|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
PHAGOCYTES
From the Department of Thoracic Medicine,
Imperial College School of Medicine at the National Heart and Lung
Institute, London, United Kingdom; EST-Biology, AstraZeneca
Pharmaceuticals, Alderley Park, Cheshire, United Kingdom; and the
Immunopharmacology Research Group, Medical School, University of
Tampere, Finland and Department of Respiratory Medicine, Tampere
University Hospital, Tampere, Finland.
We have examined the role of caspase-mediated cleavage of the
Ste20-like kinases, mammalian sterile 20-like 1 and 2 (Mst1/Mst2), in
the mechanism of human eosinophil and neutrophil apoptosis. Initial
measurements of kinase activity, using myelin basic protein (MBP) as a
substrate in "in-gel" renaturation assays, showed that constitutive
eosinophil and neutrophil apoptosis were associated temporally with the
activation of a 36-kd MBP kinase (p36 MBPK) and a 34-kd MBP kinase (p34
MBPK), respectively. A constitutively active 63-kd MBP kinase (p63
MBPK) was also detected in freshly prepared eosinophils but not
neutrophils, whose activity was transiently augmented during
spontaneous apoptosis. Immunoblotting studies demonstrated the
expression of Mst1 and Mst2 in eosinophils but not neutrophils whereas
immunoprecipitation studies identified the p63 MBPK activity as
being Mst1 and Mst2 and showed that the p36 MBPK activity
represented the N-terminal catalytic fragment of Mst1. A role for
the p36 MBPK in eosinophil cell death was supported by studies showing
increased activation upon exposure to the proapoptotic
Fas/CD95-activating antibody, CH-11, and attenuation in the presence of
the survival-promoting cytokine, interleukin-5. Furthermore, spontaneous and Fas-induced activation of p36 MBPK was inhibited by catalase and the general caspase inhibitor,
z-Asp-CH2-DCB, at concentrations that suppressed eosinophil
apoptosis. These studies therefore implicate a role for caspase-
and H2O2-mediated cleavage of the Mst1 and the
subsequent release of the 36-kd catalytic fragment in the
mechanism of eosinophil apoptosis. In contrast, neutrophil apoptosis
occurs independently of Mst1 and Mst2 but instead is correlated with
the activation of an as-yet-unidentified 34-kd MBPK.
(Blood. 2002;99:3432-3438) The accumulation of eosinophils and neutrophils
within the airways has been implicated in the etiology of a range of
acute and chronic inflammatory responses.1-3 The number of
eosinophils and neutrophils in the airways reflects a balance between
their rate of accumulation and removal. Whereas emigration of
leukocytes from the circulation is mediated by a host of chemotactic
factors released from various cells, the removal of tissue-associated cells can occur by the process of apoptosis that is regulated by a
variety of factors, including cell-cell/cell-matrix interactions, as
well as direct stimulation by cytokines. Apoptosis is a highly controlled nonphlogistic process of cell death, during which apoptotic cells shrink to form apoptotic bodies. These may then be recognized and
removed from the tissue by phagocytes such as macrophages and
epithelial cells, without discharge of their cytotoxic contents. This process is distinct from necrosis, during which the cell lyses and
the uncontrolled release of cellular contents occurs, which may
subsequently produce an inflammatory response. For this reason it has
been speculated that the therapeutic induction of apoptosis may be
desirable in the treatment of inflammatory
diseases.2,3
Although little is known of the intracellular mechanisms that regulate
cell death, recent investigations have suggested that the initiator
events during apoptosis may be broadly divided into intrinsic and
extrinsic pathways.4 Intrinsic apoptosis is centrally dependent upon the release into the cytosol of mitochondrial cytochrome c, as a consequence of an imbalance of expression of proapoptotic and
antiapoptotic Bcl2 family members. Once in the cytosol,
mitochondrial-derived cytochrome c may form a complex (known as the
apoptosome) with apoptotic protease activating factor 1 (APAF1) and
procaspase-9, thus catalyzing the autoprocessing and generation of
active caspase-9. In contrast, extrinsic apoptosis is dependent upon
the agonism of death receptors such as Fas/CD95. Fas is a type-1
transmembrane receptor belonging to the tumor necrosis receptor
superfamily of proteins,5 which induces apoptosis in
numerous cell types on ligation with its natural ligand, FasL, or with
agonistic anti-Fas monoclonal antibodies. The intracellular domain of
Fas contains a sequence required for induction of apoptosis, known as
the death domain,6 which is conserved among several
proteins including Fas-associated death domain
(FADD)/MORT1.7 Thus the downstream signals propagated by
Fas recruit FADD/MORT1 via interactions between the death domains of
these 2 proteins.8 Subsequently, procaspase-8 is also
recruited to Fas through an interaction between the death effector
domains of FADD and procaspase-8,9,10 so initializing the
caspase cascade that is crucial to the apoptotic signaling pathway. It
has been suggested that the activation of initiator caspases by the
extrinsic and intrinsic pathways converge with the activation of
caspase-3 and caspase-7 which are the executioners of apoptosis. It is
thought that these caspases may mediate their effects by cleavage of
several proteins. Cleavage of some substrates renders them inactive,
such as poly (ADP-ribose) polymerase (PARP), which has a role in DNA
repair. Conversely, caspase-mediated cleavage of other targets,
including caspases themselves, as well as protein kinases MEKK1 and
PAK2, results in their activation.
