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*
Department of Clinical and Laboratory Medicine, Akita University School of Medicine, Akita, Japan; and
Department of Internal Medicine, Division of Allergy and Immunology, University of Texas Medical Branch, Galveston, TX 77555
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Abstract |
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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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The p21 G protein Rho and its targets, Rho-associated coiled-coil
forming protein kinases (p160ROCK/ROCK I/ROK
and Rho kinase/ROCK II/ROK
), play a crucial role in actin
cytoskeleton reorganization (7). One example of this
function could be observed in smooth muscle contraction that is
regulated by the cytosolic Ca2+ concentration and
by the Ca2+ sensitivity of myosin/actin filament
(8). The former activates myosin light-chain kinase,
whereas Rho-ROCK is responsible for the latter through the inhibition
of myosin phosphatase or the direct phosphorylation of myosin light
chain (9, 10, 11). Y-27632, a selective inhibitor for ROCK I
and ROCK II, effectively suppresses the contraction of arterial smooth
muscle and corrects hypertension in hypertensive rat models
(12). The compound also inhibited guanosine
5'-O-(3-thiotriphosphate)-induced
Ca2+ sensitization of rabbit trachea, suggesting
that ROCK is involved in the regulation of bronchial hyperreactivity
(13). Another important role of ROCK is its involvement in
focal adhesion and stress fiber formation (14, 15, 16).
Transfection with Rho-kinase/p160ROCK
induces focal adhesions and stress fibers, whereas these phenomena are
not observed in cells with the kinase-defective mutant. The assembly
and disassembly of stress fibers and focal adhesions are critical
processes for cell scattering. The microinjection of exoenzyme C3
transferase, a specific Rho inhibitor, or dominant negative Rho-kinase
inhibits wound-induced migration of NRK49F cells (17). The
role of Rho-ROCK in cell adherence and migration has also been shown in
neutrophils. Inactivation of Rho by exoenzyme C3 blocks fMLP-, IL-8-,
or PMA-induced neutrophil adhesion to fibronectin (18).
Furthermore, Y-27632 suppresses myosin light-chain phosphorylation and
chemotactic peptide-induced chemotaxis of neutrophils
(19). However, little is known about the functional role
of Rho-ROCK in eosinophils.
In the present study, we investigated the activation and functional relevance of Rho and ROCK in eosinophils after eotaxin stimulation. We found that eotaxin stimulates activation of Rho and ROCK in eosinophils, and that there is a dichotomy of downstream signaling pathways of Rho, namely, Rho-ROCK and Rho-ERK pathways. Both Rho and ROCK are critical for eosinophil chemotaxis and production of reactive oxygen species (ROS).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RPMI 1640 medium and Gey’s buffer were obtained from Life
Technologies (Grand Island, NY) and FCS from Filtron (Brooklyn,
Australia). Percoll was purchased from Pharmacia Biotech (Uppsala,
Sweden). Myelin basic protein (MBP), streptolysin-O, cold ATP, and Ca
ionophore A23187 were obtained from Sigma (St. Louis, MO). The mouse
mAb against phospho-ERK, rabbit polyclonal anti-ERK2, anti-p38
Abs, HRP-conjugated goat anti-mouse and anti-rabbit Abs, and
protein A/G Plus agarose were obtained from Santa Cruz Biotechnology
(Santa Cruz, CA). The mouse monoclonal anti-ROK Ab was purchased
from Transduction Laboratories (Lexington, KY). The polyclonal
anti-phospho-p38 Ab was obtained from New England Biolabs (Beverly,
MA). The source of exoenzyme C3 was Upstate Biotechnology (Lake Placid,
NY). Y-27632 was a gift from Welfide (Hirakata, Japan). Anti-CD16
immunomagnetic beads were obtained from Miltenyi Biotec (Bergisch
Gladbach, Germany). Human eotaxin was purchased from R&D Systems
(Minneapolis, MN). ECL detection system and Hybond ECL nitrocellulose
membrane were obtained from Amersham (Arlington Heights, IL).
[
-32P]ATP and guanosine-5'
[-32P]triphosphate [] 4-azidoanilide
([-32P]GTPAA) were purchased from NEN
Life Science Products (Boston, MA) and Amersham Pharmacia Biotech
(Piscataway, NJ), respectively.
