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Vol. 15, Issue 2, 922-933, February 2004
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Mitogen-activated Protein Kinase Sensitizes Cells to Apoptosis Induced by Different Stimuli
* Departamento de Bioquimica y Biologia Molecular II (Centro Mixto UCM/CSIC), UCM, Ciudad Universitaria, 28040 Madrid, Spain;
|| Centro de Citometría de Flujo y Microscopía Confocal, Facultad de Farmacia, UCM, Ciudad Universitaria, 28040 Madrid, Spain; and
European Molecular Biology Laboratory, 69117 Heidelberg, Germany
Submitted August 14, 2003;
Accepted October 26, 2003
Monitoring Editor: Carl-Henrik Heldin
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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mitogen-activated protein (MAP) kinase is a broadly expressed signaling molecule that participates in the regulation of cellular responses to stress as well as in the control of proliferation and survival of many cell types. We have used cell lines derived from p38 knockout mice to study the role of this signaling pathway in the regulation of apoptosis. Here, we show that cardiomyocytes and fibroblasts lacking p38 are more resistant to apoptosis induced by different stimuli. The reduced apoptosis of p38-deficient cells correlates with decreased expression of the mitochondrial proapoptotic protein Bax and the apoptosis-inducing receptor Fas/CD-95. Cells lacking p38 also have increased extracellular signal-regulated kinase (ERKs) MAP kinase activity, and the up-regulation of this survival pathway seems to be at least partially responsible for the reduced levels of apoptosis in the absence of p38. Phosphorylation of the transcription factor STAT3 on Ser-727, mediated by the extracellular signal-regulated kinase MAP kinase pathway, may contribute to the decrease in both Bax and Fas expression in p38-/- cells. Thus, p38 seems to sensitize cells to apoptosis via both up-regulation of proapoptotic proteins and down-regulation of survival pathways. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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, 
, 
, and 
, also known as stress-activated kinase (SAPK)2a, SAPK2b, SAPK3, and SAPK4, respectively, which may have both overlapping and specific functions (reviewed by Cohen, 1997
; Nebreda and Porras, 2000; Ono and Han, 2000; Kyriakis and Avruch, 2001). p38 is broadly expressed and is also the most abundant p38 family member present in most cell types. Targeted inactivation of the mouse p38 gene results in embryonic death due to a placental defect (Adams et al., 2000; Mudgett et al., 2000; Tamura et al., 2000). Studies using cell lines have suggested an important role for p38 MAPKs in the regulation of cardiomyocyte differentiation, hypertrophy, and apoptosis (Wang et al., 1998; Zechner et al., 1998; Mackay and Mochly-Rosen, 1999; Saurin et al., 2000; Stephanou et al., 2000). However, p38 does not seem to play a critical role in the development of the embryonic heart in mice (Adams et al., 2000), although heterozygous p38+/- mice have been reported to be less sensitive to myocardial cell death caused by ischemia-reperfusion (Otsu et al., 2003). In addition, cardiac-specific expression of dominant negative forms of p38 and their activators MKK3 and MKK6 enhances cardiac hypertrophy in transgenic mice and/or makes the mice resistant to cardiac fibrosis (Braz et al., 2003; Zhang et al., 2003b).
The role of p38 MAPKs in apoptosis depends on the cell type and the stimuli (reviewed by Nebreda and Porras, 2000). Most of the data available are based on the effect of either chemical inhibitors such as SB203580 and SB202190 (which can inhibit both p38 and p38) or the overexpression of mutant forms of the p38 MAPKs and their activators.
Anti-apoptotic roles of p38 MAPKs have been described in cardiomyocytes (Zechner et al., 1998), DNA-damaged fibroblasts (Héron-Milhavet et al., 2002), endothelial cells exposed to anoxia-reoxygenation (Zhang et al., 2003a), differentiating neurons (Okamato et al., 2000), and activated macrophages (Park et al., 2002). In some cases, prevention of apoptosis by p38 MAPKs has been associated with the regulation of transcription factor activity (Okamato et al., 2000; Park et al., 2002). On the other hand, there is also good evidence for a role of p38 MAPKs as mediators of apoptosis, for example, in neurons (Le-Niculescu et al., 1999; Ghatan et al., 2000; Ciesielski-Treska et al., 2001; DeZutter and Davis, 2001) and cardiac cells (Wang et al., 1998; Mackay and Mochly-Rosen, 1999; Saurin et al., 2000). In other cell types, p38 MAPKs can also have proapoptotic roles, for example, upon stimulation with tumor necrosis factor- (Valladares et al., 2000), transforming growth factor- (Edlund et al., 2003), or in response to oxidative stress (Zhuang et al., 2000). The mechanisms by which p38 can contribute to an enhanced apoptotic response include the phosphorylation and/or translocation of proteins from the Bcl-2 family, which leads to the release of cytochrome c from the mitochondria (Ghatan et al., 2000; Zhuang et al., 2000; Torcia et al., 2001), the transforming growth factor--induced activation of caspase 8 (Schrantz et al., 2001) as well as the regulation of membrane blebbing and nuclear condensation (Deschesnes et al., 2001). At the transcriptional level, expression of monoamine oxidase (DeZutter and Davis, 2001) or growth arrest and DNA damage (GADD)-inducible genes (Sarkar et al., 2002) have both been shown to mediate proapoptotic effects of p38 MAPKs.
