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Departments of
*
Physiology and
Clinical Hematology, Osaka City University Medical School, Asahi-machi, Abeno-ku, Osaka, Japan
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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During apoptosis, a restricted set of cellular proteins is cleaved and degraded in a caspase-dependent manner, and proteolysis of specific proteins promotes the apoptotic pathways or induces the characteristic morphological changes such as cytoplasmic shrinkage, membrane blebbing, nuclear condensation, and DNA fragmentation (4). Most previous studies about the cleavage of proteins during apoptosis addressed the proteins related to cell growth, cell survival, or cell death, but not cell functions. For example, the cleavage of an inhibitor of caspase-activated deoxyribonuclease results in activation of caspase-activated deoxyribonuclease leading to internucleosomal DNA degradation (5). The cleavage of p21-activated kinase results in activation of the kinase (6), which in turn promotes the apoptotic pathways. Caspase-3-mediated cleavage of Bcl-2 may further activate downstream caspases and contribute to amplification of the caspase cascade (7). Among the molecules involved in the MAPK cascade, Raf-1, Ras GTPase-activating protein, and MAPK/ERK kinase (MEK) kinase-1 have been reported to be cleaved in a caspase-dependent manner (8, 9). The cleavage of Raf-1 and Ras GTPase-activating protein results in inactivation of these proteins, which possibly promotes the process of apoptosis by turning off the ERK-mediated survival pathway (8). The cleaved kinase domain of MEK kinase-1 is active and may stimulate caspase activity leading to apoptosis (9). On the other hand, the cleavage of ERK and p38 MAPK has not been previously described in any types of cells undergoing apoptosis, and the recent study shows that neither ERK nor p38 MAPK are cleaved in Jurkat and U937 cells undergoing apoptosis (8).
In this paper, we studied the fate of ERK and p38 MAPK in human neutrophils undergoing apoptosis and their roles in neutrophil functions. The results show that both ERK and p38 MAPK are involved in O2- release and adherence in neutrophils stimulated by cytokines (GM-CSF and TNF) and that both signaling molecules are cleaved and degraded in a caspase-dependent manner during apoptosis. The results suggest that the cleavage of ERK and p38 MAPK in neutrophils undergoing apoptosis may be partly responsible for decreased responsiveness of these cells to inflammatory cytokines and may be physiologically important for preventing excessive or undesired tissue damage by infiltrating neutrophils.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Highly purified recombinant human G-CSF, GM-CSF, and TNF produced by Escherichia coli were provided by Kirin Brewery (Tokyo, Japan), Schering-Plough (Osaka, Japan), and Dainippon Pharmaceutical (Osaka, Japan), respectively. The specific activity of TNF was 3 x 106 U/mg protein. Endotoxin contamination of each preparation was <100 pg/mg protein. Cytochrome c type III, FMLP, PMA, superoxide dismutase, cycloheximide, and mouse mAb against gelsolin were purchased from Sigma (St. Louis, MO); benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk) and benzyloxycarbonyl-Ileu-Glu-Thr-Asp-fluoromethylketone (zIETD-fmk) were purchased from Calbiochem (La Jolla, CA); Ficoll was purchased from Pharmacia (Piscataway, NJ); and Conray was purchased from Mallinckrodt (St. Louis, MO). PD98059 (MEK inhibitor) and rabbit polyclonal Abs against ERK1/ERK2, Thr202/Tyr204-phosphorylated ERK1/ERK2, p38 MAPK, and Thr180/Tyr182-phosphorylated p38 MAPK were purchased from New England Biolabs (Beverley, MA). The Abs against ERK1/ERK2 and p38 MAPK recognized the C-terminal portion of each protein. For immunoprecipitation of p38 MAPK, rabbit polyclonal Ab (H-147) from Santa Cruz Biotechnology (Santa Cruz, CA) was used. Recombinant human active caspase-3 and caspase-8 were purchased from PharMingen (San Diego, CA). The ECL Western blotting system was purchased from Amersham (Arlington Heights, IL). SB203580 (p38 MAPK inhibitor) was provided by SmithKline Beecham Pharmaceuticals (King of Prussia, PA).
