anti-p21^WAF1 (6B6; Becton Dickinson, Oxford, U.K.); mouse monoclonal anti-heat shock protein 27 (HSP27; G31), rabbit polyclonal anti-phospho(Ser82)-HSP27, anti-p38, anti-phospho(Thr180/Tyr182)-p38 (Cell Signaling, New England BioLabs, Hitchin, U.K.). An enhanced chemiluminescence kit (Amersham, Little Chalfont, Buckinghamshire, U.K.) was used for visualization using horseradish peroxidase-coupled goat secondary antibodies. After use, the filter was stained with India ink. Quantification of the specific signal and the amount of protein loaded for the immunoblot was performed using a Bio-Rad (Hemel Hempstead, U.K.) imaging densitometer with Molecular Analyst (Bio-Rad) software.

Immunofluorescence Microscopy
Actin staining for immunofluorescence microscopy was performed essentially as described (14). Briefly, the cells were plated into 35-mm plastic dishes in DMEM and allowed to settle for 48 hours. The cells were then washed in phosphate-buffered saline (PBS), fixed in 3.7% paraformaldehyde for 20 minutes, and permeabilized with 0.1% Triton X-100 for 20 minutes. F-actin was detected using fluorescein isothiocyanate-conjugated phalloidin (33 µg/ml), diluted 1:50 in PBS for 30 minutes in the dark, followed by washing in PBS.

	RESULTS

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Altered Growth Kinetics and Morphology in WS Cells Treated With SB203580
To assess the effect of treatment of primary WS fibroblasts with SB203580, cells were grown continuously in the presence or absence of the drug until they reached M1 senescence. As shown in Figure 1, control (untreated) AG05229 WS fibroblasts reached M1 after 18.2 PD and had a growth rate of 0.13 PD/day (Figure 1A, Table 1). This is typical for WS fibroblast cultures: By comparison, normal adult fibroblasts typically show a growth rate of >0.4 PD/day and a life span of 30–60 PD (7–9). Drug treatment had a striking and immediate effect on WS cell growth; AG05229 cultures supplemented with SB203580 had an increased growth rate of 0.34 PD/day and achieved a total of 43.7 PD (Table 1), which is now within the range expected for normal fibroblasts. The effect of SB203580 on the growth rate and life span of AG05229 fibroblasts was reversible as removal of SB203580 reduced the growth rate to that seen for untreated cells, with the cells having an intermediate life span (Figure 1A). Restoration of drug supplementation resulted in an increased growth rate and life span; however, the life span was shorter than that seen in cells treated continuously with SB203580. The effects on cell growth were inversely proportional to the replicative age of the cultures when drug treatment began, with SB203580 having no effect on senescent WS fibroblasts (not shown). These results are not restricted to AG05229 cells; a similar effect of SB203580 on growth rate and replicative capability was found for the WS strain AG03141 (Figure 1B), and a smaller effect was seen for the WS strain AG12795 (Figure 1C). The effects of SB203580 on both the growth rate and life span were highly statistically significant and reversible in all three WS strains (Table 1). In contrast, SB203580 had a statistically insignificant effect on the growth of normal MRC5 fibroblasts (Figure 1D, Table 1). In addition, single cultures of WI38 cells had a similar growth rate and replicative life span in the absence (0.44 PD/day and 65 PD) or presence (0.41 PD/day and 62.5 PD) of SB203580.

View larger version (65K): [in this window] [in a new window]   Figure 1. Growth parameters of Werner Syndrome (WS) cells with or without treatment with SB203580. A, AG05229 cells; B, AG03141 cells; C, AG12795 cells; D, MRC5 cells. Fibroblasts were grown in standard Dulbecco's modified Eagle medium with no supplementation (), with continual daily supplementation with SB203580 (•), with SB203580 before drug removal [point a] (), and with SB203580 for 29 days followed by drug removal [point a] for 14 days and then restoration of the supplementation regime [point b] (). Growth measured as population doublings (PD) versus days. Rate of exit from the cell cycle for (E) AG05229 cells and (F) AG03141 cells, measured as the percentage of cells that label in a 1-hour bromodeoxyuridine (BrdU) pulse, versus population doublings. Untreated cells (), SB203580-treated cells (•). G and H, Phase contrast pictures of AG05229 WS fibroblasts grown in the absence or presence of SB203580; bar = 100 µm. I, AG03141.hTERT clone 8 cells grown in standard Dulbecco's modified Eagle medium with no supplementation (), with continual daily supplementation with SB203580 (•), or with SB203580 before drug removal [point a] ()