We have investigated the apoptotic response in different types of p38-deficient cells: primary fibroblasts and immortalized cardiomyocytes and fibroblasts. We have found that p38-deficient cells were all more resistant to apoptosis induced by different stimuli. Reduced apoptosis in the absence of p38 correlates with the down-regulation of the proapoptotic proteins Fas and Bax as well as enhanced activity of the extracellular signal-regulated kinase (ERK) MAPKs survival pathway.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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) or by transfection with simian virus 40 (SV40) large T antigen constructs (Benito et al., 1993). Cells expressing large T were selected with G-418.
Cardiomyocytes were isolated from E9.5 embryos obtained by intercrossing animals heterozygous for both p38 and the immorto transgene allele, which drives expression of a temperature-sensitive large T under the control of the interferon- (IFN-)-inducible H-2K promoter (Jat et al., 1991). Cardiomyocytes were isolated by trypsinization of embryonic hearts as described previously (Adams et al., 2000). Cardiomyocytes were grown on collagen-coated tissue culture plates in DMEM containing 10% fetal bovine serum (Invitrogen), IFN- (10 U/ml; Sigma-Aldrich, St. Louis, MO), and cardiotrophin-1 (0.2 ng/ml; R&D Systems, Minneapolis, MN) at 33°C in a humidified atmosphere of 5% CO2 or, when indicated, at 37°C in the absence of IFN-.
Primary MEFs were derived from E11.5 and E12.5 embryos obtained by crossing heterozygous p38+/- mice, as described previously (Ambrosino et al., 2003). For the rescue experiments, two rounds of retroviral infection were performed as described in Ambrosino et al. (2003).
Transfection of Bax and p38 Constructs
The mouse Bax and human p38 cDNAs were cloned into the EcoRI site of the pEFmlink expression vector. Transient transfections with the Bax and p38 constructs were performed using the MBS transfection kit (2003-88-51; Stratagene, La Jolla, CA), a modified calcium phosphate method. The protocol supplied by the manufacturer was followed using 10 µg of DNA per dish (1.5 x 106 cells). Cells were analyzed 24-48 h after transfection.
Induction of Apoptosis
To induce apoptosis, cells were treated as follows: UV stimulated for 10 min followed by 30 min - 3h in the 37°C incubator; serum deprived for 24-48 h; and incubated either with 1-2 µg/ml of the agonist anti-Fas antibody Jo2 (15401D; BD Biosciences PharMingen, San Diego, CA) or with 2 µM of the Ca2+ ionophore A23187
[GenBank]
for different periods. Before these treatments, cardiomyocytes were transferred to 37°C and maintained without IFN- for 16-18 h.
For 4,6-diamidino-2-phenylindole (DAPI) staining, cells were washed with phosphate-buffered saline (PBS), fixed in 1.5% paraformaldehyde (prepared in PBS at pH 7.2) for 20-30 min, washed with PBS, and stained with 1 µM DAPI for 15 min in the dark. After washing with PBS, nuclear morphology was analyzed by fluorescence microscopy.
Apoptotic cells were quantified by flow cytometry. Cells were trypsinized, washed with PBS, and fixed with cold ethanol (70% vol/vol). The cells were then washed and resuspended in PBS and incubated with RNase (25 µg/106 cells) for 30 min at 37°C. After addition of 0.05% propidium iodide, cells were analyzed in the cytometer.
Fluorometric Analysis of Caspase 3 Activity
Caspase 3 activity was determined by fluorometric quantification of the fluorogenic substrate Ac-DEVD-AMC (66081; BD Biosciences PharMingen). Cells were lysed by incubation for 10 min at 4°C in a buffer (125 µl/6-cm plate) containing 10 mM Tris (pH 7.5), 130 mM NaCl, 1% Triton X-100, and 10 mM Na F. Cell lysates (2-10 µg of total protein) were incubated at 37°C for 1.5 h in a buffer containing 20 mM HEPES (pH 7.5), 10% glycerol, 2 mM dithiothreitol, and 20 µM Ac-DEVD-AMC. A blank without lysate was prepared in parallel. The fluorescent AMC liberated by caspase 3 activity was quantified in a fluorometer with a fixed excitation wavelength of 380 nm and an emission wavelength range of 430-460 nm.