Preparation of cells
Human neutrophils and mononuclear cells (PBMC) were prepared from healthy adult donors as described previously (10) using dextran sedimentation, centrifugation with Conray-Ficoll, and hypotonic lysis of contaminated erythrocytes. Neutrophil fractions contained >98% neutrophils. PBMC fractions contained 75–85% lymphocytes, 15–25% monocytes, and <1% neutrophils. Cells were suspended in HBSS containing 10 mM HEPES (pH 7.4) and 0.1% human serum albumin. For the experiments with cell cultivation, neutrophils were suspended in RPMI 1640 supplemented with 10% FCS.
Cell culture and determination of apoptosis
For the experiment with cell survival, neutrophils (5 x 106/ml) were placed in each well of a 96-well plate (Falcon 3072, Falcon Labware; Becton Dickinson, Mountain View, CA) and were cultivated in 5% CO2/95% humidified air at 37°C. Viable cells were counted using the trypan blue dye exclusion test. For the other experiments, neutrophils were cultivated in a polypropylene tube (Falcon 2059). HL-60 and Jurkat cells were grown in RPMI 1640 supplemented with 10% FCS, penicillin (100 U/ml), and streptomycin (100 µg/ml). For the experiments with apoptosis, HL-60 and Jurkat cells suspended in RPMI 1640 supplemented with 2% FCS were cultivated in the presence or absence of cycloheximide (10 µg/ml) plus TNF (100 U/ml). DNA fragmentation was determined by propidium iodide staining and flow cytometry with FACScalibur (Becton Dickinson) as described elsewhere (11). For determination of DNA fragmentation, cells (1 x 106) were placed in 70% ethanol in PBS and stored at -20°C until use. Cells were treated with DNase-free RNase (50 µg/ml) and propidium iodide (50 µg/ml) for 15 min at room temperature. Samples were kept at 4°C in the dark until analysis.
Determination of O2- release and cell adherence
O2- was assayed by superoxide dismutase-inhibitable reduction of ferricytochrome c as described previously (3, 12). The cell suspension in HBSS was added to each FCS-coated well of a 48-well plate (Falcon 3078) containing 100 µM ferricytochrome c with or without superoxide dismutase (200 U/ml) to obtain a final volume of 0.2 ml. The final cell concentration was 3 x 105 cells/0.2 ml. When required, cells were pretreated with PD98059 (10 or 50 µM) or SB203580 (1 or 10 µM) for 20 min at 37°C. After incubation with appropriate stimuli for 3 h at 37°C, the reduction of ferricytochrome c was measured at 550 nm with a reference wavelength at 540 nm. Using the same plate, neutrophil adherence was assayed by measuring the protein content of adherent neutrophils in each well after washing each well thoroughly with warm HBSS (12). The protein content was measured according to the method of Lowry et al. (13) with BSA as standard.
