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p38 mitogen-activated protein kinase plays a key role in regulating MAPKAPK2 expression
BBRC Biochemical and Biophysical Research Communications 337 (2005) 415–421 www.elsevier.com/locate/ybbrc
p38 mitogen-activated protein kinase plays a key role in regulating MAPKAPK2 expression q Tatsuhiko Sudo a,*, Kayoko Kawai a,b, Hiroshi Matsuzaki b, Hiroyuki Osada a,b a b
Antibiotics Laboratory and Bioarchitect Research Group, DRI, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan Graduate School of Science and Engineering, Saitama University, 255 Shimo-okubo, Saitama, Saitama 338-8570, Japan Received 5 September 2005 Available online 20 September 2005
Abstract One of three major families of the mitogen-activated kinases (MAPK), p38 as well as JNK, has been shown to transduce extracellular stress stimuli into cellular responses by phospho-relay cascades. Among p38 families, p38a is a widely characterized isoform and the biological phenomena are explained by its kinase activity regulating functions of its downstream substrates. However, its specific contributions to each phenomenon are yet not fully elucidated. For better understanding of the role of MAPKs, especially p38a, we utilized newly established mouse fibroblast cell lines originated from a p38a null mouse, namely, a parental cell line without p38a gene locus, knockout of p38a (KOP), Zeosin-resistant (ZKOP), revertant of p38a (RKOP), and Exip revertant (EKOP). EKOP is smaller in size but grows faster than the others. Although comparable amounts of ERK and JNK are expressed in each cell line, ERK is highly phosphorylated in EKOP even in normal culture conditions. Serum stimulation after serum starvation led to ERK phosphorylation in RKOP and ZKOP, but not in EKOP as much. On the contrary, relative phosphorylation level of JNK to total JNK in response to UV was low in RKOP. And its phosphorylation as well as total JNK is slightly lower in EKOP. RKOP is less sensitive to UV irradiation as judged by the survival rate. Stress response upon UV or sorbitol stimuli, leading to mitogen activate protein kinase activated kinase 2 (MAPKAPK2) phosphorylation, was only observed in RKOP. Further experiments reveal that MAPKAPK2 expression is largely suppressed in ZKOP and EKOP. Its expression was recovered by re-introduction of p38a. The loss of MAPKAPK2 expression accompanied by the defect of p38a is confirmed in an embryonic extract prepared from p38a null mice. These data demonstrate that p38 signal pathway is regulated not only by phosphorylation but also by modulation of the expression of its component. Together, we have established cell lines that can be used in analyzing the functions of MAPKs, especially p38a, and show that p38 is indispensable for MAPKAPK2 expression. 2005 Elsevier Inc. All rights reserved. Keywords: p38; Exip; MAPKAPK2 expression; p38 null cell line; p38 revertant; RKOP; EKOP
p38 MAP kinases (p38s) were simultaneously identified as either an anti-inflammatory drug binding protein, a lipopolysaccharide-activated protein kinase or a stress-responsive protein kinase [1–3]. Considerable knowledge has been accumulated in the last decade regarding the roles of p38 in converting extracellular stimuli into cellular response. q
Abbreviations: MAP kinase, mitogen-activated protein kinase; KOP, knockout of p38; ZKOP, Zeocin-resistant; RKOP, revertant of p38a; EKOP, Exip revertant. * Corresponding author. Fax: +81 48 462 4669. E-mail address: [email protected] (T. Sudo). 0006-291X/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.09.063
Major efforts have been made to identify the upstream kinases and downstream substrates to link the cell surface and the nucleus under various stress conditions. That is, upstream kinases such as MKK6, sometimes MKK3 or MKK4, specifically activate p38 by phosphorylating both threonine and tyrosine residues in the TGY motif in the activation loop. Then, transcription factors represented by ATF-2, MEF2C, and CHOP as well as cytosolic kinases such as MAPKAPK2/3 are phosphorylated by p38 to become active and transduce signals to the downstream targets (reviewed in [4–6]). Moreover, we have shown the involvement of p62, recently identified as a causal gene
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for PagetÕs disease of bone 3 (PDB3, [7]), in the selective regulation of p38 activity [8]. This type of selective regulation with extra factor(s) may hint how various environmental stimuli exert different responses in cell type- and/or in the stimulation-dependent fashions by using a limited number of signal transducers. In addition, four isoforms of p38, namely a, b, c, and d, have been identified in mammals thus far. Among them, p38a and b show relatively broad expressions and are sensitive to p38-specific inhibitors, such as SB202190 [1] and SB203580 [9]. In addition to p38a (CSBP2) itself, CSBP1 [1], Mxi2, and Exip [10] were identified as alternative splicing variants of the gene. However, these variants share 100% identity in NH2-terminal domains and maintain amino acids responsible for the binding of p38-specific inhibitors [11,12], p38a has been shown to phosphorylate various substrates using its common docking domain located in the COOH-terminus. On the contrary, Exip, which lost conserved subdomain XI of the protein kinase family, is the only member that does not have any kinase activity to date [10]. Exip, but not p38a, has been shown to form a complex with Tollip and/or IRAK to down-regulate NF-jB pathway [13]. Together with the preferential expression of Exip in cell lines such as HL-60 and THP-1, Exip may play a role in innate immune response in the way different from p38a. These variants with diverse functions may account for multi-functions of p38. In addition to in vitro analyses, recent reports have shown that the targeted disruption of p38a gene locus results in homozygous embryonic lethality caused by defects in erythropoiesis or in placental organogenesis [14–17]. These findings unveil new physiological roles that p38a functions not only in stress responses but also in development. Although, major roles attributed to the specific inhibitor sensitive functions of p38 might be carried out by p38a, which is the most abundant and the best characterized variant, we still have many questions to answer in the p38 signal transduction pathway. Here, we report the establishment and characterization of mouse fibroblast cell lines which could be used in analyzing the functions of p38a, and provide a new evidence by analyzing these cell lines that p38a is indispensable for the regulation of MAPKAPK2 expression. Materials and methods Cell lines. Mouse embryonic fibroblasts (MEFs) were taken from E12.5 embryos which were obtained by intercrossing mice heterozygous for p38a [14], trypsinized, plated, and grown initially in DMEM (Sigma– Aldrich, Japan) supplemented with 10% fetal bovine serum. Genotypes were examined by PCR to isolate p38a null cells [14]. The culture medium was changed every 3 days, and an immortalized clone was isolated after passages and designated as KOP (knockout of p38a). Thereafter, cells were grown in a-MEM (Sigma–Aldrich, Japan) with 10% fetal bovine serum. Then, to obtain the stable clones expressing p38a (RKOP for revertant of p38) or Exip (EKOP for revertant of Exip), transfection with Flag-tagged human p38a or Flag-tagged human Exip was conducted using Effectene (Qiagen) as described [13]. The plasmid encoding human p38a was constructed by excising HindIII–ApaI fragment of pcDNA3Flag-hp38 [10] and the fragment was then cloned into pZeoSV2. The same strategy