Western Blot Analysis
Western blot analysis was carried out as described previously (Adams et al., 2000). Proteins were separated by electrophoresis by using Anderson gels and transferred to nitrocellulose membranes that were probed with the following antibodies: active cleaved caspase 3 (9661; Cell Signaling Technology, Beverley, MA), Bax (sc-526; Santa Cruz Biotechnology, Santa Cruz, CA), BclxL (sc-634; Santa Cruz Biotechnology), Bid (AF860; R&D Systems), p38 (sc-535; Santa Cruz Biotechnology), p53 (antibody-1 0P03; Calbiochem, San Diego, CA), phospho-Akt (9271; Cell Signaling Technology), phospho-ERK1/2 (9101S; Cell Signaling Technology), STAT1 (sc-464; Santa Cruz Biotechnology), phospho-Ser727-STAT3 (sc-8001R; Santa Cruz Biotechnology), and phospho-Tyr705-STAT3 (9131; Cell Signaling Technology).
Total cell extracts were obtained as described previously (Adams et al., 2000) except for the analysis of the levels of active caspase 3 and Bid proteolytic fragment, which were prepared as described above for the fluorometric analysis of caspase 3 activity. Nuclear extracts were prepared as described previously (Valladares et al., 2000).
Cytometric Quantification of Fas Expression
Fas expression in cardiomyocytes was quantified by flow cytometric analysis. Cells were grown at 33°C, and trypsinized, washed, resuspended in PBS (1-1.5 x 106 cells per assay), and incubated for 30 min at 4°C with 2.5 µg of the anti-Fas antibody Jo2 conjugated with fluorescein isothiocyanate (15404D; BD Biosciences PharMingen). After washing, cells were incubated with propidium iodide (0.005%) and analyzed in the cytometer. The percentage of cells labeled with the anti-Fas antibody was considered positive for Fas expression, whereas the relative fluorescence intensity was recorded as a measure of the amount of Fas expression per cell.
Semiquantitative Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and Northern Blot
Total RNA (2 µg) was isolated with the TRIzol reagent (15596-026; Invitrogen) and used for cDNA synthesis with the SuperScript II RT kit (1098-018; Invitrogen). The following primers were used for PCR: Fas-5' ATC CGA GCT AGG AGG CGG GTT CAT GAA AC and Fas-3' GGT TCT AGA TTC AGG GTC ATC CTG (Hsu et al., 1999); p38-5' TGC ATA ATT TTC TGA ATT TTG and p38-3' TCC TAT GGC ATA CCA GAT TAC; and GAPDH-5' CAG TAT GAC TCC ACT CAC GGC and GAPDH-3' GAG GGG CCA TCC ACA GTC TTC.
Total RNA was analyzed by Northern blotting as described previously (Valladares et al., 2000).
Statistical Analysis
Statistical analysis was carried out by Student's t test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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in apoptosis, we first compared the effect of serum withdrawal in wild-type (wt) and p38-deficient cardiomyocytic cell lines. After 48 h of serum deprivation, phase-contrast microscopy evidenced the presence of a high number of wild-type (wt) cardiomyocytes with the characteristic morphology of apoptotic cells. This included the loss of cellular contacts, appearance of cellular blebbing, and finally detachment of many cells from the plate (Figure 1A, left). In contrast, most of the p38-/- cells remained attached to the plate, and the apoptotic phenotype was only observed in a few cells (Figure 1A, right). Analysis of nuclear morphology by fluorescence microscopy after DAPI staining also indicated the presence of a higher number of condensed and/or fragmented nuclei in wt cardiomyocytes (18%) than in p38-/- cells (5%) (Figure 1B). In addition, the percentage of cells with DNA content lower than 2C (as determined by flow cytometry analysis) was also higher in wt than in p38-deficient cardiomyocytes, either serum deprived or maintained in 10% serum (Figure 1C). Treatment with the p38 MAPK inhibitor SB203580 strongly reduced the number of apoptotic wt cells, in particular upon serum withdrawal, whereas having little effect on p38-/- cells. These results support a connection between p38 and the increased levels of apoptosis observed in wt cells.
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p38-deficient Cells Are More Resistant to Apoptosis Induced by Different Stimuli
The reduced susceptibility of p38-lacking cardiomyocytes to apoptosis upon serum deprivation prompted us to investigate other proapoptotic stimuli. Quantification by cytometry of the percentage of cells with DNA content lower than 2C also showed that treatment with UV or the Ca2+ ionophore A23187
[GenBank]
also induced a higher number of apoptotic cells in wt than in p38-/- cardiomyocytes (Figure 2A). p38-/- cells were also more resistant to other proapoptotic stimuli such as tumor necrosis factor- or staurosporine (our unpublished data). Together, these data indicate that the presence of p38 sensitizes cardiomyocytes to apoptosis, both under basal conditions and in response to different apoptotic stimuli. It should be mentioned, however, that basal apoptosis varied depending on cell density, being significantly higher (around twofold) in wt confluent cells than in cells maintained at a lower density (our unpublished data).