Western blotting
Cells were suspended in HBSS containing 10 mM HEPES (pH 7.4). When required, cells were prewarmed for 10 min at 37°C and were then stimulated with GM-CSF (5 ng/ml) or TNF (100 U/ml) for 10 min at 37°C. The reactions were terminated by rapid centrifugation, and the pellets were frozen in liquid nitrogen, resuspended in ice-cold extraction buffer containing 50 mM HEPES (pH 7.4), 1% Triton X-100, 2 mM sodium orthovanadate, 100 mM sodium fluoride, 1 mM EDTA, 1 mM EGTA, 1 mM PMSF, 100 µg/ml aprotinin, and 10 µg/ml leupeptin, and were lysed for 60 min at 4°C. After rapid centrifugation, the supernatant was mixed 1:1 with 2x sample buffer (4% SDS, 20% glycerol, 10% 2-ME, and a trace amount of bromphenol blue dye in 125 mM Tris-HCl, pH 6.8), heated at 100°C for 5 min, and then frozen at -80°C until use. Samples were subjected to 10% SDS gel electrophoresis. After electrophoresis, proteins were electrophoretically transferred from the gel onto a nitrocellulose membrane in a buffer containing 25 mM Tris, 192 mM glycine, and 20% methanol at 2 mA/cm2 for 1.5 h at 25°C. Residual binding sites on the membrane was blocked by incubating the membrane in TBS (pH 7.6) containing 0.1% Tween 20 and 5% nonfat dry milk for 2 h at 25°C. The blots were washed in TBS containing 0.1% Tween 20 (TBST) and then incubated with appropriate Ab overnight at 4°C. After washing three times with TBST, the membrane was incubated with anti-rabbit or anti-mouse IgG Ab conjugated with HRP, and the Ab complexes were visualized by the ECL detection system (Amersham) as directed by the manufacturer. Immunoreactive bands were quantified by a NIH Image program on a Macintosh computer.
Cleavage of ERK and p38 MAPK in a cell-free system
Freshly prepared neutrophils (5 x 106) or PBMC (5 x 106) were suspended in the buffer (50 µl) containing 50 mM PIPES (pH 7.4), 50 mM KCl, 10 mM EGTA, 2 mM MgCl2, 1 mM DTT, 1 mM PMSF, 10 µg/ml leupeptin, and 100 µg/ml aprotinin and were lysed for 60 min at 4°C. After centrifugation, the supernatants were used as the cell extracts. In another experiment, ERK and p38 MAPK were immunoprecipitated by using Ab against each protein. For immunoprecipitation, cell lysates were incubated with appropriate Ab for 2 h at 4°C. The immune complexes were collected using protein A-Sepharose. The resulting immunoprecipitates were washed three times with the extraction buffer, and suspended in the buffer mentioned above. The immunoprecipitates or the cell extracts were incubated with recombinant active caspase-3 (2 µg/ml) or caspase-8 (10 µg/ml) for 60 min at 37°C (14), and thereafter the Western blotting was performed.
Statistical analysis
An ANOVA followed by a multiple comparison test was done to determine statistical significance.
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Results |
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Human neutrophils underwent spontaneous cell death when cultivated
in vitro (Fig. 1
). Neutrophil death was
accelerated by cycloheximide or TNF and was markedly accelerated by the
combination of cycloheximide and TNF (15). By contrast,
G-CSF prolonged neutrophil survival (16), and this effect
of G-CSF was completely abolished by cycloheximide (Fig. 1). The effect
of these agents on neutrophil survival was mediated through their
effect on apoptosis, as evidenced by the findings in DNA fragmentation.
The cultivation of cells in the medium alone for 3 h resulted in
minimal increase in DNA fragmentation, which was significantly enhanced
by cycloheximide or TNF and was markedly enhanced by the combination of
cycloheximide and TNF (Fig. 2). The
cultivation of cells in the medium alone for 24 h resulted in a
significant increase in DNA fragmentation, which was inhibited by the
presence of G-CSF in the medium. G-CSF-mediated inhibition of DNA
fragmentation was abolished by cycloheximide (Fig. 2).
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After cultivation for 6–48 h in the presence or absence of G-CSF,
the cell lysates from an equal number of viable cells were analyzed by
immunoblotting using Abs against ERK and p38 MAPK. Cultivation of
neutrophils for 6 h in the medium alone resulted in a decreased
intensity of the ERK band (42 kDa) with concomitant appearance of lower
molecular mass bands, suggesting that ERK is cleaved during culture
(Fig. 3A). The major bands of
the cleavage products were 40- and 36-kDa proteins. The maximal
intensity of the bands of the cleavage products was detected at 12
h, and thereafter the intensity was decreased, suggesting further
degradation. The cleavage of ERK was inhibited and delayed by the
presence of G-CSF in the medium (Fig. 3A). In the presence
of G-CSF, the maximal intensity of the bands of cleavage products was
detected at 24 h and thereafter the intensity was decreased.