Eosinophil purification
Peripheral blood was obtained from subjects with mild to moderate eosinophilia. Eosinophils were isolated by sedimentation with 6% dextran followed by centrifugation on 1.088 Percoll density gradients according to the method of Hansel et al. (20). The cells were further purified by negative selection using anti-CD16 immunomagnetic beads and a MACS system (Miltenyi Biotec). Eosinophils (>99% pure) were then suspended in HBSS with 1% FCS in tubes coated with 3% human serum albumin.
Membrane isolation
Purified eosinophils were rinsed twice in cold PBS followed by incubation for 10 min on ice in Dounce homogenizing buffer containing 50 mM Tris-HCl, pH 8.0, 5 mM EDTA, 5% sucrose, 0.1 mM PMSF, and 1 µg/ml aprotinin, leupeptin, and pepstatin. The cells were homogenized at 2 x 106 cells/ml with 30 strokes in a Dounce homogenizer. Intact cells were then pelleted by centrifugation at 400 x g for 5 min at 4°C. The supernatant was further centrifuged at 400,000 x g for 45 min at 4°C. The pellets were resuspended in TE buffer (20 mM Tris, 1 mM EDTA) and kept at -80°C.
Cell permeabilization
The permeabilization of eosinophils was performed as described elsewhere, with slight modification (21). The eosinophils were permeabilized for 2 min at 37°C in a buffer containing 137 mM sodium glutamate, 2 mM MgCl2, 1 mg/ml BSA, 20 mM NaPIPES, pH 6.8 with 3 mM EGTA (glutamate buffer-EGTA) and 0.4 U/ml streptolysin-O. After permeabilization, the cells were washed twice with cold glutamate buffer-EGTA and resuspended in RPMI 1640 with 10% FCS.
Preparation of cytosolic cell extracts and immunoprecipitation
The permeabilized eosinophils (106 cells) were cultured with and without exoenzyme C3 for 24 h at 37°C. In some experiments, purified eosinophils (106 cells) were incubated with and without Y-27632 for 1 h. The cell viability was determined by trypan blue to exclude a possibility of toxic effects of the inhibitors. After the incubation, the cells were stimulated with 10 nM human eotaxin. The reaction was terminated by the addition of 9 volumes of ice-cold HBSS containing 1 mM Na3VO4. The cells were pelleted by centrifugation and lysed in a lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM Na3VO4, 1 mM NaF, 1 mM EDTA, 1 mM EGTA, 1 mM PMSF, 1% Triton X-100, 10% glycerol, and 1 µg/ml aprotinin, leupeptin, and pepstatin). After 20 min on ice, detergent-insoluble materials were removed by centrifugation at 4°C at 12,000 x g. The whole cell lysates were boiled in 2x Laemmli reducing buffer for 4 min.
For immunoprecipitation, the cell lysates were prepared using the above described lysis buffer without glycerol. The cell lysates were incubated with the appropriate Ab (1–2 µg for each sample) for 1 h, then incubated with 20 µl of protein A/G Plus agarose for 2 h at 4°C. The beads were washed three times with the cold lysis buffer.
[-32P]GTPAA labeling
The labeling of cell membranes with
[-32P]GTPAA was performed as described
elsewhere, with slight modifications (22). Frozen membrane
samples were thawed and pelleted by centrifugation at 14,000 x
g for 10 min at room temperature. The membranes were then
incubated in TE buffer (20 mM Tris-HCl, 1 mM EDTA, pH 7.4) containing
50 µM GDP, 0.2 mM EGTA, 2 mM MgCl2, and 50 mM
CH3CO2Na for 30 min at room
temperature. The sample was stimulated with eotaxin in the presence of
2 µCi of [-32P]GTPAA. The reaction was
terminated by the addition of ice-cold buffer and the placement of
samples on ice for 10 min. After centrifugation at 14,000 x
g for 10 min, the pellets were resuspended in TE buffer with
1 mM DTT. The suspension was then UV irradiated using a hand-held UV
lamp at 254 nm for 10 min in a 24-well plate. The samples were
collected and the wells were rinsed with buffer followed by
centrifugation at 14,000 x g for 10 min. The pellets
were dissolved in 20 µl of 2% SDS, and then 400 µl of the lysis
buffer was added to each sample. After measuring the protein
concentration using bicinchoninic acid assay (Pierce, Rockford, IL) to
standardize the samples, the lysates were subjected to
immunoprecipitation.