was employed for Exip. Cells expressing these proteins were selected by 800 lg/ml Zeocin (Invitrogen). Microscopic analysis. Cells grown on coverslips were fixed with 3.7% formaldehyde in PBS for 5 min. After one washing with PBS followed by soaking in 0.2% Triton X-100 in PBS for 5 min, they were then washed once with PBS and incubated in rhodamine–phalloidin at 37 C for 20 min. Then, the cells were washed three times with PBS and were examined by using a phase-contrast microscope and a fluorescent microscope. Growth kinetics. Cell growth kinetics were obtained by a standard technique. Briefly, 1 · 104 cells were seeded into 12-well culture dishes and the adherent cells were counted at the day indicated. Western blot analysis. Expressions of proteins were examined using Western analysis with the following antibodies: anti-p38 [18], anti-p38 N-20 (Santa Cruz), anti-ERK (Cell Signaling), anti-JNK (Cell Signaling), anti-phospho-p38 (Cell Signaling), anti-phospho-JNK (Cell Signaling), anti-phospho-ERK (Cell Signaling), anti-MAPKAPK2 (Cell Signaling), anti-phospho-MAPKAPK2 (Cell Signaling), and anti-actin (Sigma). Sample preparation was done by normalizing cell numbers, namely 0.5 · 105 cells plated in the previous day were washed twice with PBS and lysed by 40 ll of the SDS sample buffer. Then, 20 ll of each sample was separated by 12% SDS–PAGE followed by immuno-blotting using each single antibody described above. Embryonic extracts were prepared as previously described [18]. Cell adhesion assay using crystal violet. Cell viability after stress stimuli was evaluated by dye extraction of adherent cells as described [19], with modifications. Briefly, 0.25 · 105 cells were grown in 24-well plate and stimulated by UV or sorbitol, followed by additional 48 h incubation. They were then washed once with PBS and fixed by cold methanol. The cells were then stained with 0.5% crystal violet for 5 min. After removing the solution, they were washed twice with deionized water and air-dried. The adsorbed dye was extracted by 1 mM HCl in 30% ethanol. Absorbance readings at 590 nm of these extracts were measured in triplicate. The readings were then normalized to those of control samples, namely without any stimuli, as 100% in each cell line. RT-PCR analysis. We performed RT-PCR analysis to examine MAPKAPK2 expressions in each cell line. mRNA was prepared and cDNA was synthesized as mentioned previously [10]. We designed primers corresponding to the nucleotide between 7 and 14 (5 0 -GGGCACCATG CTGTCGGGCTC-3 0 ), and between 424 and 403 (5 0 -CGAGACACT CCATGACAATCAGC-3 0 ) to obtain 431 bp PCR product. GAPDH was used as a control.
Results and discussion Establishment of KOPs To establish p38a null cell lines, E12.5 embryos were obtained from intercrossing p38+/ mice. Genotyping was done to select p38 null embryo as described previously [14], and MEFs derived from the embryo were maintained in DMEM supplemented with 10% fetal bovine serum. The medium was changed every 3 days. Then, the cell clone which was spontaneously immortalized was isolated and designated KOP (knockout of p38). Genotyping at this moment reconfirmed its complete loss of p38a gene locus (data not shown). In addition, KOP was revealed to be a female embryo derived MEF by PCR [20]. Thereafter, a-MEM supplemented with 10% fetal bovine serum was used for growing cells. To establish p38a revertant (RKOP), a plasmid encoding Flag-tagged human p38, pZeoSV2-Flag-hp38 was re-introduced into KOP and selected by Zeocin. DNA sequence analyses clearly show that human p38a was integrated in the mouse genome
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(data not shown). ZKOP (Zeocin-resistant) and EKOP (Exip revertant) were established as well. Since there is a strong nonspecific signal in MEF overlapping to Flaghp38 and Flag-hExip when we used anti-Flag M2 antibody (Sigma), the specific expression of p38a or Exip was confirmed by Western analysis using anti-p38 antibody or N-20 antibody which recognizes N-terminus peptides of p38 families, respectively (Fig. 1A). Microscopic analyses reveal that EKOP is smaller in size and attaches to the dish
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surface differently (Fig. 1B). Higher sensitivity of EKOP to trypsinization, or rather the cells easily detached from a culture dish with PBS alone, during cell transfer process can be partly explained by microscopic observation that EKOP seemed to attach to a culture dish less tightly. Adhesion machinery seemed to be affected by the expression of Exip, but not by p38. Once cells become confluent, RKOP lines up as myoblast does (Fig. 1B-b) supporting the possible function of p38 in differentiation as previously described (reviewed in [21]). Their growth rates were examined by counting cell numbers in each time point. EKOP grows faster than the others and grows to twice as much as RKOP and ZKOP in 4 days (Fig. 1C). Although, we demonstrated in the earlier study that transient over-expression of Exip can lead to apoptosis in HeLa cells [10], stable expression of Exip accelerates cell cycle indicating that its contribution to cell fates must be either dose dependent, cell type specific or dependent on p38 expression. No clear difference can be observed in the appearances and in the growth kinetics between ZKOP and RKOP. These data suggest that p38 does not have any critical function in the proliferation under normal growth conditions in this system. ERKs are highly phosphorylated in EKOP
Fig. 1. Characterization of established cell lines. (A) Immuno-blot experiments reveal specific expressions of p38 and Exip in RKOP and EKOP, respectively (*, nonspecific signal). (B) Phase-contrast images of cells are shown (a–c). Bars, 200 lm. Actin structures are shown (d–f). Bars, 50 lm. (C) Growth kinetics are drawn.