Although phosphorylation of ERK1/2 was detected at 10 min after
stimulation with G-CSF (3), phosphorylated bands of ERK1/2
were not detected 6 h after stimulation with G-CSF (Fig. 3A). Thus, the stability of ERK protein in the presence of
G-CSF is unlikely to be ascribed to sustained phosphorylation of the
protein.
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A). The major bands of
the cleavage products were 35- and 31-kDa proteins. The maximal
intensity of the bands of the cleavage products was detected at 12
h, and thereafter the intensity was decreased, suggesting further
degradation. The cleavage of p38 MAPK was inhibited and delayed by the
presence of G-CSF in the medium (Fig. 3A). In the presence
of G-CSF, the maximal intensity of the bands of cleavage products was
detected at 24 h and thereafter the intensity was decreased. Thus,
the kinetics of the cleavage of ERK and p38 MAPK in the presence or
absence of G-CSF was essentially identical to each other. In contrast
to ERK, p38 MAPK was not phosphorylated by stimulation with G-CSF
(3) (Fig. 3A). Phosphorylated p38 MAPK was
sometimes detected in freshly isolated neutrophils, and the intensity
of the phosphorylated p38 MAPK band was decreased, maintained, or
sometimes increased during culture regardless of the presence of G-CSF
(Fig. 3A; see also Fig. 7). The increased phosphorylation of
p38 MAPK sometimes observed during culture might be ascribed to cell
stresses caused by the cultivation itself or the cell preparation
procedures. The maintained intensity of phosphorylated p38 MAPK band
observed during culture with G-CSF (Fig. 3A) may primarily
reflect preserved p38 MAPK protein with some contribution of increased
phosphorylation. As observed in the G-CSF effect on cell survival and
DNA fragmentation, G-CSF-mediated inhibition of cleavage of ERK and p38
MAPK was abolished by cycloheximide (Fig. 3B).
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The cleavage of ERK and p38 MAPK during culture was enhanced by
cycloheximide or TNF and was markedly enhanced by the combination of
cycloheximide and TNF (Fig. 4).
Therefore, the kinetics and the regulation in cleavage of ERK and p38
MAPK paralleled those in neutrophil apoptosis (
Figs. 1–4). It has been
recently demonstrated that treatment of human neutrophils with TNF plus
cycloheximide results in activation of caspase-8, which in turn
activates caspase-3 and an additional caspase (possibly caspase-10)
(14). Processing of pro-caspase-8 in neutrophils is
blocked by zVAD-fmk, resulting in inhibition of caspase-3 activation
(14). To determine whether ERK and p38 MAPK are cleaved in
a caspase-dependent manner, cells were cultivated in the presence or
absence of zVAD-fmk. As shown in Fig. 4, the cleavage of ERK and p38
MAPK induced by cycloheximide, TNF, or TNF plus cycloheximide was
almost completely inhibited by zVAD-fmk with concomitant disappearance
of the cleavage products. Similar findings were observed when zIETD-fmk
(50 µM), a specific inhibitor of caspase-8, was used instead of
zVAD-fmk (data not shown). Thus, ERK and p38 MAPK appear to be
specifically cleaved in a caspase-dependent manner in close association
with neutrophil apoptosis.
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B). Consistent with this,
gelsolin was cleaved in neutrophils undergoing apoptosis induced by TNF
plus cycloheximide (Fig. 5B). In contrast, neither ERK nor
p38 MAPK was cleaved by the addition of caspase-3 or caspase-8 to the
extracts from neutrophils or mononuclear cells (Fig. 5A).