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Similar results were observed by comparing wt and p38-/- immortalized MEFs. The percentage of apoptotic cells, as determined by flow cytometry analysis of DNA content, was always higher in wt immortalized MEFs, either maintained in 10% serum or upon serum withdrawal or incubation with A23187
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(Figure 2B). Therefore, the proapoptotic effect of p38 seems to be neither cell type nor stimulus specific.
Cardiomyocytes were usually grown at 33°C with IFN- to induce large T expression. To investigate the effect of different culture conditions, cardiomyocytes were grown at 37°C in the absence of IFN- for 18 h before the induction of apoptosis. Quantification of caspase 3 activity by using a fluorogenic substrate showed that it was approximately twofold higher in wt than in p38-/- cells maintained in the absence of serum for 48 h (Figure 3A, histogram). As expected, the levels of caspase 3 activity were reduced in cells maintained in 10% serum, but this activity was also 
2-3 times higher in wt than in p38-/- cells. In cells maintained at 33°C, caspase 3 activity was generally reduced, but the differences between wt and p38-/- cells were also maintained. These results were confirmed by immunoblots with an antibody that specifically detected active (cleaved) caspase 3 (Figure 3A, top) and cytometric quantification of the percentage of hypodiploid cells (Figure 3B). Therefore, the reduced levels of apoptosis in cardiomyocytes lacking p38 compared with wt cells is affected neither by the presence of IFN- nor by the growth temperature. MEFs lacking p38 also show reduced levels of caspase 3 activity (measured by using a fluorometric substrate) upon treatment with UV or A23187
[GenBank]
(our unpublished data).
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Down-Regulation of Bax Expression in p38-deficient Cells
To investigate the molecular basis for the reduced apoptosis observed in cells lacking p38, we analyzed the expression levels of several pro- and antiapoptotic proteins. We first studied these differences in cardiomyocytes, where apoptosis levels were higher than in MEFs. No differences were found in the expression levels of BclxL and p53 proteins, either in the presence or absence of serum for 48 h (Figure 4A). Likewise, Bcl-2 and phospho-Bad levels were similar in wt and p38-/- cells (our unpublished data), whereas Bim protein expression was detected neither in wt nor in p38-deficient cells. In contrast, the levels of the proapoptotic protein Bax were higher in wt cells, either with or without serum deprivation (Figure 4A). Consistent with the idea that p38 positively regulates Bax expression, incubation with the p38 inhibitor SB203580 decreased Bax protein levels in wt cells (Figure 4B). Moreover, Bax mRNA expression was also increased in wt cells compared with p38-deficient cells (Figure 4C), suggesting that p38 up-regulates Bax gene expression, either at the level of transcription or mRNA stability.
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To characterize the effect that different levels of Bax expression might have on apoptosis, wt and p38-deficient MEFs were transiently transfected with a Bax expression construct. We found that forced expression of Bax in p38-deficient cells, to a similar level of that in wt cells (Figure 5A), increased the number of apoptotic cells, either in the absence or in the presence of serum, up to a similar level as in wt cells (Figure 5B). These results correlate low expression levels of Bax protein with reduced apoptosis and place p38 upstream of Bax in the proapoptotic pathway.
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Down-Regulation of Fas Expression in p38-deficient Cells
Another important proapoptotic molecule is the receptor Fas/CD-95, which can be induced by p38 MAPKs (Hsu et al., 1999; Stephanou et al., 2001) and can mediate cardiomyocyte apoptosis (Jeremias et al., 2000; Stephanou et al., 2001). We therefore quantified Fas expression on the cell surface of wt and p38-/- cardiomyocytes by flow cytometry by using a fluorescein isothiocyanate-labeled anti-Fas antibody. As shown in Figure 6A, the percentage of cells positive for Fas expression was more than double in wt than in p38-/- cardiomyocytes under basal conditions (in the presence of serum). In addition, the anti-Fas fluorescence intensity was also higher in wt cells (Figure 6A), indicating the presence of more Fas protein per cell in wt cardiomyocytes than in those deficient in p38. Therefore, not only the percentage of cardiomyocytes expressing Fas but also the number of Fas molecules per cell is reduced in the absence of p38.
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The differences in Fas expression at the cell surface might be due to changes in the cellular distribution of the receptor or to changes in Fas gene expression. To address these possibilities, the levels of Fas mRNA were analyzed by RT-PCR. The results revealed reduced expression levels of Fas mRNA in p38-/- cardiomyocytes (Figure 6B).
Fas has been implicated in apoptosis induced by many stimuli, suggesting that differences in Fas expression could also explain the different levels of apoptosis found in p38-/- cells under basal conditions. Moreover, it is expected that apoptosis induced by FasL, which interacts with and activates Fas receptor, should be also reduced in p38-deficient cardiomyocytes. To confirm this hypothesis, wt and p38-/- cardiomyocytes were incubated with the anti-Fas antibody Jo2, which mimics the effect of FasL. Figure 7A shows that incubation with Jo2 strongly increased the levels of active caspase 3 in wt cardiomyocytes, similar to that induced by serum withdrawal. In contrast, active cleaved caspase 3 was undetectable in p38-deficient cardiomyocytes treated with anti-Fas antibody, and serum deprivation also induced a lower increase in cleaved caspase 3 than in wt cells. Therefore, cardiomyocytes lacking p38 are more resistant to Fas-induced apoptosis.