Apoptosis appears to be the default state, during in vitro culture of
both eosinophils and neutrophils in the absence of the hemopoietic
cytokines (intrinsic death), that can be increased following agonism of
the Fas (CD95) receptor (extrinsic death).11 Interestingly, there appear to be significant differences in the mechanisms that mediate eosinophil and neutrophil apoptosis, which has
raised the possibility of therapeutic intervention to induce selective
cell death. It has previously been shown that although glucocorticoids
attenuate neutrophil apoptosis, they induce cell death in
eosinophils.12 Similarly, we have recently demonstrated that SB203580, an inhibitor of p38 mitogen-activated protein (MAP) kinase, promotes intrinsic eosinophil apoptosis,13 whereas
other studies showed that these inhibitors either
attenuate14 or have no effect on neutrophil
apoptosis.15
Recently, investigations in hemopoietic-derived cell lines have
suggested a possible role for members of the Ste20-related family of
serine/threonine kinases in stress responses and
apoptosis.16 Based on their structure, mammalian
Ste20-homologues can be divided into either the germinal center kinase
(GCK) or the p21-activated kinase (PAK) subfamilies.17 Of
these, members of the GCK family can further be divided into 2 broad
groups based on differences in their structure and
function.17 Group I enzymes comprise GCK itself, together
with GCK-related kinase, GCK-like kinase, hematopoietic progenitor
kinase-1, Nck-interacting kinase, and Drosophilia misshapen.
Group II GCKs include Ste20-like oxidant stress-activated kinase-1,
mammalian sterile 20-like-1 (Mst1, also known as kinase responsive to
stress-2 [Krs2]), Mst2 (also called Krs1), Mst3,
lymphocyte-orientated kinase, and severin kinase. The functional roles
of GCKs are currently obscure although it appears that group I members
function as important regulators of stress-activated MAP kinase
cascades whereas group II enzymes may be involved in the regulation of
cellular responses to environmental stress and longevity. Thus, early
studies showed that caspase-catalyzed cleavage of the group II enzyme
Mst1 and the subsequent release of an active N-terminal 36-kd catalytic
subunit is associated with Fas-induced apoptosis of the human
B-lymphoma cell line, BJAB,18 and human thyoma-derived
human peripheral blood-acute lymphoblastic leukemia
cells.19 Proteolytic activation of Mst was also implicated
in MT-21- and cytotrienin A-induced apoptosis of human leukemia HL-60
cells.20,21 Recently, extensive overexpression studies in
a variety of cell lines have demonstrated that caspase-mediated Mst1
cleavage and the subsequent release of the 36-kd catalytic fragment are
capable of inducing cell death.18,20,22-25
Although most of these investigations of Mst1 have been reported in
cell lines, only one has described this kinase in a primary cell Drugs and analytical reagents
Isolation and culture of human eosinophils
Isolation and culture of human neutrophils Neutrophils were isolated from the peripheral blood of healthy donors. Briefly, red blood cells were allowed to sediment on Elohaes and the supernatant was layered onto a Percoll gradient. After centrifugation the neutrophil rich fraction, at the 70% to 80% interface, was collected, washed, resuspended at 5 × 106 cells/mL in Iscoves modified Dulbecco medium (IMDM), supplemented with 10% FCS and antibiotics, and cultured upon FCS-coated 6-well plates for up to 24 hours.Determination of apoptosis Eosinophil and neutrophil apoptosis were determined by PI-staining of DNA fragmentation and flow cytometry (FACScan; Becton Dickinson, San Jose, CA). Cells showing decreased relative DNA content were considered apoptotic. With eosinophils, the cells were gently resuspended in 300 µL hypotonic PI (25 µg/mL in 0.1% sodium citrate and 0.1% Triton X-100) and stored in the dark at 4°C before flow cytometric analysis. In contrast, neutrophils were washed in phosphate buffered saline (PBS) solution, fixed by 70% ethanol, and incubated on ice for 30 minutes. The cell pellet was then resuspended in 200 µL PI (25 µg/mL in PBS), incubated for 1 hour at 4°C, and analyzed by flow cytometric analysis.Extraction of cytosolic proteins for Western blotting and "in gel" kinase assays Eosinophils (1.2 × 106 cells/mL) and neutrophils (5 × 106 cells/mL) were harvested, washed with cold Hanks buffered saline, and resuspended in 100 µL and 200 µL immunoprecipitation buffer, respectively (final concentration: 10 mM Tris base, pH 7.4, 1% Triton X-100, 0.5% Na-deoxycholate, 0.1% sodium dodecyl sulfate [SDS], 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid [EDTA], 0.5 mM phenylmethylsulfonyl fluoride, 2 mM Na-orthovanadate, 10 µg/mL leupeptin, 25 µg/mL aprotinin, 10 µg/mL pepstatin A, 1.25 mM NaF, and 1 mM Na-pyrophosphate). After vortex mixing, cells were incubated on ice for 30 minutes before centrifuging at 12000 rpm for 10 minutes to remove cell debris. Laemmli buffer (× 6) was then added to eosinophil (20 µL) and neutrophil (40 µL) supernatants. Samples were then boiled and stored at 20°C until immunoblotting and "in gel" renaturation assay analysis.
Immunoprecipitation Eosinophils (2.5 × 106 cells/mL) and neutrophils (5 × 106 cells/mL) were lysed in 100 µL and 200 µL immunoprecipitation buffer, respectively. Following cytosolic extraction, lysates were incubated with 1 µg per 106 cells of the relevant antibody or control IgG overnight at 4°C before addition of 2.5 µL per 106 cells of protein G PLUS agarose beads and incubation at 4°C for 2 hours. Samples were then centrifuged, supernatants retained, and made up to 200 µL with immunoprecipitation buffer. Agarose pellets were washed 3 times with immunoprecipitation buffer and finally resuspended in 200 µL immunoprecipitation buffer. Laemmli buffer (40 µL) was then added to supernatant and immunoprecipitated samples before boiling and storing at20°C for "in gel" kinase assays.
Immunoblotting The expression and cleavage of Mst1 and Mst2 were assessed by Western immunoblot analysis. Protein samples (containing ~ 2.0 × 105 eosinophils or ~ 5 × 105 neutrophils) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 10% polyacrylamide gels. Following transfer to nitrocellulose (HybondECL), proteins were labeled using antibodies (1:500) raised to the catalytic fragments and subsequently detected using horseradish peroxide-linked anti-goat IgG (diluted 1:4000) and ECL Western blotting detection agents on ECL hyperfilm. Relevant bands intensities were quantified by laser-scanning densitometry.Determination of protein kinase activity by "in gel" renaturation assay Protein kinase activity was determined using an "in gel" renaturation assay employing myelin basic protein (MBP) as a substrate. Briefly, eosinophil and neutrophil samples were separated on 10% SDS polyacrylamide gels containing 0.2 mg/mL MBP. After electrophoresis, SDS was removed by 3 × 20 minutes (50 mL) washes with 20% isopropyl alcohol in 50 mM TrisHCl, pH 8.0, followed by 3 × 20 minutes (50 mL) washes in 50 mM TrisHCl, pH 8.0, 5 mM 2-mercaptoethanol. Proteins were denatured by 2 × 30 minutes (50 mL) incubations with 6 M guanidineHCl, 5 mM 2-mercaptoethanol, 50 mM TrisHCl, pH 8.0, and subsequently renatured by 5 washes (50 mL) over 12 to 18 hours with 5 mM 2-mercaptoethanol, 0.04% Tween 40, and 50 mM TrisHCl, pH 8.0. Gels were then re-equilibrated by washing 2 × 30 minutes (50 mL) with 40 mM HEPES, pH 8.0, 10 mM MgCl2, and 2 mM dithiothreitol (DTT). For the kinase reaction the gels were incubated for 3 hours with 10 mL of 40 mM HEPES, pH 8.0, 10 mM MgCl2, 0.055 mM ATP, 0.1 µM PKA inhibitor, and 25 µCi (925 KBq) [32P]ATP. The reaction was stopped and excess [32P]ATP removed by 5 washes in 5% trichloroacetic acid and 1% sodium pyrophosphate over 12 to 18 hours, the gels dried, and radioactivity detected by autoradiography.Statistical analysis Data points, and values in the text and figure legends, represent the mean ± SEM of 3 to 4 independent determinations taken from different cell preparations. When statistical evaluation was required, data were analyzed parametrically by 2-tailed t test for paired data. The null hypothesis was rejected when P < .05.