Immunocomplex kinase assay
The immunoprecipitates were assayed for ROCK (ROK) by
measuring the phosphotransferase activity for MBP. The kinase reaction
was performed by incubating the immunoprecipitates in 40 µl of kinase
buffer (10 mM HEPES, 50 mM NaCl, 10 mM MgCl2, 100
µM Na3VO4, 500 µM DTT,
25 mM -glycerophosphate) containing 2.5 µM ATP, 10 µCi of
[-32P]ATP, and 50 µg/ml MBP for 30 min at
30°C. After centrifugation, the reaction was stopped by boiling the
supernatant with an equal amount of 2x Laemmli buffer. The kinase
reaction products were then applied to SDS-PAGE and
autoradiography.
Gel electrophoresis and Western blotting
SDS-polyacrylamide gels were prepared according to the Laemmli protocol and used for Western blotting. In some experiments, gel electrophoresis was performed using Ready Gels J (Bio-Rad, Hercules, CA). The concentration of polyacrylamide was 10–15% depending on the m.w. of the protein in which we were interested. Gels were blotted onto Hybond membranes for Western blotting using the ECL system. Blots were incubated in a blocking buffer containing 10% BSA in TBST buffer (20 mM Tris-HCl, 137 mM NaCl, pH 7.6, 0.05% Tween 20) for 1 h followed by incubation in the primary Ab (0.1 µg/ml) for 1–2 h. After washing three times in TBST buffer, blots were incubated for 30 min with a HRP-conjugated secondary Ab (0.04 µg/ml) directed against the primary Ab. The blots were developed with the ECL substrate according to the manufacturer’s instructions. In some experiments, blots were reprobed with another Ab after stripping in a buffer of 62.5 mM Tris-HCl (pH 6.7), 100 mM 2-ME, and 2% SDS at 50°C for 30 min.
Chemotaxis assay
The chemotaxis assay was performed in a 48-well Boyden microchamber (NeuroProbe, Gaithersburg, MD) as described previously (6). Briefly, eotaxin was diluted in Gey’s buffer with 0.02% BSA and placed in the lower wells (100 µl) at a 10 nM concentration. After incubation of eosinophils at 37°C with and without the inhibitors for indicated times, 100 µl of the cell suspension at 2 x 106 cells/ml was added to the upper well of the chamber, which was separated from the lower well by a 5-µm pore size polycarbonate, polyvinylpyrolidone-free membrane (Nuclepore, Pleasanton, CA). The chamber was incubated for 60 min at 37°C. Then, the membrane was removed followed by fixation and staining for 5 min in May-Grünwald solution. The cells that migrated and adhered to the lower surface of the membrane were counted from 10 fields by light microscopy. The chemotactic response to buffer (<40/10 fields) was subtracted from that induced with eotaxin with or without the inhibitors.
ROS production from eosinophils
ROS production from eosinophils was analyzed by luminol-dependent chemiluminescence. The stimulation of eosinophils was performed by adding 50 µl of 30 µM Ca ionophore A23187 to 100 µl of eosinophil suspension (5 x 104 cells) containing 0.25 mM luminol. The luminol-dependent maximal and integral intensity counts were determined for 60 min using an ARGUS-50/2D luminometer/MP (Hamamatsu Photonics, Hamamatsu, Japan). All experiments were performed in duplicate or triplicate.