Since, each member of MAPK tightly links to each other and shares their roles in many phenomena, we first tested their expression in these cell lines. Comparable amounts of ERK (Fig. 2A, lanes 1–3) and JNK (Fig. 2B, lanes 1– 3) were expressed in all cell lines when the samples were normalized by cell numbers. It should be also mentioned that the relatively poor reproducibility was observed, especially in the case of phospho-proteins, when we prepared samples by normalizing protein concentrations as follows. That is, cells were harvested and lysed in a buffer containing 50 mM Hepes–KOH (pH 7.9), 250 mM NaCl, 1% Triton X-100, 20 mM b-glycerophosphate, 1 mM sodium orthovanadate, 1 mM PMSF, and 10 lg/ml leupeptin. Then, the samples were sonicated for 5 s and rotated for 1 h followed by 15 min centrifugation at 15,000 rpm at 4 C. The lysates containing 30 lg of total protein were examined by 12% SDS–PAGE. These observations suggest that expressions of ERK and JNK are basically independent of p38 expression, but there must be some effects of p38 expression either on the localization or on the complex formation of ERK and JNK to alter solubility to the buffer. Since ERK plays a major role in proliferation, we then tested the phosphorylation status of ERKs in each cell line. As shown in Fig. 2A, ERK phosphorylation is obvious in EKOP without serum stimulation. This sustained activation of ERK supports the result that EKOP grows faster than the others. Mxi2, another splicing variant of p38a, was also shown to lead to sustained phosphorylation of ERK induced by epidermal growth factor by direct binding to ERK [22]. Since EKOP showed hyper-phosphorylation
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Fig. 2. Signal transduction through MAPK families in the cell lines. (A) Western analysis of ERK phosphorylation upon serum stimulation. Z, ZKOP; R, RKOP; E, EKOP. Normal culture conditions (lanes 1–3). After 24 h serum starvation, cell cultures were stimulated with (+) or without serum () (lanes 4–9). (B) Western analysis of JNK phosphorylation induced by UV-C irradiation (200 J/m2). (C) Western analysis of p38 phosphorylation induced by UVC irradiation (200 J/m2).
of ERK even without serum (Fig. 2A, lanes 4–6), the mechanisms of sustained phosphorylation of ERK by Mxi2 or Exip should be somehow different. Upon serum stimulation after 24 h starvation, phosphorylation of ERK is dramatically induced in ZKOP and RKOP, demonstrating that activation of ERK in short range is independent of p38. Although, a cross-talk between p38 and ERK is often described [4–6], a direct cross-talk from p38 to ERK cannot be observed in this system. We cannot rule out the possible cross-talk between p38 and ERK in a long range. Moreover, a moderate induction of ERK phosphorylation upon serum stimulation after serum starvation in EKOP indicates that the phosphorylation of ERK has already reached at maximum level or Exip functions as a brake in the hyperproliferative environment. JNK compensates p38 in response to stress stimuli JNK, as well as p38, is responsible for stress responses and it is generally hard to discriminate their roles. Then, to explore the connection between p38 and JNK under stress conditions, we tested activation status of JNK in these cell lines. As shown in Fig. 2B, JNK phosphorylation by UV was comparable in the absence of p38. In the presence of p38, in RKOP, its relative phosphorylation to total JNK is lower indicating that p38 compensates JNK for some reasons. This was also observed when we used sorbitol instead of UV as a stimulus (data not shown). The observation that phosphorylation level of JNK upon stress stimuli was affected by p38 expression may be explained by their coordinated functions in signal transduction. The upstream kinase, such as MKK4, sometimes is responsible for both JNK and p38 activation [23], MKK4 signals originated by these stresses might have accumulated into JNK in the absence of p38. Sum activities of JNK and p38 may be required for coping with this type of stress. RKOP is more resistant to apoptosis Since p38 is generally accepted as a key mediator of apoptosis signal, we then assessed their sensitivities to
UV irradiation by using the cell lines. Even though, the same number of cells was plated and irradiated, RKOP is more resistant to UV (Fig. 3A). Since RKOP grows as fast as ZKOP (Fig. 1C), clear difference in the numbers of adherent cells between RKOP and ZKOP observed in Fig. 3A suggests that p38 plays important role(s) in survival under stress conditions. RKOPÕs insensitivity to UV was also assessed by measuring incorporation of crystal violet dye. The survival rate of RKOP is almost 150% to that of ZKOP (Fig. 3B). Since the initial activation of p38 was only emerged in RKOP upon UV stimulation (Fig. 2C), p38 preferentially plays a role in survival in this system. This is the case in the previous study in which p38a plays a critical role in the cardiomyocyte survival pathway in response to pressure overload [24]. However, this is the exact reverse phenomenon reported in previous study, where p38 is required for apoptosis in response to stress stimuli in cells [25] and in the heart caused by ischemia–reperfusion [26]. Furthermore, ERK phosphorylation without p38 observed in a previous study [25] is not obvious in this study. These differences may reflect the origin of the cells and the method to establish p38 null cell. In addition, in the absence of p38, irrespective of Exip, cells were more susceptible to UV irradiation. Together, these cell lines established in this study may be useful tools to analyze the function of p38 for survival under stress conditions. p38a modulates MAPKAPK2 expression Since p38a is only expressed in RKOP, it is quite natural that the stress signal originated by UV or sorbitol reached p38 only in RKOP judging from immuno-blot analysis using the antibody specific for TGY dual phosphorylated p38 (Fig. 2C). This result also indicates that other isoforms of p38, including p38b, c, and d, did not play a principal role in this signal transduction. Then, we examined the signal transduction downstream of p38 under stress conditions in these cell lines. Successful transfer of the stress to the downstream of p38 was assessed by detecting MAPKAPK2 phosphorylation. p38 undoubtedly transduced upstream signal to a downstream target (Fig. 4A). Moreover,
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Fig. 3. p38 plays a key role in survival signal under stress conditions. (A) Twenty-four hours after UV-C irradiation (200 J/m2), cells were stained with rhodamine–phalloidin and photographs taken under microscope. (B) The effect of UV on their survival was estimated by measuring crystal violet dye incorporation 48 h after UV-C irradiation (200 J/m2). The survival rates of each cell line without UV treatment were regarded as 100%. An average of three independent experiments is shown.