Following in vitro culture in the absence of hematopoietic
cytokines, eosinophils and neutrophils underwent constitutive
apoptosis, as measured by PI-staining of DNA fragmentation, resulting
in 47% and 61% cell death at 24 hours, respectively (Figure
1). At 24 hours, this process was
significantly attenuated by the survival-promoting cytokines, IL-5 (10 picomolar [pM]; eosinophils: 19% death,
P < .05), and GM-CSF (1 ng/mL; neutrophils: 23% death,
P < .05) and enhanced by the proapoptotic ligand CH-11
(100 ng/mL), which activates cell-surface Fas receptors (eosinophils:
78% death, P < .01; neutrophils: 79% death,
P < .005) (Figure 1).
To investigate the possible role of Mst1 and Mst2 in eosinophil and
neutrophil apoptosis, preliminary experiments were undertaken using
antibodies directed toward the catalytic N-terminal domain. Western
immunoblotting detected the presence at approximately 63 kd of Mst1 and
Mst2 in eosinophils but not neutrophils (Figure 2), even when the total protein loading
of the latter was increased by 4 times (data not shown).
Activation of the Ste20-like kinases was determined using an MBP "in
gel" renaturation assay. In freshly isolated eosinophils, we detected
the presence of a 63-kd MBP kinase (p63 MBPK) whose activity was
elevated at 4 hours and gradually returned to basal activity by 24 hours (Figure 3). In addition, in
eosinophils aged for 4 hours, we detected a 36-kd MBP kinase (p36
MBPK), whose activity increased in a time-dependent manner up to 24 hours (Figure 3). Parallel studies with neutrophils failed to detect
the p63 MBPK but identified the activation of a 34-kd MBP kinase (p34 MBPK) which correlated with constitutive apoptosis (Figure 3).
In an attempt to identify the MBP kinases activated during cell death,
we undertook immunoprecipitation studies using lysates of freshly
purified cells and those undergoing constitutive apoptosis (ie, after
24-hour culture). As would be expected following our inability to
detect these proteins by Western blotting, antibodies to Mst1 and Mst2
failed to immunoprecipitate the p34 MBPK activity from neutrophil
lysates (data not shown). In contrast, anti-Mst1, and to a lesser
extent anti-Mst2, antibodies immunoprecipitated the p63 MBPK activity
from both freshly isolated and apoptotic eosinophils, as detected by
MBP "in gel" kinase assay. Moreover, anti-Mst1, but not anti-Mst2,
also precipitated the p36 MBPK in lysates from apoptotic eosinophils at
24 hours (Figure 4A), suggesting that
this represented the cleaved catalytic fragment of Mst1. A comparison
of the activity of p63 MBPK and p36 MBPK recovered in the pellet and
supernatant of eosinophil lysates at 24 hours following
immunoprecipitation showed that the anti-Mst1 antibody only partially
precipitated these activities. However, this appeared to be due to low
avidity of the antibody for the enzyme since subsequent rounds of
precipitation continued to pull down significant p36 and p63 MBP
activity (Figure 4B).
These studies therefore identified a possible role for the cleavage and
release of a catalytic 36 kd of Mst1 in the mechanism of eosinophil
apoptosis. To provide further evidence of a role of p36 MBPK and to
investigate its mechanism of activation we examined the action of a
number of proapoptotic and antiapoptotic stimuli. The role of caspases
in the mechanism of Mst1 cleavage and spontaneous eosinophil apoptosis
was investigated using the broad specificity caspase inhibitor,
z-Asp-CH2-DCB. As shown in Figure
5, incubation with
z-Asp-CH2-DCB caused dose-dependent suppression of both
constitutive apoptosis and activation of p36 MBPK at 24 hours, with 200 µM producing 5.4 ± 0.8% and 12 ± 8% of control, respectively.
In addition, the finding that the activation of p36 MBPK was augmented
to 338 ± 102% of control at 24 hours by the proapoptotic
Fas/CD95-activating antibody, CH-11 (100 ng/mL; Figure
6B), while it was attenuated to
15 ± 4% of control by the antiapoptotic IL-5 (10 pM;
Figure 6A) further implicated p36 MBPK in eosinophil apoptosis.