The buffer used for this reaction contained several serine protease
inhibitors, including PMSF, leupeptin, and aprotinin. These serine
protease inhibitors might inhibit novel serine protease(s) responsible
for the cleavage of ERK and p38 MAPK. Then, the analysis was performed
using the buffer lacking serine protease inhibitors. Under the
condition without any serine protease inhibitors, both ERK and p38 MAPK
were completely degraded and no cleavage products were detected (Fig. 5A). The addition of leupeptin and aprotinin to the buffer
failed to prevent the degradation of both molecules (data not shown).
Although the addition of PMSF alone to the buffer was sufficient to
prevent the degradation of ERK and p38 MAPK, the cleavage products
observed in neutrophils undergoing apoptosis were not detected under
this condition (Fig. 5A). Thus, one possibility is that
caspase(s) may activate PMSF-sensitive serine proteases, which in turn
may cleave ERK and p38 MAPK.
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and 4 apparently show that ERK was
primarily cleaved to 40- and 36-kDa proteins and that p38 MAPK was
primarily cleaved to 35- and 31-kDa proteins, respectively. The
densitometric analysis of the data shown in Fig. 4 confirmed that the
band intensity of intact ERK1/2 in control cells was essentially
identical to the sum of the band intensity of residual intact ERK1/2
and the band intensity of cleavage products in cells treated with
cycloheximide, TNF, or TNF plus cycloheximide (data not shown). Similar
results were obtained for p38 MAPK (data not shown). These findings and
the fact that the Abs used in this study recognize the C-terminal
portion of each protein indicate that the cleaved sites of each protein
are located at the N-terminal portion.
The recent study shows that neither ERK nor p38 MAPK is cleaved in
Jurkat and U937 cells undergoing apoptosis (8), in
remarkable contrast to the present findings observed in human
neutrophils. These differences may reflect the differences in the type
of cells or the differentiation stage of cells. Then we analyzed the
cleavage of ERK and p38 MAPK in HL-60 and Jurkat cells undergoing
apoptosis induced by TNF plus cycloheximide. The results demonstrated
that neither ERK nor p38 MAPK was cleaved in these cells undergoing
apoptosis (Fig. 6), in agreement with the
previous report (8). Under the same conditions, gelsolin
was cleaved in both types of cells (Fig. 6). Thus, in HL-60 and Jurkat
cells undergoing apoptosis, ERK and p38 MAPK may be left uncleaved
despite activation of caspase-3. These findings also support the idea
that ERK and p38 MAPK are not the substrates for caspase-3 or caspase-8
and both molecules are cleaved indirectly by activation of caspases in
neutrophils undergoing apoptosis.
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Neutrophils cultivated for 12 h responded with increased
phosphorylation of ERK and p38 MAPK when challenged with GM-CSF or TNF.
However, the net effect of GM-CSF or TNF on phosphorylation of ERK or
p38 MAPK was significantly decreased in these aged neutrophils
undergoing spontaneous apoptosis as compared with freshly prepared
neutrophils (Fig. 7). Using neutrophils
cultivated for 6 or 12 h, the functional response to cytokines was
assessed. Spontaneous release of
O2- was not altered, whereas
spontaneous adherence was significantly increased in these aged
neutrophils (Fig. 8). When challenged
with GM-CSF or TNF, aged neutrophils showed decreased release of
O2- in a culture time-dependent
manner. Similar findings were obtained in GM-CSF- and TNF-induced
adherence when the net effects were assessed (Fig. 8). The decreased
functional response of neutrophils undergoing apoptosis appears to be
obvious when GM-CSF and TNF were used as stimuli, as these cells showed
preserved or rather enhanced release of
O2- and adherence to
stimulation with PMA or FMLP (Fig. 8).