Fig. 4. p38 regulates MAPKAPK2 expression. (A) Western analysis of MAPKAPK2 phosphorylation induced by UV-C irradiation (200 J/m2). (B) MAPKAPK2 mRNA expressions in the cell lines. GAPDH was used as a control. (C) MAPKAPK2 expression in p38 null mouse embryo (W, p38+/+; H, p38+/; and KO, p38/). Thirty micrograms of embryonic extracts was subjected and examined by each antibody.
Exip failed to substitute p38 in the stress signal pathway, suggesting its unique features as expected in earlier studies [10,13]. Then, we examined its expression in these cell lines as a control. Surprisingly, MAPKAPK2 expressions were dramatically induced by re-introduction of p38 (Fig. 4A). We then examined its mRNA expression and detected comparable but a slightly higher expression in RKOP (Fig. 4B, lanes 7–9). This cannot account for the drastic reduction of MAPKAPK2 protein without p38. These results suggest that p38 modulates MAPKAPK2 expression by regulating
both transcriptionally and post-transcriptionally. Reduced expression of MAPKAPK2 in response to loss of p38 was confirmed by testing its expression in mouse E9.5 embryo derived from p38/ (Fig. 4C). Only p38a, but not Exip which lost CD domain indispensable for specific interactions with upstream kinases or downstream substrates [27], can recover the expression of MAPKAPK2 indicating that their interaction plays a role maintaining MAPKAPK2 stability. Together with the previous report in which MAPKAPK2 regulates p38 expression [28], the re-
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sults obtained in this work indicate that p38 mutually modulates MAPKAPK2 expression in cells and in mice. These data reveal that newly established cell lines well reflect the p38 signal transduction machinery in the mouse embryo. Although, a previous study strongly suggested that p38 is involved in actin cyto-skeleton via MAPKAPK2 and HSP27 [29], defects in both p38 and MAPKAPK2 did not affect much to destroy the actin skeleton in normal culture conditions as judged by microscopy (Fig. 1B). Further studies are required to answer these discrepancies. In general, to solve the questions of the p38 signal transduction pathway left behind, we have to pay more attention to the expressions of components in the pathway in addition to their phosphorylation status. In summary, our study utilizing newly generated p38a null MEF cell line demonstrates that cells without p38a barely express MAPKAPK2. Although, most of our knowledge in terms of p38 signal transduction machinery comes from kinase activity itself, the results obtained in this study show that long-term depletion of p38 also results in the reduction of its specific substrate with unidentified mechanism(s). In addition, we show that established cell lines are promising tools for analyzing functions of p38, especially survival roles under stress conditions. Acknowledgments We are grateful to Amy Chan for reviewing manuscripts, to Rie Nakazawa and Yasue Ichikawa for sequencing. This work was supported by ‘‘Bioarchitect Research Project’’ from RIKEN to T.S. References [1] J. Lee, J.T. Laydon, P.C. Mcdonnell, T.F. Gallagher, S. Kumar, D. Green, D. McNulty, M.J. Blumenthal, J.R. heys, S.W. Landvatter, J.E. Strickler, M. McLaughlin, I.R. Siemens, S.M. Fisher, G.P. Livi, J.R. White, J.L. Adams, P.R. Young, A protein kinase involved in the regulation of inflammatory cytokine biosynthesis, Nature 372 (1994) 739–746. [2] J. Han, J.D. Lee, L. Biggs, R.J. Ulevitch, A MAP kinase targeted by endotoxin and hyperosmolarity in mammalian cells, Science 265 (1994) 808–811. [3] J. Rouse, P. Cophen, S. Trigon, M. Morange, A. Alonso-Llamazares, D. Zamanillo, T. Hunt, A.R. Nebreda, A novel kinase cascade triggered by stress and heat shock that stimulates MAPKAP kinase-2 and phosphorylation of small heat shock proteins, Cell 78 (1994) 1027–1037. [4] P. Cohen, The search for physiological substrates of MAP and SAP kinases in mammalian cells, Trends Cell Biol. 7 (1997) 353–361. [5] T. Zarubin, J. Han, Activation and signaling of the p38 MAP kinase pathway, Cell Res. 15 (2005) 11–18. [6] J.M. Kyriakis, J. Avruch, Mammalian mitogen-activated protein kinase signal transduction pathways activated by stress and inflammation, Physiol. Rev. 81 (2001) 807–869. [7] N. Laurin, J.P. Brown, J. Morissette, V. Raymond, Recurrent mutation of the gene encoding sequestosome 1 (SQSTM1/p62) in Paget disease of bone, Am. J. Hum. Genet. 70 (2002) 1582–1588. [8] T. Sudo, M. Maruyama, H. Osada, p62 functions as a p38 MAP kinase regulator, Biochem. Biophys. Res. Commun. 2 (69) (2000) 521–525.