Similarly, GM-CSF (1 ng/mL; not shown) attenuated eosinophil apoptosis
as well as abolished p36 MBPK activity by 24 hours. Furthermore, the
antioxidant catalase (0.3 mg/mL and 3 mg/mL), which prolonged
eosinophil survival (Figure 7A),
attenuated p36 MBPK activity during both constitutive and CH-11-induced cell death (30 ± 11% and 1 ± 1% of
controls at 0.3 mg/mL, respectively; Figure 7B), implicating
H2O2 as having a role in p36 MBPK
activation. Interestingly, p63 MBPK activation did not correlate
with eosinophil cell death since exposure to CH-11 (Figure 6B) and IL-5
(Figure 6A) resulted in a similar activation profile as that observed
during constitutive apoptosis (Figure 3) whereas catalase (3 mg/mL)
augmented the activity of p63 MBP at 24 hours (Figure 7B).
Caspase-mediated cleavage of full-length 63-kd Mst1 and Mst2 and the subsequent release of a catalytically active 36-kd kinase during apoptosis has been demonstrated in several investigations using hematopoietic cell lines, implicating these kinases in this mechanism of cell death.18,22,23 Thus, studies in the human promyelocytic leukemia HL-60 cells showed that induction of apoptosis, using a range of anticancer drugs, is associated with caspase-mediated cleavage and release of the catalytic domain of Mst1, which was detected as a 36-kd activity by "in gel" kinase assay employing MBP as a substrate.20,21,27 In this report, we have extended these observations by examining the role of Ste20-like kinases in the intracellular pathways that determine apoptosis in the primary, nondividing hemopoietic cells, eosinophils and neutrophils. Surprisingly, although immunoblot studies identified the presence of Mst1 and Mst2 in eosinophils, these enzymes were not expressed in neutrophil lysates. This observation was supported by measurement of kinase activity by an MBP "in gel" renaturation assay, which showed the presence of constitutive activity at 63 kd in eosinophils, but was absent in neutrophils. It might be envisioned that the failure to identify Mst1 results from its protease cleavage following neutrophil lysis. However, we believe that this is improbable given the large excess of protease inhibitors included in these experiments and the absence of this phenomenon in the protease-rich eosinophils. Subsequent studies suggested a role for Mst1 but not Mst2 cleavage during constitutive eosinophil cell death, since p36 MBPK could only be immunoprecipitated with antibodies to the N-terminal catalytic domain of Mst1. In contrast, neutrophil constitutive apoptosis was correlated with activation of an as-yet-unidentified 34-kd MBPK. Since ERK-1/2 and p38 activation can be detected by MBP "in gel" renaturation assay, it might be imagined that the p36 MBPK activity represents these MAP kinases. However, it was while investigating the role of ERK-1/2 and p38 MAPK in eosinophil apoptosis that we first identified the activation of an unidentified p36 MBPK. During these studies, 2 pieces of evidence indicated that the p36 MBPK did not represent ERK-1/2 and p38. First, MBP "in gel" renaturation assays showed that the p36 MBPK activity was detected at a lower molecular weight than activated ERK-1/2 and p38 MAPK controls. Second, Western blot studies using polyclonal antibodies to the inactive and activated (phosphoantibodies) ERK-1/2 and p38 did not label the p36 MAPK activity.13 Due to the lack of selective pharmacologic inhibitors, we attempted to identify the functional role of p36 MBPK by demonstrating that enzyme activation correlates with eosinophil cell death following exposure to a range of proapoptotic and antiapoptotic mediators. Thus, the time-course of constitutive-induced p36 MBPK activation paralleled spontaneous apoptosis and both of these events could be augmented upon agonism of the proapoptotic Fas/CD95 receptor. In contrast, incubation with the survival-promoting cytokines, GM-CSF (data not shown) and IL-5, attenuated activation of p36 MBPK. Similarly, catalase, an enzyme that inhibits apoptosis by catalyzing the conversion of endogenous H2O2 to H2O and O2, also inhibited p36 MBPK activity. Indeed, the latter result implicates H2O2 in the mechanism of eosinophil apoptosis and is in agreement with a recent report by Wedi et al,28 which showed that both constitutive and Fas-mediated apoptosis is associated with intracellular H2O2 production and that antioxidants such as glutathione (GSH) and N-acetyl-cysteine (NAC) could inhibit eosinophil cell death. In addition, our finding that H2O2 acts either upstream or in parallel to p36 MBPK is in accordance with 2 recent studies of MT-21- and H2O2-induced apoptosis in leukemic HL-60 cells.20,21 Since the appearance of the p36 MBPK and apoptosis could be attenuated in the presence of z-Asp-CH2-DCB, this suggested that this kinase represented a