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Originally published online as doi:10.1189/jlb.0907664 on May 1, 2008

Published online before print May 1, 2008
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(Journal of Leukocyte Biology. 2008;84:255-263.)
© 2008 by Society for Leukocyte Biology

Calpain-mediated regulation of the distinct signaling pathways and cell migration in human neutrophils

Masataka Katsube, Takayuki Kato, Maki Kitagawa, Haruyoshi Noma, Hisakazu Fujita and Seiichi Kitagawa1

Department of Physiology, Osaka City University Graduate School of Medicine, Osaka, Japan

1Correspondence: Department of Physiology, Osaka City University Graduate School of Medicine, Asahi-machi, Abeno-ku, Osaka 545-8585, Japan. E-mail: kitagawas{at}med.osaka-cu.ac.jp


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ABSTRACT
 
We studied the mechanisms underlying calpain inhibition-mediated human neutrophil migration. MAPKs, including ERK, p38, and JNK, MEK1/2, MAPK kinase 3/6 (MKK3/6), PI-3K/Akt, c-Raf, and p21-activated kinase (PAK; an effector molecule of Rac) were rapidly (within 30 s) activated in neutrophils upon exposure to calpain inhibitors (PD150606 and N-acetyl-Leu-Leu-Nle-CHO) but not PD145305 (inactive analog of PD150606). Following activation of these pathways, neutrophils displayed active migration (chemotaxis), which was sustained for more than 45 min. The studies with pharmacological inhibitors suggest that calpain inhibition-mediated neutrophil migration is mediated by activation of MEK/ERK, p38, JNK, PI-3K/Akt, and Rac. NSC23766 (Rac inhibitor) and pertussis toxin (PTX) suppressed calpain inhibitor-induced phosphorylation of distinct signaling molecules (PAK, c-Raf, MEK1/2, ERK, MKK3/6, p38, JNK, and Akt) as well as cell migration, suggesting that the PTX-sensitive G protein and Rac axis may be a possible key target of calpain inhibitors. Differentiated neutrophil-like HL-60 cells but not undifferentiated cells displayed cell migration and activation of MAPKs and PI-3K/Akt on calpain inhibition. These findings suggest that constitutively active calpain negatively regulates activation of the distinct signaling pathways and cell migration in resting neutrophils, and this regulatory system develops during differentiation into mature neutrophils.

Key Words: mitogen-activated protein kinases • pertussis toxin • phosphatidylinositol 3-kinase • Rac


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INTRODUCTION
 
Calpain, the calcium-dependent cysteine protease, has been implicated as a regulator of the actin cytoskeleton and cell migration [1 , 2 ]. In all cell types except human neutrophils, inhibition of calpain reduces cell migration rates and invasiveness. Calpain promotes the turnover of adhesion complexes by cleaving focal adhesion proteins such as focal adhesion kinase, talin, and paxillin. Calpain-deficient fibroblasts (Capn4–/–) exhibit reduced migration, extended numerous thin projections, and delayed retraction of membrane protrusions and filopodia [3 ]. In contrast to all other cell types examined, inhibition of calpain I (µ-calpain) activity promotes polarization and random migration (chemokinesis) in human neutrophils [4 ]. Calpain is constitutively active in resting neutrophils, and the calpain activity in resting neutrophils may be primarily ascribed to calpain I [4 5 6 7 ]. Neutrophil migration induced by calpain inhibition may be associated with activation of Rac and Cdc42 [4 ]. By contrast, a recent study shows that chemoattractants induce asymmetric recruitment of calpain II (m-calpain) but not calpain I to the leading edge of polarized human neutrophils, and calpain II may play an important role in regulating pseudopodia formation [7 ]. These previous studies suggest that calpain plays an important role in regulation of neutrophil migration in an isoform-specific manner. However, the mechanisms by which global inhibition of calpain activity promotes neutrophil migration in the absence of any exogenous stimuli remain to be elucidated.

Our recent study shows that G-CSF induces human neutrophil random migration via activation of ERK and PI-3K [8 ]. Human neutrophils also display random migration in response to TLR agonists such as LPS (TLR4 agonist) and N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)- propyl]-(R)-cysteinyl-seryl-(lysyl)(3)-lysine (TLR2 agonist), and these TLR agonists induce neutrophil migration via activation of ERK and p38 MAPK [9 ]. It has also been reported that fMLP-induced human neutrophil chemotaxis is dependent on activation of p38, but not ERK, and that IL-8-induced human neutrophil chemotaxis is independent of activation of ERK and p38 [10 , 11 ]. These findings suggest that MAPK subtypes play an important role in human neutrophil migration according to the agonists used. These previous studies raise the possibility that the MAPK cascades might be activated in human neutrophils on calpain inhibition.