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To further characterize the differences in Fas-induced apoptosis between wt and p38-/- cardiomyocytes, a time-course study was performed. In this experiment, high levels of active caspase 3 were already present in wt cardiomyocytes maintained with serum but were further increased upon treatment with anti-Fas (Figure 7B). In contrast, p38-/- cells presented a low basal level of cleaved caspase 3 that only slightly increased after 38 h of treatment with anti-Fas (Figure 7B). We also found that caspase 3 activation correlated with the appearance of the proteolytic fragment of Bid in wt cells (Figure 7B, bottom). In p38-/- cardiomyocytes, the level of the proteolytic fragment of Bid was very low and did not significantly increase with time, which correlates well with the low levels of cleaved caspase 3 and the decreased apoptosis. The results suggest a possible role for Bid cleavage in Fas-induced apoptosis of these cells, although it is unclear whether Bid cleavage is produced before the first peak of caspase 3 activation or later, leading to the amplification of the apoptotic response. In either case, cleaved Bid is important as it facilitates the proapoptotic effect of Bax.
Reintroduction of p38 Sensitizes p38-/- Cardiomyocytes to Apoptosis Induced by Fas and Serum Withdrawal
The data presented above suggest that p38 sensitizes cells to apoptosis by a mechanism involving enhanced Bax and Fas expression. To confirm that the reduced apoptosis of p38-/- was directly due to the absence of p38, we infected these cells with a p38-expressing retrovirus. Figure 8A shows that the level of active caspase 3 induced by anti-Fas treatment was increased upon reintroduction of p38 in the p38-/- cardiomyocytes, to a similar level as in wt cells. Accordingly, the number of apoptotic cells determined by flow cytometry was also higher in the rescued cardiomyocytes, either treated with anti-Fas or serum deprived, compared with cardiomyocytes deficient in p38 (Figure 8B). The increased levels of apoptosis in rescued cells correlated with the level of p38 expression and are consistent with the idea that p38 sensitizes cells to Fas and serum withdrawal-induced apoptosis. The increased level of basal apoptosis (no induction) normally seen in wt cells was also observed in the rescued p38-/- cells expressing ectopic p38. Bax expression was also up-regulated upon reintroduction of p38 in p38-deficient cardiomyocytes (Figure 8C).
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In addition, transient transfection of p38 into p38-/- MEFs was able to increase both Bax expression and the basal level of apoptosis of these cells (as determined by cleaved caspase 3 levels) up to a similar level as in wt cells (Figure 8D).
Fas and Serum Withdrawal-induced Apoptosis Are Reduced in Primary p38-/- MEFs
To investigate whether the resistance to Fas-induced apoptosis in p38-deficient cardiomyocytes was cell type specific, we used primary MEFs. Both the Fas-induced activation of caspase 3 and the percentage of apoptotic cells, as determined by flow cytometry, were lower in p38-/- MEFs (Figure 9A). This also correlated with higher expression of Fas mRNA in wt cells (Figure 9B, inset). Therefore, the decreased sensitivity of p38-deficient cells to Fas-induced apoptosis is not particular to the cardiomyocytic cell line.
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To exclude the possibility that some of the results described above could be a consequence of large T expression, we also measured the apoptotic response and the expression of Bax protein in primary MEFs. As shown in Figure 9B, both the basal level of apoptosis and the apoptosis induced upon serum deprivation were higher in wt than in p38-deficient primary MEFs. Bax protein levels were also higher in wt MEFs and were further increased in cells maintained without serum for 48 h (Figure 9C). Therefore, similarly to the cardiomyocyte-derived cell lines, there is also a correlation between Bax protein expression and apoptosis in primary MEFs.
Up-Regulation of ERK MAPKs and Increased Levels of Nuclear Phospho-Ser-STAT3 in p38-/- Cardiomyocytes
Searching for potential mechanisms that could account for the proapoptotic effect of p38, we analyzed the activation state and nuclear levels of the transcription factors STAT1 and STAT3, which are potential candidates to regulate Fas and Bax expression (Naka et al., 1998; Ivanov et al., 2001; Stephanou et al., 2001). As shown in Figure 10A, treatment of p38-deficient cardiomyocytes with 10% serum strongly increased the nuclear levels of STAT3 phosphorylated on Ser-727, the active form of STAT3. However, the nuclear levels of STAT1 were not significantly modified. We did not observe significant changes in the total level of tyrosine-phosphorylated STAT3 between wt and p38-/- cells (Figure 10A).