caspase-cleaved catalytic product and that this mechanism was important to eosinophil cell death. Indeed, this result is in agreement with early studies that identified the presence of a caspase-cleavage sequence, DEMD326S, between the catalytic and regulatory domain of Mst1, which is cleaved during apoptosis to release the 36-kd catalytic subunit.18,29 Recently, 2 reports have identified the presence of an additional caspase site, TMTD349G, whose cleavage releases a 40-kd to 41-kd MBPK activity.22,23 Of note, we also observed the activation of an MBPK of approximately 41 kd during eosinophil apoptosis. Although there is strong evidence suggesting a role for caspase 3 in mediating Mst1 cleavage, a recent in vitro study suggests that the 2 sites may be selectively cleaved since the DEMD326S site is acted upon by caspases 3, 6, 7, and 9 whereas TMTD349G is cleaved only by caspases 6 and 7.22 Examination of the profile of p63 MBPK activation showed that it was constitutively active and appeared to be regulated independently of apoptosis and p36 MBPK release. Analysis of the regulatory domain of Mst1 has demonstrated the presence of a kinase inhibitory domain within the central region and a C-terminal self-association sequence.30,31 It has therefore been proposed that Mst1 exists in vivo as an autoinhibitory homodimer that is activated following posttranslational modification such as phosphorylation and/or proteolytic cleavage. Strong evidence now exists demonstrating that phosphorylation is required for the activation of both the 63-kd Mst1 and its 36-kd cleavage products.22,23 Indeed, Graves et al have identified an autophosphorylation site at Ser 327 that attenuates caspase cleavage at the DEMD326S site.22 Following caspase cleavage, the Mst1 catalytic subunit has been shown to migrate from the cytosol to the nucleus where it mediates its proapoptotic responses.23,24 Recently, Ura et al have demonstrated that full-length Mst1 is excluded from the nucleus by 2 nuclear export signals (NESs) located at 361-370 and 441-451 within the C-terminal regulatory domain.24 They show that removal of these NCSs following caspase cleavage results in the translocation of the catalytic Mst1 fragment into the nucleus where it promotes chromatin condensation.24 Unfortunately, because we have been unable to detect Mst1 by immunohistochemistry either as a result of the low antibody affinity and/or low protein expression levels, it has not been possible to examine whether Mst nuclear translocation occurs during eosinophil apoptosis. In conclusion, we have implicated a possible role for caspase-mediated cleavage Mst1 and subsequent release of the N-terminal catalytic fragment in the mechanism of constitutive and Fas-mediated eosinophil apoptosis that appears to be at least partially dependent upon H2O2 production. In contrast, neutrophils do not express either Mst1 or Mst2, although cell death is temporally associated with the activation of an as-yet-unidentified 34-kd MBPK. These differences in the mechanism of apoptosis may be useful for the selective induction of eosinophil and neutrophil apoptosis. Several recent studies have suggested that a number of antitumor drugs such as cytotrienin A, camptothecin, taxol, and 5-fluorouracil may mediate their actions through Mst1 cleavage and the subsequent induction of apoptosis.21,27 Therefore, our demonstration of a similar role for Mst1 in eosinophil apoptosis implies that this could provide a possible selective therapeutic regimen in the resolution of eosinophilic inflammation.
Submitted February 28, 2001; accepted December 28, 2001.
Supported by grants from AstraZeneca Pharmaceuticals, Wellcome Trust (grant no. 056814), GlaxoWellcome United Kingdom, Medical Research Fund of Tampere University Hospital, Finland and the Jalmari and Rauha Ahokas Foundation Academy, Finland.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Mark A. Lindsay, EST-Biology, AstraZeneca Pharmaceuticals, Alderley Park, Cheshire, United Kingdom, SK10 4TG; e-mail: mark.lindsay{at}astrazeneca.com.
1.
Giembycz MA, Lindsay MA.
Pharmacology of the eosinophil.
Pharmacol Rev.
1999;51:213-340 2. Ward I, Dransfield I, Chilvers ER, Haslett I, Rossi AG. Pharmacological manipulation of granulocyte apoptosis: potential therapeutic targets. Trends Pharmacol Sci. 1999;20:503-509[CrossRef][Medline] [Order article via Infotrieve].
3.
Haslett C.
Granulocyte apoptosis and its role in the resolution and control of lung inflammation.
Am J Respir Crit Care Med.
1999;160:S5-S11 4. Roy S, Nicholson DW. Cross-talk in cell death signaling. J Exp Med. 2000;192:21-26. 5. Itoh N, Yonehara S, Ishii A, et al. The polypeptide encoded by the cDNA for human cell surface antigen Fas can mediate apoptosis. Cell. 1991;66:233-243[CrossRef][Medline] [Order article via Infotrieve].