Statistical analysis
Data were analyzed for statistical significance using ANOVA and paired Student’s t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To study the activation of Rho in CCR3 signaling, we first
determined the presence of Rho in eosinophils. Using Western blotting,
we demonstrated that the p21 Rho A is present in the lysates of
eosinophils (Fig. 1
A). The Rho
activation was analyzed based on the level of GTP binding by the
activated G proteins. With the difficulties in internalizing a
nonhydrolyzable form of GTP and maintaining the ability to stimulate
eosinophils with chemokines, we used the cell membranes that do not
require the internalization. The study of membrane-bound G proteins
using cell membranes has been demonstrated (22). Prepared
eosinophil membranes were labeled with
[-32P]GTPAA, a nonhydrolyzable form of
GTP, to detect Rho activity. Additionally,
[-32P]GTPAA forms a covalent bond when UV
irradiated, resulting in a strong binding between the GTP and the G
protein. Immunoprecipitation for Rho A was performed after eotaxin
stimulation of the labeled membranes. The bound GTP was examined by
SDS-PAGE and autoradiography. We found that the Rho A activity was
enhanced by eotaxin in a dose-dependent manner (Fig. 1B).
The densitometric analysis revealed that 10 nM eotaxin stimulates
Rho activity (n = 3) (Fig. 1C).
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Next, the expression of ROCK in eosinophils was studied. We found
that eosinophils express ROCK II (ROK) (Fig. 2A) but not ROCK I (data not
shown). To investigate the ROCK activity, eosinophils were stimulated
with eotaxin (10 nM) and then immunoprecipitated with the
anti-ROK Ab. The kinase activity of ROCK II was then determined
by measuring the phosphotransferase activity for MBP. As shown in Fig. 2B, the ROCK activity in eosinophils was enhanced by
eotaxin. The pretreatment of exoenzyme C3 (25 µg/ml) inhibited the
ROCK activity, suggesting that ROCK II is exactly downstream of Rho A
in eotaxin signaling. As expected, Y-27632 (1–100 µM) blocked the
phosphorylation of MBP by ROCK in a dose-dependent manner (Fig. 2C).
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Rho and ROCK are critical for the formation of stress fibers and
focal adhesions (14, 15, 16), and consequently regulate the
cell scattering (17). Therefore, we studied their
involvement in eotaxin-induced eosinophil chemotaxis. Eosinophils were
incubated with the inhibitors and then applied in the upper chambers.
The lower chambers contained 10 nM eotaxin. The cells that migrated and
adhered to the lower surface of the membrane were counted by light
microscopy. The pretreatment of exoenzyme C3 (25 µg/ml) significantly
reduced the number of migrated eosinophils stimulated with eotaxin
(n = 3) (Fig. 3A). Y-27632 (0.3–100 µM)
also inhibited eosinophil chemotaxis in a dose-dependent manner
(n = 3) (Fig. 3B).
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We have previously found that ERK1/2 and p38 MAP kinases are
activated by eotaxin, and that these kinases are critical for
eosinophil chemotaxis (5, 6). Because Rho and ROCK are
also important for the function, we investigated whether the MAP
kinases are downstream of Rho or ROCK. After incubation with exoenzyme
C3 or Y-27632, eosinophils were stimulated with eotaxin followed by
electrophoresis and Western blotting with the anti-p-ERK or
anti-p-p38 Ab. Exoenzyme C3 (25 µg/ml) partially inhibited
eotaxin-induced phosphorylation of ERK1/2 (Fig. 4A), but not p38 (Fig. 4B). In contrast, neither ERK1/2 nor p38 phosphorylation was
abrogated by Y-27632 (Fig. 5, A and B). Reprobing the membranes with the
anti-ERK2 or anti-p38 Ab indicated that the same amounts of
protein were loaded on the gels. Taken together, ERK1/2 appears
to be downstream molecules of Rho in eotaxin signaling.