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Decreased functional response of apoptotic neutrophils to GM-CSF
or TNF could be caused by the cleavage of p38 MAPK, since p38 MAPK may
be involved in GM-CSF- and TNF-induced
O2- release in human
neutrophils (3). In fact, as shown in Table I, GM-CSF- and TNF-induced
O2- release and adherence were
inhibited by SB203580 (p38 MAPK inhibitor) (18), although
the inhibitory effect of SB203580 on adherence was less than that on
O2- release. The role of ERK in
neutrophil functions was assessed using PD98059 (MEK inhibitor)
(19). Unexpectedly, GM-CSF- and TNF-induced
O2- release and adherence were
also inhibited by PD98059 (10 and 50 µM) (Table I), which
specifically inhibits phosphorylation of ERK without affecting
phosphorylation of p38 MAPK at the concentrations used
(3). These findings suggest that not only p38 MAPK but
also ERK is involved in GM-CSF- and TNF-induced
O2- release and adherence, and
decreased functional responses of apoptotic neutrophils to GM-CSF and
TNF could be ascribed to the cleavage of ERK as well as p38 MAPK. The
cell responses induced by GM-CSF were more sensitive to the inhibitory
effect of PD98059 than those induced by TNF. On the other hand, the
cell responses induced by TNF were more sensitive to the inhibitory
effect of SB203580 than those induced by GM-CSF. These differences
might reflect the fact that GM-CSF and TNF predominantly activate the
ERK pathway and the p38 MAPK pathway, respectively, in human
neutrophils (3).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The cleavage of ERK and p38 MAPK in neutrophils undergoing apoptosis
was accompanied with a decrease in cytokine-induced phosphorylation of
both molecules and decreased functional responses to GM-CSF or TNF
stimulation. These findings and a critical role of ERK and p38 MAPK in
GM-CSF- and TNF-induced functional responses taken together suggest
that the cleavage of ERK and p38 MAPK may be, at least in part,
responsible for decreased responsiveness of apoptotic neutrophils to
GM-CSF or TNF. Decreased functional responsiveness of apoptotic
neutrophils to cytokines may be physiologically important, and the
altered responsiveness may contribute to preventing excessive or
undesired tissue damage by infiltrating neutrophils. The cleavage of
p38 MAPK may also result in decreased synthesis of TNF in apoptotic
neutrophils stimulated by LPS, since p38 MAPK may mediate LPS-induced
NF-
B activation and synthesis of TNF in human neutrophils
(22).
It has been reported that aged neutrophils cultivated for 24 h show impaired response to FMLP (23), and apoptotic neutrophils treated with TNF plus cycloheximide for 3 h show impaired response to FMLP or PMA (14). The present experiments reveal that aged neutrophils cultivated for 6 h already show impaired response to GM-CSF or TNF, but not FMLP or PMA, with apparent cleavage of ERK and p38 MAPK. These findings suggest that cytokine-induced activation of neutrophils is highly affected by the cleavage of ERK and p38 MAPK, in accordance with high sensitivity of cytokine-induced activation of neutrophils to the inhibitory effect of PD98059 or SB203580 (3). In fact, it has been reported that SB203580 does not inhibit PMA-induced responses (H2O2 release and adherence) in human neutrophils and this compound is less potent in blocking the response to a formyl peptide than it is in blocking that for TNF (24). Furthermore, it has been reported that PD98059 does not affect FMLP-induced O2- release in human neutrophils (25). These findings also suggest that activation of ERK and p38 MAPK plays a critical role in cytokine-induced activation of neutrophils.
It has been reported that several proteins, including protein kinase
C
, gelsolin, and actin, are cleaved in human neutrophils undergoing
apoptosis (17, 26, 27, 28). Caspase-3-mediated cleavage of
protein kinase C results in activation of this protein, which may
enhance neutrophil apoptosis (26, 27). The cleavage
product of gelsolin produced by caspase-3 severs actin filaments in a
Ca2+-independent manner (17).