In the present study, we examined the signaling molecules activated in human neutrophils upon calpain inhibition. We found that MAPKs, including ERK, p38, and JNK, c-Raf, MEK1/2, MAPK kinase 3/6 (MKK3/6), PI-3K/Akt, and p21-activated kinase (PAK; an effector molecule of Rac and Cdc42) were rapidly activated in human neutrophils upon exposure to calpain inhibitors. The results suggest that calpain inhibition-mediated neutrophil migration is mediated by activation of MEK/ERK, p38, JNK, PI-3K/Akt, Rac, and a pertussis toxin (PTX)-sensitive G protein, and the PTX-sensitive G protein and Rac axis may be a possible key target of calpain inhibitors. We also found that the cell responsiveness to calpain inhibition developed during differentiation of HL-60 cells into mature neutrophils.


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MATERIALS AND METHODS
 
Reagents
PD150606, PD145305, N-acetyl-Leu-Leu-Nle-CHO (ALLN), calpastatin peptide, SB203580, U0126, SP600125, and NSC23766 were purchased from Calbiochem (San Diego, CA, USA). fMLP, PTX, and LY294002 were purchased from Sigma Chemical Co. (St Louis, MO, USA). IL-8 was purchased from PeproTech (Rocky Hill, NJ, USA). Recombinant human G-CSF and GM-CSF produced by Escherichia coli were provided by Kirin Brewery (Tokyo, Japan) and Schering-Plough (Osaka, Japan), respectively. Alexa Fluor 546-conjugated phalloidin was purchased from Molecular Probes (Eugene, OR, USA). Conray was purchased from Mallinckrodt (St. Louis, MO, USA). Ficoll and the ECL Western blotting system were purchased from Amersham Pharmacia Biotech (Buckinghamshire, UK). Rabbit polyclonal antibodies against ERK1/2, Thr202/Tyr204-phosphorylated ERK1/2, p38, Thr180/Tyr182-phosphorylated p38, JNK, Thr183/Tyr185-phosphorylated JNK, Akt, Ser473-phosphorylated Akt, Ser338-phosphorylated c-Raf, Ser217/221-phosphorylated MEK1/2, Ser189/207-phosphorylated MKK3/6, Thr423-phosphorylated PAK1/Thr402-phosphorylated PAK2, Tyr705-phosphorylated STAT3, and goat anti-rabbit IgG antibody conjugated with HRP were purchased from Cell Signaling Technology (Beverley, MA, USA).

Cell culture
Human myelogenous leukemia cell line HL-60 cells were grown in RPMI-1640 medium supplemented with 10% heat-inactivated FCS, penicillin (100 U/ml), and streptomycin (100 µg/ml). For induction of differentiation of HL-60 to granulocytic cells, cells were seeded at 2 x 105 cells/ml and grown in the presence of 1.3% DMSO for 6 days [12 ].

Preparation of cells
Human peripheral blood neutrophils were prepared from healthy adult donors as described previously [13 ], using dextran sedimentation, centrifugation with Conray-Ficoll, and hypotonic lysis of contaminating erythrocytes. Neutrophil fractions were suspended in HBSS containing 10 mM HEPES (pH 7.4) and contained more than 95% neutrophils, and contaminating cells were almost exclusively eosinophils. Informed consent was obtained from all subjects. Differentiated and undifferentiated HL-60 cells were suspended in HBSS containing 10 mM HEPES (pH 7.4).

Time-lapse recording and analysis of cell migration
Cells (3.5x105/ml) suspended in HBSS were placed in a glass-bottom dish (MatTek Co., Ashland, MA, USA), the surface of which was precoated with FCS to prevent spontaneous neutrophil adherence to the glass surface. The cell suspensions were kept at 37°C, a warmer being placed under the dish. Cells were monitored under a microscope (Olympus, Tokyo, Japan) with a 40x phase-contrast objective lens, and the videotape recording was performed as described previously [8 , 9 ]. The videotape recording was converted to digital imaging for analysis. The migration distance and speed were determined using an image analysis software MOVE-TR/2D (Library Co., Tokyo, Japan). The center of the cell body was traced every 10 s with the software to determine the migration distance during the period required. The migration speed was determined by measuring the distances that cells traveled during each 5 min period. When required, cells were pretreated with various inhibitors (U0126, SB203580, SP600125, LY294002, and NSC23766) for 30 min at 37°C before stimulation with calpain inhibitors. Cell viability was determined by the trypan blue exclusion test, and the inhibitors used were not toxic to neutrophils.

Migration assay with the micropipette method
Cells were placed in a glass-bottom dish as described above. PD150606 (500 µM or 2.5 mM), ALLN (6.5 mM), G-CSF (2.5 µg/ml), or fMLP (1 µM) was loaded into a sterile injection capillary, Femtotip II (Eppendorf, Hamburg, Germany). A gradient was formed by slow release of the stimuli from the tip into the medium by using an Eppendorf microinjector, CellTram vario. Cells were monitored under a microscope, and the videotape recording was performed as described above.