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p38 mitogen-activated protein kinase plays a key role in regulating MAPKAPK2 expression q Tatsuhiko Sudo a,*, Kayoko Kawai a,b, Hiroshi Matsuzaki b, Hiroyuki Osada a,b a b
Antibiotics Laboratory and Bioarchitect Research Group, DRI, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan Graduate School of Science and Engineering, Saitama University, 255 Shimo-okubo, Saitama, Saitama 338-8570, Japan Received 5 September 2005 Available online 20 September 2005
Abstract One of three major families of the mitogen-activated kinases (MAPK), p38 as well as JNK, has been shown to transduce extracellular stress stimuli into cellular responses by phospho-relay cascades. Among p38 families, p38a is a widely characterized isoform and the biological phenomena are explained by its kinase activity regulating functions of its downstream substrates. However, its specific contributions to each phenomenon are yet not fully elucidated. For better understanding of the role of MAPKs, especially p38a, we utilized newly established mouse fibroblast cell lines originated from a p38a null mouse, namely, a parental cell line without p38a gene locus, knockout of p38a (KOP), Zeosin-resistant (ZKOP), revertant of p38a (RKOP), and Exip revertant (EKOP). EKOP is smaller in size but grows faster than the others. Although comparable amounts of ERK and JNK are expressed in each cell line, ERK is highly phosphorylated in EKOP even in normal culture conditions. Serum stimulation after serum starvation led to ERK phosphorylation in RKOP and ZKOP, but not in EKOP as much. On the contrary, relative phosphorylation level of JNK to total JNK in response to UV was low in RKOP. And its phosphorylation as well as total JNK is slightly lower in EKOP. RKOP is less sensitive to UV irradiation as judged by the survival rate. Stress response upon UV or sorbitol stimuli, leading to mitogen activate protein kinase activated kinase 2 (MAPKAPK2) phosphorylation, was only observed in RKOP. Further experiments reveal that MAPKAPK2 expression is largely suppressed in ZKOP and EKOP. Its expression was recovered by re-introduction of p38a. The loss of MAPKAPK2 expression accompanied by the defect of p38a is confirmed in an embryonic extract prepared from p38a null mice. These data demonstrate that p38 signal pathway is regulated not only by phosphorylation but also by modulation of the expression of its component. Together, we have established cell lines that can be used in analyzing the functions of MAPKs, especially p38a, and show that p38 is indispensable for MAPKAPK2 expression. 2005 Elsevier Inc. All rights reserved. Keywords: p38; Exip; MAPKAPK2 expression; p38 null cell line; p38 revertant; RKOP; EKOP
p38 MAP kinases (p38s) were simultaneously identified as either an anti-inflammatory drug binding protein, a lipopolysaccharide-activated protein kinase or a stress-responsive protein kinase [1–3]. Considerable knowledge has been accumulated in the last decade regarding the roles of p38 in converting extracellular stimuli into cellular response. q
Abbreviations: MAP kinase, mitogen-activated protein kinase; KOP, knockout of p38; ZKOP, Zeocin-resistant; RKOP, revertant of p38a; EKOP, Exip revertant. * Corresponding author. Fax: +81 48 462 4669. E-mail address: [email protected] (T. Sudo). 0006-291X/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.09.063
Major efforts have been made to identify the upstream kinases and downstream substrates to link the cell surface and the nucleus under various stress conditions. That is, upstream kinases such as MKK6, sometimes MKK3 or MKK4, specifically activate p38 by phosphorylating both threonine and tyrosine residues in the TGY motif in the activation loop. Then, transcription factors represented by ATF-2, MEF2C, and CHOP as well as cytosolic kinases such as MAPKAPK2/3 are phosphorylated by p38 to become active and transduce signals to the downstream targets (reviewed in [4–6]). Moreover, we have shown the involvement of p62, recently identified as a causal gene
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for PagetÕs disease of bone 3 (PDB3, [7]), in the selective regulation of p38 activity [8]. This type of selective regulation with extra factor(s) may hint how various environmental stimuli exert different responses in cell type- and/or in the stimulation-dependent fashions by using a limited number of signal transducers. In addition, four isoforms of p38, namely a, b, c, and d, have been identified in mammals thus far. Among them, p38a and b show relatively broad expressions and are sensitive to p38-specific inhibitors, such as SB202190 [1] and SB203580 [9]. In addition to p38a (CSBP2) itself, CSBP1 [1], Mxi2, and Exip [10] were identified as alternative splicing variants of the gene. However, these variants share 100% identity in NH2-terminal domains and maintain amino acids responsible for the binding of p38-specific inhibitors [11,12], p38a has been shown to phosphorylate various substrates using its common docking domain located in the COOH-terminus. On the contrary, Exip, which lost conserved subdomain XI of the protein kinase family, is the only member that does not have any kinase activity to date [10]. Exip, but not p38a, has been shown to form a complex with Tollip and/or IRAK to down-regulate NF-jB pathway [13]. Together with the preferential expression of Exip in cell lines such as HL-60 and THP-1, Exip may play a role in innate immune response in the way different from p38a. These variants with diverse functions may account for multi-functions of p38. In addition to in vitro analyses, recent reports have shown that the targeted disruption of p38a gene locus results in homozygous embryonic lethality caused by defects in erythropoiesis or in placental organogenesis [14–17]. These findings unveil new physiological roles that p38a functions not only in stress responses but also in development. Although, major roles attributed to the specific inhibitor sensitive functions of p38 might be carried out by p38a, which is the most abundant and the best characterized variant, we still have many questions to answer in the p38 signal transduction pathway. Here, we report the establishment and characterization of mouse fibroblast cell lines which could be used in analyzing the functions of p38a, and provide a new evidence by analyzing these cell lines that p38a is indispensable for the regulation of MAPKAPK2 expression. Materials and methods Cell lines. Mouse embryonic fibroblasts (MEFs) were taken from E12.5 embryos which were obtained by intercrossing mice heterozygous for p38a [14], trypsinized, plated, and grown initially in DMEM (Sigma– Aldrich, Japan) supplemented with 10% fetal bovine serum. Genotypes were examined by PCR to isolate p38a null cells [14]. The culture medium was changed every 3 days, and an immortalized clone was isolated after passages and designated as KOP (knockout of p38a). Thereafter, cells were grown in a-MEM (Sigma–Aldrich, Japan) with 10% fetal bovine serum. Then, to obtain the stable clones expressing p38a (RKOP for revertant of p38) or Exip (EKOP for revertant of Exip), transfection with Flag-tagged human p38a or Flag-tagged human Exip was conducted using Effectene (Qiagen) as described [13]. The plasmid encoding human p38a was constructed by excising HindIII–ApaI fragment of pcDNA3Flag-hp38 [10] and the fragment was then cloned into pZeoSV2. The same strategy