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STAT transcription factors can be phosphorylated by the MAPKs ERK and p38 (Kovarik et al., 1999; Stephanou et al., 2001; Zhang et al., 2001; Ramsauer et al., 2002) and by other protein kinases such as Akt (Nguyen et al., 2001). We found that incubation with the mitogen-activated protein kinase kinase (MEK) inhibitor PD98059 strongly inhibited STAT3 phosphorylation on Ser-727, whereas the phosphatidylinositol 3-kinase inhibitor LY29400 abolished Akt activation but did not affect STAT3 phosphorylation (Figure 10B). Moreover, the levels of active ERK were found to be higher in p38-deficient cells either in serum-deprived medium or after serum stimulation for 10 min (Figure 11A). In addition, treatment with the p38 inhibitor SB203580 also induced an up-regulation of the active form of ERKs in wt cells (Figure 11B), indicating that p38 negatively regulates ERK activity. Thus, increased ERK activity may account for the increased phosphorylation of Ser-727-STAT3, which in turn could participate in the negative regulation of Bax and Fas, leading to increased survival of p38-deficient cells. In agreement with this hypothesis, inhibition of ERK activity with PD98059 increased the number of apoptotic cells (Figure 11C). This effect of ERK inhibition was particularly important in serum-deprived p38-deficient cells.
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The up-regulation of ERK activity described above does not seem to be stimulus specific, because it was also observed upon serum deprivation for 24 h and during Fas-induced apoptosis (our unpublished data).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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are more resistant to apoptosis induced by different stimuli (including activators of both the death receptor pathway and the mitochondrial pathway) and also have reduced levels of basal apoptosis under normal proliferating conditions. In contrast, a previous study reported no differences in apoptosis between wt and p38-deficient embryonic stem cells (Allen et al., 2000). This confirms that the effect of p38 MAPKs on apoptosis depends on the cellular context, as suggested by previous studies using chemical inhibitors or mutated components of the p38 MAPK pathway (see INTRODUCTION). Our results also indicate that p38 can sensitize cells to apoptosis through the positive regulation of Fas/CD-95 and Bax expression.
Cardiomyocytes lacking p38 are particularly resistant to Fas-induced apoptosis, and this correlates with decreased expression of the receptor in these cells. Fas receptor has been implicated in apoptosis induced not only by its ligand (FasL) but also by other stimuli such as UV (Leverkus et al., 1997), survival factor withdrawal (Le-Nicolescu et al., 1998), ischemia (Jeremias et al., 2000; Stephanou et al., 2001), and cisplatin treatment (Yuan et al., 2003). Therefore, higher cell surface expression of Fas receptor may sensitize wt cells to apoptosis induced by several treatments.
Fas receptor is constitutively present in a variety of cell types (Leithauser et al., 1993), and its expression can be increased by several extracellular signals such as UV (Leverkus et al., 1997), hypoxia (Tanaka et al., 1994), or cytokines (Stephanou et al., 2001) through the regulation of different transcription factors. p38 MAPKs have been implicated in the regulation of Fas expression, although their precise role is still controversial. In human melanoma cells, for example, p38 down-regulates Fas expression through inhibition of nuclear factor-
B (Ivanov and Ronai, 2000). In contrast, p38 was shown to contribute to the anti-CD3-induced up-regulation of Fas and FasL expression in T cells, although p38 alone was unable to increase Fas expression (Hsu et al., 1999). In cardiac myocytes exposed to ischemia/reperfusion, p38 activation also mediates Fas expression through phosphorylation of STAT1 in Ser-727 (Stephanou et al., 2001). Thus, Fas expression could be induced in wt cardiomyocytes by a p38-dependent mechanism. Factors present in the serum and/or autocrine or paracrine signals could be responsible for the stimulation of p38 leading to Fas expression, whereas the absence of p38 would impair this mechanism. It should be noted that cardiomyocytes are normally grown in the presence of IFN-, which might also trigger the induction of Fas expression (Ruiz-Ruiz et al., 2000; Stephanou et al., 2001). However, wt primary MEFs or cardiomyocytes that are grown in the absence of IFN- still show an enhanced response to Fas-induced apoptosis compared with p38-deficient cells. A possible transcription factor candidate to account for the differences in apoptosis and Fas expression produced by p38 deficiency is p53, which can be phosphorylated on different residues by p38 MAPKs (Bulavin et al., 1999; Huang et al., 1999; Sánchez-Prieto et al., 2000; Bulavin et al., 2002; Kwon et al., 2002) and can induce Fas expression (Munsch et al., 2000). In addition, the increased levels of nuclear STAT3 phosphorylated on Ser-727 that we have found in p38-deficient cardiomyocytes could play a role because it has been shown to down-regulate Fas expression through cooperation with c-Jun (Ivanov et al., 2001).