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We studied the functional relevance of Rho and ROCK in ROS
production from eosinophils. Eotaxin alone has a minimal effect on ROS
production (23). As eotaxin induces
Ca2+ influx in eosinophils (24, 25),
we stimulated eosinophils with Ca ionophore A23187. ROS production from
eosinophils was analyzed by luminol-dependent chemiluminescence. Both
exoenzyme C3 (25 µg/ml) and Y-27632 (100 µM) reduced ROS production
by 45% (n = 3) and 58% (n = 5),
respectively (Fig. 6, A and
B). We next performed similar experiments using
eotaxin-primed eosinophils because eotaxin has been shown to possess a
priming effect on ROS production evoked by Ca ionophore
(23). However, after culturing eosinophils for 24 h
in case of the experiments using exoenzyme C3, the Ca ionophore-induced
ROS production was much less than that of freshly purified eosinophils,
and the priming effect of eotaxin was barely observed. For this reason,
we simply investigated the effect of Y-27632 on ROS production from
eosinophils primed with eotaxin. As shown in Fig. 6C, the
priming of eosinophils with 100 nM eotaxin augmented the Ca
ionophore-induced ROS production that was inhibited by Y-27632 (1–100
µM) in a dose-dependent manner (n = 3).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The Rho GTPase family, including Rho, Rac, and Cdc42, regulates
actomyosin-based cellular function such as cell adhesion and migration
(26). Rac regulates the assembly of actin filaments at the
cell periphery to form lamellipodia and membrane ruffles, whereas Cdc42
induces actin-rich surface protrusions called filopodia. The
activation of Rho leads to the formation of stress fibers and
associated focal adhesion complexes. Although several targets of Rho
have been identified, the details of Rho downstream signaling are
complicated. Ishizaki et al. (16) have initially shown
that the phenotype of stress fibers induced by ROK is different from
that induced by Rho A. The dominant active mutant of RhoA induces the
formation of parallel stress fibers, whereas the dominant active mutant
of ROCK induces the formation of stellate stress fibers. Furthermore,
the transfection of HeLa cells with Rho A and
p160ROCK defective in kinase and Rho-binding
activities exhibits an increase in the amount of F-actin along with the
reduction of stress fiber formation, indicating that Rho-induced actin
polymerization is mediated by different effectors other than ROCK. mDia
has subsequently been identified as another target protein for Rho that
regulates stress fiber formation through profilin (27).
Interestingly, however, the roles of ROCK and mDia in the regulation of
actin cytoskeleton appear to be different (28, 29). The
mDia1-induced formation of thin actin stress fibers is disorganized in
the absence of ROCK activity. Moreover, active mDia1 transforms
ROCK-induced condensed actin fibers into structures similar to those
regulated by Rho. According to these results, the thickness and density
of Rho-induced stress fibers depends on the balance between ROCK and
mDia signals. Although ROCK regulates actin cytoskeleton by inhibiting
myosin phosphatase activity, two groups have identified an alternative
pathway that transduces signals through ROCK-LIM kinase-cofilin
(30, 31). In the present study, we showed that the
Rho-ROCK pathway is activated by eotaxin in eosinophils. Several
studies have shown that the actin polymerization is induced by eotaxin
(5, 24, 25, 32). Thus it is possible that the eotaxin
signaling pathway involves molecules other than Rho-ROCK, e.g., mDia
and profilin.
The involvement of MAP kinases in the downstream of the small GTPase Rho family has been well studied (33). Frost et al. (34) have investigated the molecular action of Rho family proteins on MAP kinase activation using a kidney epithelial cell line, 293 cells. The expression of either activated Cdc42 or Rho A does not increase ERK activity. However, the ERK2 activity, which can be stimulated with the active form of Raf, is greatly enhanced when the constitutively active Rac2, Cdc42, or Rho A is cotransfected with Raf. Consistent with the results, our data revealed that ERK1/2 activity is partially regulated by Rho in eotaxin signaling. Angkachatchai et al. (35) have also shown that the pretreatment of T cells with exoenzyme C3 partially reduced the ERK activity following TCR ligation, which supports our results. We did not see any inhibition of p38 MAP kinase phosphorylation by exoenzyme C3. One possible reason for this observation is that the activation of p38 MAP kinase is regulated by Rac or Cdc42 (36) through p21-activated kinase (PAK) activation (34, 37). Although the details remain to be clarified, we preliminarily found that Rac and PAK are activated by eotaxin in eosinophils (R. Vita, S. Stafford, and R. Alam, unpublished data).