Morphologically, even at low passages, untreated AG05229 fibroblasts appeared aged and similar to aged normal fibroblasts, in that they were enlarged and granular (Figure 1G). Growth in SB203580 restored the morphology of AG05229 fibroblasts to that seen for young normal fibroblasts (Figure 1H) until the point when the culture finally senesced, when they assumed an irregular "senescent" morphology (not shown). SB203580 had a similar effect on the WS AG03141 and AG12795 cells, but had no effect on the morphology of MRC5 or WI38 cells (not shown).

Visualization of F-Actin Stress Fibers in WS Fibroblasts
Cells growing under conditions of stress, e.g., during exposure to H₂O₂ or arsenite, are often enlarged with the presence of prominent F-actin stress fibers (14,15). In addition, cells that have reached M1 have a high level of such fibers, whereas young growing cells do not (25). We compared normal cells at medium and high PD values stained with phalloidin-fluorescein isothiocyanate to visualize the F-actin fibers. Young MRC5 cells were small in morphology with few actin fibers visible (Figure 2A). At M1, MRC5 cells were enlarged with numerous actin stress fibers visible (Figure 2C). MRC5 cells grown in the presence of SB203580 were similar to untreated cells (Figure 2, B and D). Similar results were seen using WI38 cells (not shown). In contrast, many of the WS cells were enlarged with numerous prominent stress fibers even at the lowest PD obtainable, as illustrated for both AG03141 and AG05229 cells (Figure 2, E and I). Indeed, the young WS cells had more prominent stress fibers than did normal cells at M1, even though the WS cells were from growing cultures; however, some additional cells were present that did have a typical young-looking morphology (arrow in Figure 2I). When the WS cells were grown in the presence of SB203580, the morphology resembled that of young normal cells, although some stress fibers were still visible (Figure 2, F and J). WS cells at M1 resembled normal senescent cells, irrespective of SB203580 treatment (Figure 2, G, H, K, and L).

View larger version (79K): [in this window] [in a new window]   Figure 2. Observation of actin stress fibers in Werner Syndrome (WS) and normal cells. Young, senescent, and SB203580-treated cells were fixed and stained with fluorescein isothiocyanate-conjugated-phalloidin. A–D, MRC5 cells; E–H, AG03141 cells; I–L, AG05229 cells. The actual population doubling (PD) values for the cell samples are indicated below each panel. Bar = 50 µm. Arrow in (I) indicates a WS cell with a normal morphology

The ability of ectopic expression of telomerase to immortalize WS fibroblasts indicates that WS cultures can senesce as a result of telomere erosion. However, when compared to normal cells, telomerized WS cells still exhibit the slow growth characteristic of primary WS cells (21). The growth rate for a typical telomerized WS culture (AG03141.hTERT clone 8) was 0.31 ± 0.01 PD/day (Figure 1I). In the presence of SB203580, this rate significantly increased to 0.53 ± 0.02 PD/day (p <.0037), a rate that was not significantly different to control telomerized HCA2 cells (0.55 ± 0.07 PD/day; p <.65) (21). Upon drug removal, the growth rate of AG03141.hTERT clone 8 reverted to that seen in untreated cells (0.30 ± 0.05 PD/day; p >.86).

Characterization of p38 Effector Pathways in SB203580-Treated Cells
To assess the activation status of p38 in normal and WS fibroblasts, proteins were extracted from young and senescent AG03141 and MRC5 cells grown in the presence or absence of SB203580, and were probed with antibodies specific for p38 and its activating phosphorylation (Figure 3A). Phosphorylated p38 was present in young untreated AG03141 cells indicating p38 activation in these cells. A similar level of phosphorylated p38 was seen in untreated WS cells at M1. Young AG03141 cells grown in the presence of SB203580 had a reduced level of phosphorylated p38; however, when AG03141 cells that had been continuously treated with SB203580 finally reached M1, the level of phosphorylated p38 was now similar to that seen in untreated cells at M1 (Figure 3A). In contrast, only low levels of phosphorylated p38 were seen in young MRC5 cells, and this increased when the cells reached M1, in agreement with previous work (20); SB203580 treatment did not affect this.