6.
Nagata S, Golstein P.
The Fas death factor.
Science.
1995;267:1449-1456 7. Yuan J. Transducing signals of life and death. Curr Opin Cell Biol. 1997;9:247-251[CrossRef][Medline] [Order article via Infotrieve]. 8. Chinnaiyan AM, O'Rourke K, Tewari M, Dixit VM. FADD, a novel death domain-containing protein, interacts with the death domain of Fas and initiates apoptosis. Cell. 1995;81:505-512[CrossRef][Medline] [Order article via Infotrieve]. 9. Boldin MP, Goncharov TM, Goltsev YV, Wallach D. Involvement of MACH, a novel MORT1/FADD-interacting protease, in Fas/APO-1- and TNF receptor-induced cell death. Cell. 1996;85:803-815[CrossRef][Medline] [Order article via Infotrieve]. 10. Muzio M, Chinnaiyan AM, Kischkel FC, et al. FLICE, a novel FADD-homologous ICE/CED-3-like protease, is recruited to the CD95 (Fas/APO-1) death-inducing signaling complex. Cell. 1996;85:817-827[CrossRef][Medline] [Order article via Infotrieve].
11.
Matsumoto K, Schleimer RP, Saito H, Iikura Y, Bochner BS.
Induction of apoptosis in human eosinophils by anti-Fas antibody treatment in vitro.
Blood.
1995;86:1437-1443 12. Meagher LC, Cousin JM, Seckl JR, Haslett C. Opposing effects of glucocorticoids on the rate of apoptosis in neutrophilic and eosinophil granulocytes. J Immunol. 1996;156:4422-4428[Abstract].
13.
Kankaanranta H, de Souza PM, Salmon M, Barnes PJ, Giembycz MA, Lindsay MA.
SB203580, an inhibitor of p38 mitogen-activated protein kinase, enhances constitutive apoptosis of cytokine deprived human eosinophils.
J Pharmacol Exp Ther.
1999;290:621-628
14.
Aoshiba K, Yasui S, Hayashi M, Tamaoki J, Nagai A.
Role of p38-mitogen-activated protein kinase in spontaneous apoptosis of human neutrophils.
J Immunol.
1999;162:1692-1700
15.
Courtney Frasch S, Nick JA, Fadok VA, Bratton DL, Worthen GS, Henson PM.
p38 Mitogen-activated protein kinase-dependent and -independent intracellular signal transduction pathways leading to apoptosis in human neutrophils.
J Biol Chem.
1998;273:8389-8397 16. Bokoch GM. Caspase-mediated activation of PAK2 during apoptosis: proteolytic kinase activation as a general mechanism of apoptotic signal transduction? Cell Death Differ. 1998;5:637-645[CrossRef][Medline] [Order article via Infotrieve].
17.
Kyriakis JM.
Signaling by the germinal center kinase family of protein kinases.
J Biol Chem.
1999;274:5259-5262 18. Graves JD, Gotoh Y, Draves KE, et al. Caspase-mediated activation and induction of apoptosis by the mammalian Ste20-like kinase Mst1. EMBO J. 1998;17:2224-2234[CrossRef][Medline] [Order article via Infotrieve]. 19. Lee KK, Murakawa M, Nishida E, et al. Proteolytic activation of MST/Krs, STE20-related protein kinase, by caspase during apoptosis. Oncogene. 1998;16:3029-3037[CrossRef][Medline] [Order article via Infotrieve]. 20. Watabe M, Kakeya H, Osada H. Requirement of protein kinase (Krs/MST) activation for MT-21-induced apoptosis. Oncogene. 1999;18:5211-5220[CrossRef][Medline] [Order article via Infotrieve].
21.
Kakeya H, Onose R, Osada H.
Caspase-mediated activation of a 36-kDa myelin basic protein kinase during anticancer drug-induced apoptosis.
Cancer Res.
1998;58:4888-4894
22.
Graves JD, Draves KE, Gotoh Y, Krebs EG, Clark EA.
Both phosphorylation and caspase-mediated cleavage contribute to regulation of the Ste-20-like protein kinase Mst1 during CD95/Fas-induced apoptosis.
J Biol Chem.
2001;276:14909-14915
23.
Lee K-K, Ohyama T, Yajima N, Tsubuki S, Yonehara S.
MST, a physiological caspase substrate, highly sensitizes apoptosis both upstream and downstream of caspase activation.
J Biol Chem.
2001;276:19276-19285
24.