was employed for Exip. Cells expressing these proteins were selected by 800 lg/ml Zeocin (Invitrogen). Microscopic analysis. Cells grown on coverslips were fixed with 3.7% formaldehyde in PBS for 5 min. After one washing with PBS followed by soaking in 0.2% Triton X-100 in PBS for 5 min, they were then washed once with PBS and incubated in rhodamine–phalloidin at 37 C for 20 min. Then, the cells were washed three times with PBS and were examined by using a phase-contrast microscope and a fluorescent microscope. Growth kinetics. Cell growth kinetics were obtained by a standard technique. Briefly, 1 · 104 cells were seeded into 12-well culture dishes and the adherent cells were counted at the day indicated. Western blot analysis. Expressions of proteins were examined using Western analysis with the following antibodies: anti-p38 [18], anti-p38 N-20 (Santa Cruz), anti-ERK (Cell Signaling), anti-JNK (Cell Signaling), anti-phospho-p38 (Cell Signaling), anti-phospho-JNK (Cell Signaling), anti-phospho-ERK (Cell Signaling), anti-MAPKAPK2 (Cell Signaling), anti-phospho-MAPKAPK2 (Cell Signaling), and anti-actin (Sigma). Sample preparation was done by normalizing cell numbers, namely 0.5 · 105 cells plated in the previous day were washed twice with PBS and lysed by 40 ll of the SDS sample buffer. Then, 20 ll of each sample was separated by 12% SDS–PAGE followed by immuno-blotting using each single antibody described above. Embryonic extracts were prepared as previously described [18]. Cell adhesion assay using crystal violet. Cell viability after stress stimuli was evaluated by dye extraction of adherent cells as described [19], with modifications. Briefly, 0.25 · 105 cells were grown in 24-well plate and stimulated by UV or sorbitol, followed by additional 48 h incubation. They were then washed once with PBS and fixed by cold methanol. The cells were then stained with 0.5% crystal violet for 5 min. After removing the solution, they were washed twice with deionized water and air-dried. The adsorbed dye was extracted by 1 mM HCl in 30% ethanol. Absorbance readings at 590 nm of these extracts were measured in triplicate. The readings were then normalized to those of control samples, namely without any stimuli, as 100% in each cell line. RT-PCR analysis. We performed RT-PCR analysis to examine MAPKAPK2 expressions in each cell line. mRNA was prepared and cDNA was synthesized as mentioned previously [10]. We designed primers corresponding to the nucleotide between 7 and 14 (5 0 -GGGCACCATG CTGTCGGGCTC-3 0 ), and between 424 and 403 (5 0 -CGAGACACT CCATGACAATCAGC-3 0 ) to obtain 431 bp PCR product. GAPDH was used as a control.
Results and discussion Establishment of KOPs To establish p38a null cell lines, E12.5 embryos were obtained from intercrossing p38+/ mice. Genotyping was done to select p38 null embryo as described previously [14], and MEFs derived from the embryo were maintained in DMEM supplemented with 10% fetal bovine serum. The medium was changed every 3 days. Then, the cell clone which was spontaneously immortalized was isolated and designated KOP (knockout of p38). Genotyping at this moment reconfirmed its complete loss of p38a gene locus (data not shown). In addition, KOP was revealed to be a female embryo derived MEF by PCR [20]. Thereafter, a-MEM supplemented with 10% fetal bovine serum was used for growing cells. To establish p38a revertant (RKOP), a plasmid encoding Flag-tagged human p38, pZeoSV2-Flag-hp38 was re-introduced into KOP and selected by Zeocin. DNA sequence analyses clearly show that human p38a was integrated in the mouse genome
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(data not shown). ZKOP (Zeocin-resistant) and EKOP (Exip revertant) were established as well. Since there is a strong nonspecific signal in MEF overlapping to Flaghp38 and Flag-hExip when we used anti-Flag M2 antibody (Sigma), the specific expression of p38a or Exip was confirmed by Western analysis using anti-p38 antibody or N-20 antibody which recognizes N-terminus peptides of p38 families, respectively (Fig. 1A). Microscopic analyses reveal that EKOP is smaller in size and attaches to the dish
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surface differently (Fig. 1B). Higher sensitivity of EKOP to trypsinization, or rather the cells easily detached from a culture dish with PBS alone, during cell transfer process can be partly explained by microscopic observation that EKOP seemed to attach to a culture dish less tightly. Adhesion machinery seemed to be affected by the expression of Exip, but not by p38. Once cells become confluent, RKOP lines up as myoblast does (Fig. 1B-b) supporting the possible function of p38 in differentiation as previously described (reviewed in [21]). Their growth rates were examined by counting cell numbers in each time point. EKOP grows faster than the others and grows to twice as much as RKOP and ZKOP in 4 days (Fig. 1C). Although, we demonstrated in the earlier study that transient over-expression of Exip can lead to apoptosis in HeLa cells [10], stable expression of Exip accelerates cell cycle indicating that its contribution to cell fates must be either dose dependent, cell type specific or dependent on p38 expression. No clear difference can be observed in the appearances and in the growth kinetics between ZKOP and RKOP. These data suggest that p38 does not have any critical function in the proliferation under normal growth conditions in this system. ERKs are highly phosphorylated in EKOP
Fig. 1. Characterization of established cell lines. (A) Immuno-blot experiments reveal specific expressions of p38 and Exip in RKOP and EKOP, respectively (*, nonspecific signal). (B) Phase-contrast images of cells are shown (a–c). Bars, 200 lm. Actin structures are shown (d–f). Bars, 50 lm. (C) Growth kinetics are drawn.