View larger version (69K): [in this window] [in a new window]   Figure 3. Immunoblot analysis of proteins from cells grown in the presence or absence of SB203580. Protein lysates were prepared from: (A) young (Y) and senescent (M1) primary AG03141 and MRC5 cells and (B) AG03141.hTERT clone 8 cells. Expression levels were compared for phosphorylated p38 (pp38), p38, p21WAF1, phosphorylated heat shock protein 27 (pHSP27), and HSP27. Protein loadings were normalized with regard to p38

Activated p38 is known to phosphorylate and stabilize p21^WAF1 (18). Consistent with the activation of p38 in young WS cells, a high level of total p21^WAF1 seen in untreated young AG03141 cells was reduced by SB203580 treatment (Figure 3A). p21^WAF1 was detectable in both treated and untreated AG03141 cells at M1, albeit at a lower level that that seen in untreated young AG03141 cells. With MRC5 cells, the level of p21^WAF1 was low in untreated young cells and increased as the cells reached M1, in agreement with previous reports (29). The same pattern of p21^WAF1 expression was seen in MRC5 cells treated with SB203580 (Figure 3A).

Phosphorylated p38 was also present in untreated AG03141.hTERT clone 8 cells and, as with the young primary WS cells, the level was reduced by SB203580 treatment (Figure 3B). This phosphorylated p38 was associated with high levels of p21^WAF1 that decreased upon SB203580 treatment. Despite the high p21^WAF1 levels in the telomerized cells, this was a growing culture. As the immunoblot analysis measures an average level of p21^WAF1 for the whole cell population, it is possible that some of the cells had a high p21^WAF1 level and were growth arrested, whereas the rest had low p21^WAF1 levels and were thus growing. This would provide an explanation for the slow growth rate of the culture.

	DISCUSSION

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One of the hallmarks of WS is the limited proliferative growth potential coupled with reduced growth rates of fibroblasts from WS individuals (7–9). It is this reduced growth that has been argued by several authors to underlie the accelerated aging seen in WS [reviewed in (2)].

Normal fibroblasts enter replicative senescence as a result of telomere shortening (26). The short telomeres signal via the tumor suppressor protein p53, which up-regulates p21^WAF1 and shuts down the cell cycle (30). Recent evidence has suggested that in addition to telomere shortening, stress signalling via p38 may play a role in establishing M1 in normal cells. Treatment of WI38 cells with SB203580, a p38-specific inhibitor, increased the growth rate of cells that were almost senescent, but had no effect on young cells (20). In addition, inhibition of p38 using a dominant negative form of the activating kinase MKK6 increased the replicative potential by a few PD (20). Moreover, activated p38 was present in WI38 and MRC5 cells at M1, but not in young cells. These data suggest that the short telomeres in older cells may provide a stress signal in addition to the telomere-driven p53 activating signal; this conclusion is supported by the reduction in activated p38 seen in PD-matched cells immortalized by telomerase expression (20). This activated p38 appears to be involved in cell-cycle arrest by stabilizing p21^WAF1, suggesting that the two signals may act in a coordinate fashion (18,19).

This short-telomere-induced stress signal appears to be conserved in old WS cells, as these cells have activated p38 at M1 (Figure 3). In addition, the lack of p38 activation in drug-treated telomerized WS cells is consistent with the lack of telomere shortening that is expected in a telomerase-expressing culture. These data add to the growing body of evidence that the pathways leading to M1 in old WS cells strongly resemble those in old normal cells (21,22,31,32), and indicate that the maximal life span of a WS culture is determined by telomere erosion. As the rate of telomere erosion per cell division is not increased in WS cultures (28), this then raises the question of what causes the shortened replicative life span in WS cultures.

The observation that oncogenic ras- or arsenite-induced premature senescence of fibroblasts is due to activation of p38 (14–16), and that constitutive activation of p38 can induce a proliferative arrest that is very similar to normal senescence (19), shows that p38 can play a significant role in such examples of stress-induced senescence. One of the features of senescent cells, whether they occur via stress or telomeric erosion, is an enlarged morphology and the presence of prominent stress fibers. Stress fiber formation depends on phosphorylation of HSP27, a downstream target of the p38 signalling pathway (14,15). Young WS cells show several attributes suggesting that they suffer stress, including slow growth rates and an enlarged morphology with prominent stress fibers. Indeed, many young WS cells have the hallmarks of cells that have undergone stress-induced premature senescence (16). This fact suggests that, in addition to its role at M1, p38 may be active in young WS cells leading to a proportion of cells with a premature replicative arrest.