Ura S, Masuyama N, Graves JD, Gotoh Y.
Caspase cleavage of MST1 promotes nuclear translocation and chromatin condensation.
Proc Natl Acad Sci U S A.
2001;98:10148-10153 25. Ura S, Masuyama N, Graves JD, Gotoh Y. MST1-JNK promotes apoptosis via caspase-dependent and independent pathways. Genes Cells. 2001;6:519-530[Abstract].
26.
Reszka AA, Halasy-Nagy JM, Masarachia PJ, Rodan GA.
Bisphosphonates act directly on the osteoclast to induce caspase cleavage of mst1 kinase during apoptosis: a link between inhibition of the mevalonate pathway and regulation of an apoptosis-promoting kinase.
J Biol Chem.
1999;274:34967-34973
27.
Watabe M, Kakeya H, Onose R, Osada H.
Activation of MST/Krs and c-Jun N-terminal kinases by different signaling pathways during cytotrienin A-induced apoptosis.
J Biol Chem.
2000;275:8766-8771
28.
Wedi B, Straede J, Wieland B, Kapp A.
Eosinophil apoptosis is mediated by stimulators of cellular oxidative metabolisms and inhibited by antioxidants: involvement of a thiol-sensitive redox regulation in eosinophil cell death.
Blood.
1999;94:2365-2373
29.
Taylor LK, Wang HC, Erikson RL.
Newly identified stress-responsive protein kinases, Krs-1 and Krs-2.
Proc Natl Acad Sci U S A.
1996;93:10099-10104
30.
Creasy CL, Chernoff J.
Cloning and characterization of a human protein kinase with homology to Ste20.
J Biol Chem.
1995;270:21695-21700
31.
Creasy CL, Ambrose DM, Chernoff J.
The Ste20-like protein kinase, Mst1, dimerizes and contains an inhibitory domain.
J Biol Chem.
1996;271:21049-21053
© 2002 by The American Society of Hematology.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
|
 |
|
  M. M. Perry, S. A. Moschos, A. E. Williams, N. J. Shepherd, H. M. Larner-Svensson, and M. A. Lindsay Rapid Changes in MicroRNA-146a Expression Negatively Regulate the IL-1{beta}-Induced Inflammatory Response in Human Lung Alveolar Epithelial Cells J. Immunol., April 15, 2008; 180(8): 5689 - 5698. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Kankaanranta, X. Zhang, R. Tumelius, M. Ruotsalainen, H. Haikala, E. Nissinen, and E. Moilanen Antieosinophilic Activity of Simendans J. Pharmacol. Exp. Ther., October 1, 2007; 323(1): 31 - 38. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Kankaanranta, P. Ilmarinen, X. Zhang, E. Nissinen, and E. Moilanen Antieosinophilic Activity of Orazipone Mol. Pharmacol., June 1, 2006; 69(6): 1861 - 1870. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. R. Stegert, A. Hergovich, R. Tamaskovic, S. J. Bichsel, and B. A. Hemmings Regulation of NDR Protein Kinase by Hydrophobic Motif Phosphorylation Mediated by the Mammalian Ste20-Like Kinase MST3 Mol. Cell. Biol., December 15, 2005; 25(24): 11019 - 11029. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. A. Stout, M. E. Bates, L. Y. Liu, N. N. Farrington, and P. J. Bertics IL-5 and Granulocyte-Macrophage Colony-Stimulating Factor Activate STAT3 and STAT5 and Promote Pim-1 and Cyclin D3 Protein Expression in Human Eosinophils J. Immunol., November 15, 2004; 173(10): 6409 - 6417. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Preisinger, B. Short, V. De Corte, E. Bruyneel, A. Haas, R. Kopajtich, J. Gettemans, and F. A. Barr YSK1 is activated by the Golgi matrix protein GM130 and plays a role in cell migration through its substrate 14-3-3{zeta} J. Cell Biol., March 29, 2004; 164(7): 1009 - 1020. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|||||||
 |
 |
 |
 |
 |
 |
 |
 |
||
| Copyright © 2002 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
Top
Abstract
Introduction
the
osteoclast.
[32P]-adenosine triphosphate
(
), or in the presence of the Fas-activating
monoclonal antibody (clone CH-11; 100 ng/mL; 
), IL-5 (10 pM; 
), or GM-CSF (1 ng/mL; 
) and apoptosis assessed by
flow cytometric analysis of PI-stained DNA fragmentation. Data points
show the percentage of apoptotic cells and are the mean ± SEM of
3 (A) or 6 (B) independent experiments. P versus
control: * < .05, ** < .01, and *** < .005.