Since, each member of MAPK tightly links to each other and shares their roles in many phenomena, we first tested their expression in these cell lines. Comparable amounts of ERK (Fig. 2A, lanes 1–3) and JNK (Fig. 2B, lanes 1– 3) were expressed in all cell lines when the samples were normalized by cell numbers. It should be also mentioned that the relatively poor reproducibility was observed, especially in the case of phospho-proteins, when we prepared samples by normalizing protein concentrations as follows. That is, cells were harvested and lysed in a buffer containing 50 mM Hepes–KOH (pH 7.9), 250 mM NaCl, 1% Triton X-100, 20 mM b-glycerophosphate, 1 mM sodium orthovanadate, 1 mM PMSF, and 10 lg/ml leupeptin. Then, the samples were sonicated for 5 s and rotated for 1 h followed by 15 min centrifugation at 15,000 rpm at 4 C. The lysates containing 30 lg of total protein were examined by 12% SDS–PAGE. These observations suggest that expressions of ERK and JNK are basically independent of p38 expression, but there must be some effects of p38 expression either on the localization or on the complex formation of ERK and JNK to alter solubility to the buffer. Since ERK plays a major role in proliferation, we then tested the phosphorylation status of ERKs in each cell line. As shown in Fig. 2A, ERK phosphorylation is obvious in EKOP without serum stimulation. This sustained activation of ERK supports the result that EKOP grows faster than the others. Mxi2, another splicing variant of p38a, was also shown to lead to sustained phosphorylation of ERK induced by epidermal growth factor by direct binding to ERK [22]. Since EKOP showed hyper-phosphorylation
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Fig. 2. Signal transduction through MAPK families in the cell lines. (A) Western analysis of ERK phosphorylation upon serum stimulation. Z, ZKOP; R, RKOP; E, EKOP. Normal culture conditions (lanes 1–3). After 24 h serum starvation, cell cultures were stimulated with (+) or without serum () (lanes 4–9). (B) Western analysis of JNK phosphorylation induced by UV-C irradiation (200 J/m2). (C) Western analysis of p38 phosphorylation induced by UVC irradiation (200 J/m2).
of ERK even without serum (Fig. 2A, lanes 4–6), the mechanisms of sustained phosphorylation of ERK by Mxi2 or Exip should be somehow different. Upon serum stimulation after 24 h starvation, phosphorylation of ERK is dramatically induced in ZKOP and RKOP, demonstrating that activation of ERK in short range is independent of p38. Although, a cross-talk between p38 and ERK is often described [4–6], a direct cross-talk from p38 to ERK cannot be observed in this system. We cannot rule out the possible cross-talk between p38 and ERK in a long range. Moreover, a moderate induction of ERK phosphorylation upon serum stimulation after serum starvation in EKOP indicates that the phosphorylation of ERK has already reached at maximum level or Exip functions as a brake in the hyperproliferative environment. JNK compensates p38 in response to stress stimuli JNK, as well as p38, is responsible for stress responses and it is generally hard to discriminate their roles. Then, to explore the connection between p38 and JNK under stress conditions, we tested activation status of JNK in these cell lines. As shown in Fig. 2B, JNK phosphorylation by UV was comparable in the absence of p38. In the presence of p38, in RKOP, its relative phosphorylation to total JNK is lower indicating that p38 compensates JNK for some reasons. This was also observed when we used sorbitol instead of UV as a stimulus (data not shown). The observation that phosphorylation level of JNK upon stress stimuli was affected by p38 expression may be explained by their coordinated functions in signal transduction. The upstream kinase, such as MKK4, sometimes is responsible for both JNK and p38 activation [23], MKK4 signals originated by these stresses might have accumulated into JNK in the absence of p38. Sum activities of JNK and p38 may be required for coping with this type of stress. RKOP is more resistant to apoptosis Since p38 is generally accepted as a key mediator of apoptosis signal, we then assessed their sensitivities to
UV irradiation by using the cell lines. Even though, the same number of cells was plated and irradiated, RKOP is more resistant to UV (Fig. 3A). Since RKOP grows as fast as ZKOP (Fig. 1C), clear difference in the numbers of adherent cells between RKOP and ZKOP observed in Fig. 3A suggests that p38 plays important role(s) in survival under stress conditions. RKOPÕs insensitivity to UV was also assessed by measuring incorporation of crystal violet dye. The survival rate of RKOP is almost 150% to that of ZKOP (Fig. 3B). Since the initial activation of p38 was only emerged in RKOP upon UV stimulation (Fig. 2C), p38 preferentially plays a role in survival in this system. This is the case in the previous study in which p38a plays a critical role in the cardiomyocyte survival pathway in response to pressure overload [24]. However, this is the exact reverse phenomenon reported in previous study, where p38 is required for apoptosis in response to stress stimuli in cells [25] and in the heart caused by ischemia–reperfusion [26]. Furthermore, ERK phosphorylation without p38 observed in a previous study [25] is not obvious in this study. These differences may reflect the origin of the cells and the method to establish p38 null cell. In addition, in the absence of p38, irrespective of Exip, cells were more susceptible to UV irradiation. Together, these cell lines established in this study may be useful tools to analyze the function of p38 for survival under stress conditions. p38a modulates MAPKAPK2 expression Since p38a is only expressed in RKOP, it is quite natural that the stress signal originated by UV or sorbitol reached p38 only in RKOP judging from immuno-blot analysis using the antibody specific for TGY dual phosphorylated p38 (Fig. 2C). This result also indicates that other isoforms of p38, including p38b, c, and d, did not play a principal role in this signal transduction. Then, we examined the signal transduction downstream of p38 under stress conditions in these cell lines. Successful transfer of the stress to the downstream of p38 was assessed by detecting MAPKAPK2 phosphorylation. p38 undoubtedly transduced upstream signal to a downstream target (Fig. 4A). Moreover,
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Fig. 3. p38 plays a key role in survival signal under stress conditions. (A) Twenty-four hours after UV-C irradiation (200 J/m2), cells were stained with rhodamine–phalloidin and photographs taken under microscope. (B) The effect of UV on their survival was estimated by measuring crystal violet dye incorporation 48 h after UV-C irradiation (200 J/m2). The survival rates of each cell line without UV treatment were regarded as 100%. An average of three independent experiments is shown.