To test this idea, we cultured WS cells continuously in medium supplemented with the p38 inhibitor SB203580. The growth rate and replicative life span of the WS cultures were significantly increased, in some cases to normal, with the cells (at low PD) having largely reverted to a young-looking morphology. Drug treatment had no significant effects on normal cells; this finding suggests that the abnormal growth kinetics, enlarged morphology at low PD, and premature senescence of WS cultures result from the activation of an SB203580-suppressible pathway. That the p38 stress pathway is indeed activated in young WS cells was shown by the presence of phosphorylated p38, associated with high levels of p21^WAF1 and phosphorylated HSP27. SB203580 treatment suppressed the p38 phosphorylation and p21^WAF1 levels, and resulted in a much increased growth rate. As this was not seen in control MRC5 cells, it suggests that p38 signalling via p21^WAF1 may induce a significant degree of cell-cycle arrest in young WS cells, thus causing the slow growth rate of WS cultures.

The stress signal associated with shortened telomeres is transduced via the p38 activating kinase MKK3/6 (20), which is the upstream p38-activating kinase (33). SB203580 treatment does not prevent the activation of p38 at M1, showing that it does not inhibit MKK3/6. However, the activation of p38 seen in young AG03141 cells is prevented by SB203580, suggesting that p38 activation in young AG03141 cells results from the activation of a pathway distinct from that in old AG03141 cells. This is supported by the presence of SB203580-suppressible p38 and p21^WAF1 in telomerized WS cells and indicates that the stress is still present despite the immortalization, and thus may explain the slow growth rates of telomerized WS cultures (21).

One possible transducer of this second signal is TAB1 (transforming growth factor ß-activated protein kinase 1-binding protein 1), an adaptor protein that has been reported to regulate p38 autophosphorylation. Inhibition of p38 by SB203580 would prevent its TAB1-mediated autophosphorylation (34). Alternatively, it is possible that SB203580 could inhibit a kinase that is upstream of MKK6. However, irrespective of the actual signalling pathway, it is clear that young WS cells have activated a p38 stress signal that would cause a premature cell-cycle arrest via p21^WAF1 stabilization. As telomeric erosion is the ultimate determinant of the life span of a WS culture (21), this stress pathway would be able to synergize with telomere-driven arrest to produce the shortened replicative life span seen in WS cells.

That the replicative life span of some SB203580-treated WS fibroblast cultures was within the range observed for normal fibroblasts is remarkable given that we were unable to commence drug supplementation until a significant number of PD had already elapsed (11 from a total of 16 PD for AG12795, i.e., 70% of its life span). Our data suggest that the growth rate and proliferative life span were inversely proportional to the proliferative age when treatment began, suggesting that larger increases in life span may be expected if treatment were to begin at the initiation of the WS cultures. Our calculation for AG12795 is based on the increase in experimental life span with drug treatment of 2.52-fold (5.4 to 13.6 PD based on starting at PD11). If drug treatment had commenced at the initiation of the culture, the life span of AG12795 may have been extended by 11 PD x 2.52 (= 27.7 PD) and thus may have reached 27.7 + 13.6 = 41.3 PD. The respective total life span for the AG05229 and AG03141 cultures may have been at least 59 and 48 PD, respectively. All these life-span figures are well within the range for normal human fibroblasts (7–9).

Our observations identify a small molecule treatment that can largely prevent the in vitro aging phenotype of cells from patients with a premature aging syndrome, based on a variety of cellular and molecular hallmarks. This accelerated senescence in vitro is a postulated causal mechanism for the premature aging seen in vivo (1,2); it is intriguing that p38 has been implicated in cardiovascular disease, diabetes, and osteoporosis, all of which are features of WS. If premature aging of cells and p38 activation do underlie the accelerated aging of WS individuals, then the recent development of a fully reflective mouse model of WS (35) now makes future studies possible as to whether p38 inhibitors will be effective as therapeutic agents in a premature aging disease. Some of these p38 inhibitors are undergoing Phase II and III clinical trials for inflammatory disease (36).

	Acknowledgments

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This work was funded by the Biotechnology and Biological Sciences Research Council's Experimental Research on Ageing Initiative and Research into Ageing (RiA).

	Footnotes

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Decision Editor: James R. Smith, PhD

Received May 9, 2005

Accepted June 20, 2005

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