Neutrophil functions requiring the integrity of the cytoskeleton, such
as shape change, chemotaxis, and phagocytosis, have been reported to be
impaired in aged neutrophils (23). The impairment of these
functions of apoptotic neutrophils may be related to the disruption of
cytoskeletal networks caused by proteolysis of gelsolin and actin
during apoptosis. A recent study shows that ERK mediates neutrophil
phagocytosis through activation of myosin light chain kinase
(29). Thus, it is possible that decreased phagocytosis in
aged neutrophils may be also ascribed to cleavage of ERK.
G-CSF appears to prolong neutrophil survival by inhibiting caspase activation as evidenced by inhibition of DNA fragmentation and inhibition of cleavage of ERK and p38 MAPK. It is of interest that the effects of G-CSF on neutrophil survival, DNA fragmentation, and cleavage of ERK and p38 MAPK were all abolished by cycloheximide. These findings suggest that stimulation of neutrophils with G-CSF results in the synthesis of antiapoptotic protein(s), which may inhibit the activation of the caspase cascade. Cycloheximide also enhanced spontaneous and TNF-induced apoptosis, suggesting that certain antiapoptotic protein(s) may be constitutively produced and may function against spontaneous and TNF-induced apoptosis. The antiapoptotic proteins involved in these processes remain to be elucidated. The candidates may include the Bcl-2 family proteins such as Bcl-XL, Mcl-1, A1, and Bax (30, 31, 32, 33). For example, it has been reported that G-CSF up-regulates the expression of Mcl-1 (30) and A1 (31) (antiapoptotic proteins) and down-regulates the expression of Bax (32) (an apoptotic protein). The present experiments suggest that G-CSF-induced up-regulation of certain antiapoptotic proteins may be responsible for the antiapoptotic effect of G-CSF, since the effects of G-CSF were almost completely abolished by cycloheximide.
The cleavage of ERK and p38 MAPK was almost completely inhibited by zVAD-fmk with concomitant disappearance of the cleavage products. It has been demonstrated that caspase-3, caspase-8, and possibly caspase-10 are activated in human neutrophils undergoing apoptosis, and activation of all of these caspases is inhibited by zVAD-fmk (14). Thus, it is possible that ERK and p38 MAPK are cleaved by one of these caspases or alternatively by other cellular proteases activated by caspases. The present experiments show that ERK and p38 MAPK are not directly cleaved by caspase-3 or caspase-8, but rather suggest that both molecules are cleaved by certain protease(s) activated by caspases directly or indirectly. The cleavage of ERK (42 kDa) primarily gave the cleavage products of 40- and 36-kDa proteins, whereas the cleavage of p38 MAPK (38 kDa) primarily gave the cleavage products of 35- and 31-kDa proteins. The Abs used in the present experiments recognize the C-terminal portion of each protein, indicating that both ERK and p38 MAPK may be primarily cleaved at the N-terminal portion. In addition, the cleavage of ERK and p38 MAPK shows the similar kinetics and is similarly regulated by cycloheximide, TNF, and G-CSF. These findings and the structural similarity between ERK and p38 MAPK (34, 35) suggest that both molecules may be cleaved by the same protease(s), which remain to be determined. In a cell-free system, both molecules were completely degraded regardless of the addition of caspases when PMSF was omitted in the reaction buffer. It is likely that complete degradation of both molecules under these conditions may be caused by serine proteases released from granules or cytoplasmic compartments during preparation of cell extracts. No cleavage of both molecules by the addition of caspases in the presence of PMSF raises the possibility that caspases may activate PMSF-sensitive serine proteases, which in turn cleave ERK and p38 MAPK. The C-terminal domain of ERK and p38 MAPK contains the presumed catalytic base, magnesium-binding sites and phosphorylation lip, whereas the N-terminal domain creates a binding pocket for the adenine ring of ATP (34, 35). Thus, it is conceivable that the cleavage of the N-terminal portion may result in inactivation of the kinase activity of these proteins.