Fig. 4. p38 regulates MAPKAPK2 expression. (A) Western analysis of MAPKAPK2 phosphorylation induced by UV-C irradiation (200 J/m2). (B) MAPKAPK2 mRNA expressions in the cell lines. GAPDH was used as a control. (C) MAPKAPK2 expression in p38 null mouse embryo (W, p38+/+; H, p38+/; and KO, p38/). Thirty micrograms of embryonic extracts was subjected and examined by each antibody.
Exip failed to substitute p38 in the stress signal pathway, suggesting its unique features as expected in earlier studies [10,13]. Then, we examined its expression in these cell lines as a control. Surprisingly, MAPKAPK2 expressions were dramatically induced by re-introduction of p38 (Fig. 4A). We then examined its mRNA expression and detected comparable but a slightly higher expression in RKOP (Fig. 4B, lanes 7–9). This cannot account for the drastic reduction of MAPKAPK2 protein without p38. These results suggest that p38 modulates MAPKAPK2 expression by regulating
both transcriptionally and post-transcriptionally. Reduced expression of MAPKAPK2 in response to loss of p38 was confirmed by testing its expression in mouse E9.5 embryo derived from p38/ (Fig. 4C). Only p38a, but not Exip which lost CD domain indispensable for specific interactions with upstream kinases or downstream substrates [27], can recover the expression of MAPKAPK2 indicating that their interaction plays a role maintaining MAPKAPK2 stability. Together with the previous report in which MAPKAPK2 regulates p38 expression [28], the re-
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sults obtained in this work indicate that p38 mutually modulates MAPKAPK2 expression in cells and in mice. These data reveal that newly established cell lines well reflect the p38 signal transduction machinery in the mouse embryo. Although, a previous study strongly suggested that p38 is involved in actin cyto-skeleton via MAPKAPK2 and HSP27 [29], defects in both p38 and MAPKAPK2 did not affect much to destroy the actin skeleton in normal culture conditions as judged by microscopy (Fig. 1B). Further studies are required to answer these discrepancies. In general, to solve the questions of the p38 signal transduction pathway left behind, we have to pay more attention to the expressions of components in the pathway in addition to their phosphorylation status. In summary, our study utilizing newly generated p38a null MEF cell line demonstrates that cells without p38a barely express MAPKAPK2. Although, most of our knowledge in terms of p38 signal transduction machinery comes from kinase activity itself, the results obtained in this study show that long-term depletion of p38 also results in the reduction of its specific substrate with unidentified mechanism(s). In addition, we show that established cell lines are promising tools for analyzing functions of p38, especially survival roles under stress conditions. Acknowledgments We are grateful to Amy Chan for reviewing manuscripts, to Rie Nakazawa and Yasue Ichikawa for sequencing. This work was supported by ‘‘Bioarchitect Research Project’’ from RIKEN to T.S. References [1] J. Lee, J.T. Laydon, P.C. Mcdonnell, T.F. Gallagher, S. Kumar, D. Green, D. McNulty, M.J. Blumenthal, J.R. heys, S.W. Landvatter, J.E. Strickler, M. McLaughlin, I.R. Siemens, S.M. Fisher, G.P. Livi, J.R. White, J.L. Adams, P.R. Young, A protein kinase involved in the regulation of inflammatory cytokine biosynthesis, Nature 372 (1994) 739–746. [2] J. Han, J.D. Lee, L. Biggs, R.J. Ulevitch, A MAP kinase targeted by endotoxin and hyperosmolarity in mammalian cells, Science 265 (1994) 808–811. [3] J. Rouse, P. Cophen, S. Trigon, M. Morange, A. Alonso-Llamazares, D. Zamanillo, T. Hunt, A.R. Nebreda, A novel kinase cascade triggered by stress and heat shock that stimulates MAPKAP kinase-2 and phosphorylation of small heat shock proteins, Cell 78 (1994) 1027–1037. [4] P. Cohen, The search for physiological substrates of MAP and SAP kinases in mammalian cells, Trends Cell Biol. 7 (1997) 353–361. [5] T. Zarubin, J. Han, Activation and signaling of the p38 MAP kinase pathway, Cell Res. 15 (2005) 11–18. [6] J.M. Kyriakis, J. Avruch, Mammalian mitogen-activated protein kinase signal transduction pathways activated by stress and inflammation, Physiol. Rev. 81 (2001) 807–869. [7] N. Laurin, J.P. Brown, J. Morissette, V. Raymond, Recurrent mutation of the gene encoding sequestosome 1 (SQSTM1/p62) in Paget disease of bone, Am. J. Hum. Genet. 70 (2002) 1582–1588. [8] T. Sudo, M. Maruyama, H. Osada, p62 functions as a p38 MAP kinase regulator, Biochem. Biophys. Res. Commun. 2 (69) (2000) 521–525.
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