The mechanism of cell migration involves dynamic and coordinate disassembly and reassembly of stress fibers and focal adhesions. The contribution of Rho family members to cell migration has been studied in the macrophage chemotaxis assay (38). The migration of macrophages by colony-stimulating factor-1 was completely inhibited when the cells were injected with exoenzyme C3, an inhibitor of Rho, or with the dominant-negative Rac mutant, suggesting the critical role of Rho and Rac in the function. In contrast, macrophages injected with the dominant-negative Cdc42 mutant were able to migrate but did not polarize in the direction of the gradient. Rac is required for the formation of protrusions at the front of the migrating cells and provides the major driving force for movement. Cdc42 provides a polarity signal that is required for directed migration. The activity of Rho appears to be restricted to the rear of the cells to generate the retraction force that is necessary to pull the cell body. Another study has shown that chemotactic peptide-induced neutrophil polarity and locomotion were inhibited by Y-27632, an inhibitor of ROCK (19). In support of these findings, we found that both Rho and ROCK are involved in eosinophil chemotaxis.
The role of ROCK in superoxide production from neutrophils has been
studied (39). Y-27632 reduced superoxide production from
neutrophils stimulated with PMA, but not with Ca ionophore A23187. The
discrepancy of the responsiveness between neutrophils and eosinophils
is unclear. Although our data indicate the contribution of Rho and ROCK
to ROS production from eosinophils, one cannot rule out the possibility
that other candidates are involved. A major component of superoxide
generation in inflammatory cells is the membrane-associated NADPH
oxidase complex that contains cytochrome
b558, p47phox,
p67phox, and the GTPase Rac2 (40).
Chemoattractants, such as fMLP and platelet-activating factor,
induce rapid and transient Rac activation in neutrophils
(41). The stimulation of neutrophils with PMA elicits a
respiratory burst without Rac activation. However, in bone
marrow-derived Rac2-/- neutrophils, the
superoxide production in response to IgG-coated SRBC or fMLP was
significantly abrogated, suggesting the important role of Rac2 in the
function downstream of chemoattractant and FcR (42). In
addition, Lacy et al. (43) have shown that Rac2
translocates from cytosol to plasma membrane-associated fractions along
with p47phox and p67phox in PMA-stimulated
eosinophils.
We showed that the Ca ionophore-induced ROS production from eosinophils was blocked by exoenzyme C3 and a relatively high concentration of Y-27632. In addition, a similar effect of Y-27632 was observed in eotaxin-primed eosinophils. Sullivan et al. (21) have demonstrated that Rho is downstream of Ca signaling and independent of calmodulin in mast cells. Furthermore, Rho, but not ROCK, is essential for hexosaminidase secretion despite of the ROCK activation through Rho. Our data are consistent with the results of Sullivan et al., except that both Rho and ROCK are involved in ROS production from eosinophils. Although reasons for the discrepancy between eosinophils and mast cells are unclear, signaling pathways responsible for mediator release and ROS production may be slightly different.
In conclusion, we have defined the activation of Rho and ROCK in
eotaxin signaling and its essential role in eosinophil chemotaxis and
ROS production. Fig. 7 summarizes the
evidence and shows our current model. Recruitment of eosinophils from
the bloodstream into the airways is the most characteristic feature of
asthma and allergic diseases. Chemokines, particularly eotaxin, are
important regulators of eosinophils in the foregoing conditions.
Therefore, the delineation of the signaling pathway of chemokines and
its role in various functions of eosinophils will help elucidate the
mechanism of allergic diseases and may shed light on developing a new
strategy to treat eosinophilic disorders.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Junichi Chihara, Department of Clinical and Laboratory Medicine, Akita University School of Medicine, 1-1-1, Hondo, Akita 010-8543 Japan. E-mail address: chihara{at}hos.akita-u.ac.jp
3 Abbreviations used in this paper: ERK, extracellular signal-regulated kinase; [-32P]GTPAA, guanosine-5' [-32P]triphosphate [] 4-azidoanilide; MAP, mitogen-activated protein; MBP, myelin basic protein; PAK, p21-activated kinase; ROCK, Rho-associated coiled-coil forming protein kinase; ROS, reactive oxygen species.
Received for publication May 7, 2001. Accepted for publication August 20, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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is a member of a kinase family and is involved in the reorganization of the cytoskeleton. Mol. Cell. Biol. 16:5313.[Abstract]
2-adrenergic receptor to different G proteins by protein kinase A. Nature 390:88.[Medline]
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