The present experiments also show that ERK and p38 MAPK are cleaved in a cell type- or differentiation stage-specific manner, since these molecules are not cleaved in leukemia cell lines (HL-60, U937, and Jurkat) undergoing apoptosis (8). These findings indicate that certain proteases responsible for the cleavage of ERK and p38 MAPK may develop during differentiation into mature neutrophils. These findings further imply that the proteolysis of ERK and p38 MAPK during neutrophil apoptosis plays an important role in the regulation of neutrophil functions and is specific to mature neutrophils. An alternative possibility is that no cleavage of ERK and p38 MAPK in cell lines undergoing apoptosis might reflect a nature of immortalized cells.
The present experiments show that both ERK and p38 MAPK are cleaved and degraded in human neutrophils undergoing apoptosis in a caspase-dependent manner and the cleavage of both molecules may be partly responsible for decreased functional responsiveness to inflammatory cytokines (GM-CSF and TNF). The results suggest that the cleavage of both molecules may be physiologically important for preventing excessive or undesired tissue damage by infiltrating neutrophils.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Seiichi Kitagawa, Department of Physiology, Osaka City University Medical School, Asahi-machi, Abeno-ku, Osaka 545-8585, Japan.
3 Abbreviations used in this paper: MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; MEK, MAPK/ERK kinase; TBST, TBS containing 0.1% Tween 20; zIETD-fmk, benzyloxycarbonyl-Ileu-Glu-Thr-Asp-fluoromethylketone; zVAD-fmk, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone
Received for publication August 17, 2000. Accepted for publication October 13, 2000.
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H. Kutsuna, K. Suzuki, N. Kamata, T. Kato, F. Hato, K. Mizuno, H. Kobayashi, M. Ishii, and S. Kitagawa Actin reorganization and morphological changes in human neutrophils stimulated by TNF, GM-CSF, and G-CSF: the role of MAP kinases Am J Physiol Cell Physiol, January 1, 2004; 286(1): C55 - C64. [Abstract] [Full Text] [PDF]
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 T. Hasegawa, K. Suzuki, C. Sakamoto, K. Ohta, S. Nishiki, M. Hino, N. Tatsumi, and S. Kitagawa Expression of the inhibitor of apoptosis (IAP) family members in human neutrophils: up-regulation of cIAP2 by granulocyte colony-stimulating factor and overexpression of cIAP2 in chronic neutrophilic leukemia Blood, February 1, 2003; 101(3): 1164 - 1171. [Abstract] [Full Text] [PDF]
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C.-Y. Liu, A. Takemasa, W. C. Liles, R. B. Goodman, M. Jonas, H. Rosen, E. Chi, R. K. Winn, J. M. Harlan, and P. I. Chuang Broad-spectrum caspase inhibition paradoxically augments cell death in TNF-alpha -stimulated neutrophils Blood, January 1, 2003; 101(1): 295 - 304. [Abstract] [Full Text] [PDF]
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N. Perskvist, M. Long, O. Stendahl, and L. Zheng Mycobacterium tuberculosis Promotes Apoptosis in Human Neutrophils by Activating Caspase-3 and Altering Expression of Bax/Bcl-xL Via an Oxygen-Dependent Pathway J. Immunol., June 15, 2002; 168(12): 6358 - 6365. [Abstract] [Full Text] [PDF]
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 C. Choi, O. Kutsch, J. Park, T. Zhou, D.-W. Seol, and E. N. Benveniste Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand Induces Caspase-Dependent Interleukin-8 Expression and Apoptosis in Human Astroglioma Cells Mol. Cell. Biol., February 1, 2002; 22(3): 724 - 736. [Abstract] [Full Text] [PDF]
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K. Suzuki, M. Hino, H. Kutsuna, F. Hato, C. Sakamoto, T. Takahashi, N. Tatsumi, and S. Kitagawa Selective Activation of p38 Mitogen-Activated Protein Kinase Cascade in Human Neutrophils Stimulated by IL-1{beta} J. Immunol., November 15, 2001; 167(10): 5940 - 5947. [Abstract] [Full Text] [PDF]
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