The reduced levels of proapoptotic protein Bax in p38-deficient cells may also contribute to the resistance of these cells to Fas-induced apoptosis (Ruffolo et al., 2000; Wei et al., 2001; Roth and Reed, 2002). Hepatocytes from Bax/Bak double knockout mice are resistant to death receptor-induced apoptosis in vivo (Wei et al., 2001). This is probably due to the cooperation between Bid and Bax. The activation of caspase 8 upon death receptor engagement could lead to the generation of Bid proteolytic fragment, which in turn would promote the insertion of Bax into mitochondria (Ruffolo et al., 2000). A similar situation may be occurring in our wt cell lines, because we found that Bid cleavage is produced upon Fas engagement, probably through caspase 8 activation, as it has been described for other cell types (Yin et al., 1999). In addition, down-regulation of Bax protein in p38-deficient cells might also explain the resistance of these cells to other proapoptotic stimuli as well as their lower level of basal apoptosis. In agreement with this idea, increased expression of Bax (by transfection) in p38-deficient cells can rescue serum withdrawal-induced and basal apoptosis to about the same level as in wt cells. Thus, a certain level of Bax seems to be required for apoptosis as it has been shown for c-Myc-induced apoptosis, where Bax and c-Myc cooperate to induce the release of cytochrome c from mitochondria (Juin et al., 2002). A further possible mechanism by which p38 MAPK facilitates apoptosis may be via the transmission of the Fas-triggered apoptotic signal through activation of Rb/E2F1 (Hou et al., 2002).
Regulation of Bax activity and, in particular, Bax protein levels by p38 MAPKs is not well understood. p38 MAPK can mediate both activating conformational changes in Bax (Yuan et al., 2003) and its translocation to mitochondria (Ghatan et al., 2000; Deacon et al., 2003). Recent data also suggest that activation of p38 MAPKs could mediate morphine-induced macrophage apoptosis through up-regulation of Bax, p53, and Fas (Singhal et al., 2002). However, no direct effect of p38 on Bax expression has been reported so far, although this could be mediated by p53, which has been proposed to have a positive role in Bax gene expression (Thornborrow and Manfredi, 2001; Pyrzynska et al., 2002). We have found that p53 protein is expressed to similar levels in wt and p38-deficient cardiomyocytes; the levels of p53 are high due to the expression of SV40 large T. However, as discussed above, we cannot rule out that the phosphorylation of p53 by p38 might contribute to the changes in Bax expression levels. We have preliminary results suggesting that p53 phosphorylation on Ser15 may be reduced in p38-/- cells and that incubation with the p53 inhibitor pifithrin- (Lorenzo et al., 2002) can reduce Bax protein levels, at least in cardiomyocytes. On the other hand, because STAT3 phosphorylated on Ser727 is up-regulated in p38-deficient cardiomyocytes and it is an inhibitor of Bax expression (Naka et al., 1998), it could play a role in Bax down-regulation. Hence, ERK up-regulation in p38-deficient cardiomyocytes would mediate the increase in phospho-Ser-727 STAT3, leading to decreased Bax expression. In addition, experiments done with the MEK inhibitor PD98059 indicate that upregulation of the ERK pathway in p38-/- cells is involved in cell survival, which together with the down-regulation of proapoptotic proteins would lead to reduced levels of apoptosis in p38-/- cells. In agreement with our findings, Li et al. (2003) recently showed that the proapoptotic effect of the p38 MAPK pathway relies on the dephosphorylation of MEK1/2, perhaps through PP2A, which would also result in increased ERK activity in the absence of p38.
It should be noted that up-regulation of ERKs in p38-deficient cardiomyocytes could participate in other survival mechanisms, in addition to the proposed regulation of Fas and Bax expression through STAT3. The Ras/ERK/Rsk pathway can mediate cell survival through phosphorylation of the transcription factor cAMP response element-binding protein and the proapoptotic protein Bad (Bonni et al., 1999) The ERK pathway can also increase the levels of antiapoptotic proteins such as Bcl-2, Mcl-1, Bclx-L, and c-FLIP (Boucher et al., 2000; Wang et al., 2002).
In summary, our results demonstrate that cells lacking p38 MAPK are more resistant to apoptosis induced by different stimuli, indicating that p38 is a positive regulator of apoptosis. We found that the absence of p38 correlate with lower levels of both Fas and Bax proteins, which can explain the resistance of p38-/- cells to Fas-induced apoptosis. The proapoptotic role of p38 via regulation of the expression of Bax and probably Fas is also likely to confer a higher sensitivity to other apoptotic stimuli such as serum deprivation. Up-regulation of ERK MAPKs may also contribute to the reduced apoptosis of p38-deficient cells.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Footnotes |
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Equally contributed to the experimental work. 
¶ Present address: Unidad de Citometría, Centro Nacional de Investigaciones Cardiovasculares, Instituto de Salud Carlos III, Ronda de Poniente 5, Tres Cantos 28760 Madrid, Spain.
Corresponding authors. E-mail addresses: maporras{at}farm.ucm.es or nebreda{at}EMBL-Heidelberg.de.
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A. Vertii, C. Hakim, A. Kotlyarov, and M. Gaestel Analysis of Properties of Small Heat Shock Protein Hsp25 in MAPK-activated Protein Kinase 2 (MK2)-deficient Cells: MK2-DEPENDENT INSOLUBILIZATION OF Hsp25 OLIGOMERS CORRELATES WITH SUSCEPTIBILITY TO STRESS J. Biol. Chem., September 15, 2006; 281(37): 26966 - 26975. [Abstract] [Full Text] [PDF]
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G. De Chiara, M. E. Marcocci, M. Torcia, M. Lucibello, P. Rosini, P. Bonini, Y. Higashimoto, G. Damonte, A. Armirotti, S. Amodei, et al. Bcl-2 Phosphorylation by p38 MAPK: IDENTIFICATION OF TARGET SITES AND BIOLOGIC CONSEQUENCES J. Biol. Chem., July 28, 2006; 281(30): 21353 - 21361. [Abstract] [Full Text] [PDF]
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Y. Xu, S. Huang, Z.-G. Liu, and J. Han Poly(ADP-ribose) Polymerase-1 Signaling to Mitochondria in Necrotic Cell Death Requires RIP1/TRAF2-mediated JNK1 Activation J. Biol. Chem., March 31, 2006; 281(13): 8788 - 8795. [Abstract] [Full Text] [PDF]
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 H. He, H.-T. Cho, W. Li, T. Kawakita, L. Jong, and S. C. G. Tseng Signaling-Transduction Pathways Required for Ex Vivo Expansion of Human Limbal Explants on Intact Amniotic Membrane Invest. Ophthalmol. Vis. Sci., January 1, 2006; 47(1): 151 - 157. [Abstract] [Full Text] [PDF]
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X. Li and A. Minden PAK4 Functions in Tumor Necrosis Factor (TNF) {alpha}-induced Survival Pathways by Facilitating TRADD Binding to the TNF Receptor J. Biol. Chem., December 16, 2005; 280(50): 41192 - 41200. [Abstract] [Full Text] [PDF]
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E. Kefaloyianni, E. Gourgou, V. Ferle, E. Kotsakis, C. Gaitanaki, and I. Beis Acute thermal stress and various heavy metals induce tissue-specific pro- or anti-apoptotic events via the p38-MAPK signal transduction pathway in Mytilus galloprovincialis (Lam.) J. Exp. Biol., December 1, 2005; 208(23): 4427 - 4436. [Abstract] [Full Text] [PDF]
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 G. Iaccarino, M. Ciccarelli, D. Sorriento, G. Galasso, A. Campanile, G. Santulli, E. Cipolletta, V. Cerullo, V. Cimini, G. G. Altobelli, et al. Ischemic Neoangiogenesis Enhanced by {beta}2-Adrenergic Receptor Overexpression: A Novel Role for the Endothelial Adrenergic System Circ. Res., November 25, 2005; 97(11): 1182 - 1189. [Abstract] [Full Text] [PDF]
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B. M. Emerling, L. C. Platanias, E. Black, A. R. Nebreda, R. J. Davis, and N. S. Chandel Mitochondrial Reactive Oxygen Species Activation of p38 Mitogen-Activated Protein Kinase Is Required for Hypoxia Signaling Mol. Cell. Biol., June 15, 2005; 25(12): 4853 - 4862. [Abstract] [Full Text] [PDF]
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J. J. Wu and A. M. Bennett Essential Role for Mitogen-activated Protein (MAP) Kinase Phosphatase-1 in Stress-responsive MAP Kinase and Cell Survival Signaling J. Biol. Chem., April 22, 2005; 280(16): 16461 - 16466. [Abstract] [Full Text] [PDF]
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S. Edlund, S. Y. Lee, S. Grimsby, S. Zhang, P. Aspenstrom, C.-H. Heldin, and M. Landstrom Interaction between Smad7 and {beta}-Catenin: Importance for Transforming Growth Factor {beta}-Induced Apoptosis Mol. Cell. Biol., February 15, 2005; 25(4): 1475 - 1488. [Abstract] [Full Text] [PDF]
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 L. Tourian Jr, H. Zhao, and C. B. Srikant p38{alpha}, but not p38{beta}, inhibits the phosphorylation and presence of c-FLIPS in DISC to potentiate Fas-mediated caspase-8 activation and type I apoptotic signaling J. Cell Sci., December 15, 2004; 117(26): 6459 - 6471. [Abstract] [Full Text] [PDF]
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A. J. K. Williamson, B. C. Dibling, J. R. Boyne, P. Selby, and S. A. Burchill Basic Fibroblast Growth Factor-induced Cell Death Is Effected through Sustained Activation of p38MAPK and Up-regulation of the Death Receptor p75NTR J. Biol. Chem., November 12, 2004; 279(46): 47912 - 47928. [Abstract] [Full Text] [PDF]
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