Determination of actin reorganization
Actin reorganization was analyzed using confocal laser-scanning microscopy as described previously [14 ]. Neutrophils (5x106/ml, 1 ml) suspended in HBSS were stimulated with PD150606 (10 µM), PD145305 (10 µM), or ALLN (130 µM) on FCS-coated glass coverslips for 20 min at 37°C. After incubation, cells were fixed with 4.4% paraformaldehyde and permeabilized with 0.2% Triton X-100 in PBS. Cells were incubated with Alexa Fluor 546-conjugated phalloidin (0.2 U/ml) in the dark for 30 min at room temperature. Fluorescence images were photographed with a confocal laser-scanning microscope (Zeiss LSM510, Welwyn, Garden City, UK).

Western blotting
Western blotting was performed as described previously [15 ]. Cells (5x106/ml) suspended in HBSS were stimulated with PD150606, PD145305, ALLN, calpastatin peptide, IL-8, or fMLP at 37°C. When required, cells were pretreated with various inhibitors (U0126, SB203580, SP600125, LY294002, and NSC23766) for 30 min at 37°C before stimulation with calpain inhibitors. The reactions were terminated by the addition of TCA. The final TCA concentration was 10%. The cells were washed with acetone containing 10 mM DTT and were lysed with the 1.3x sample buffer (2.6% SDS, 13% glycerol, 6.5% ME, and a trace amount of bromophenol blue dye in 62.5 mM Tris-HCl, pH 6.8), heated at 100°C for 5 min, and then frozen at –20°C until analysis. Samples were subjected to 5–20% gradient SDS gel electrophoresis. After electrophoresis, proteins were electrophoretically transferred from the gel onto a nitrocellulose membrane in a buffer containing 100 mM Tris, 192 mM glycine, and 20% methanol at 2 mA/cm2 for 1.5 h at room temperature. Residual binding sites on the membrane were blocked by incubating the membrane in Tris-buffered saline (pH 7.6) containing 0.1% Tween 20 and 5% nonfat dry milk for 2 h at room temperature. The blots were incubated with appropriate primary antibody overnight at 4°C. After washing, the membrane was incubated with appropriate secondary antibody conjugated with HRP, and the antibody complexes were visualized by the ECL detection system as directed by the manufacturer.

Statistical analysis
An ANOVA followed by a multiple comparison test or Student’s t-test was used to determine statistical significance.


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RESULTS
 
Calpain inhibition induces neutrophil chemotaxis
When neutrophils were challenged with calpain inhibitors (PD150606 and ALLN), neutrophils displayed active migration within 5 min after stimulation, and active migration was sustained for more than 45 min (Fig. 1A ). Neutrophil migration was not induced by PD145305 (an inactive analog of PD150606), which is unable to inhibit calpain activity. Consistent with induction of neutrophil migration, cell polarization with F-actin redistribution was induced by exposure to PD150606 or ALLN but not PD145305 (Fig. 1B) . These findings are consistent with the previous report demonstrating that calpain inhibitors induce neutrophil migration, presumably random migration [4 ]. However, the authors in this report presented no definite evidence supporting that neutrophils display random migration but not chemotaxis in response to calpain inhibition. Then, we studied neutrophil migration using the micropipette method [9 ], which can create a gradient of agonists (Fig. 2B ). As expected, neutrophils migrated toward the micropipette tip when fMLP, a chemotactic peptide, was used as a stimulus. When G-CSF was used as a stimulus, neutrophils displayed active migration but never migrated toward the micropipette tip (Fig. 2A) , indicating that G-CSF induces neutrophil random migration but not chemotaxis, in agreement with our previous report [8 ]. When PD150606 or ALLN was used as stimulus, neutrophils migrated toward the micropipette tip, indicating that neutrophils display chemotaxis but not random migration in response to PD150606 or ALLN (Fig. 2A) .


Figure 1
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  		Figure 1. Calpain inhibition induces neutrophil migration and polarization with F-actin redistribution. (A) Neutrophils were exposed to PD145305 (10 µM), PD150606 (10 µM), or ALLN (130 µM), and neutrophil migratory responses were monitored under a video-microscope. The migration speed represents the mean speed calculated from the migration distance that neutrophils traveled during each 5 min period. The data are expressed as the mean ± SEM, and at least 50 cells were analyzed for each point. The data shown are representative of three independent experiments. At each point, the migration speed in PD150606- or ALLN-treated cells is significantly (P<0.01) greater than that in control or PD145305-treated cells. (B) Neutrophils were treated with PD145305 (10 µM), PD150606 (10 µM), or ALLN (130 µM) for 20 min at 37°C. F-actin distribution was analyzed with confocal microscopy. The images shown are representative of three independent experiments.


Figure 2
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  		Figure 2. Calpain inhibition induces neutrophil chemotaxis. G-CSF (2.5 µg/ml), PD150606 (500 µM), ALLN (6.5 mM), fMLP (1 µM), or an Alexa Fluor 488-conjugated F(ab')2 fragment of goat anti-rabbit IgG (20 µg/ml) was loaded into a sterile injection capillary, Femtotip II. A gradient was formed by slow release of the stimuli from the tip into the medium by using an Eppendorf microinjector. (A) The images at the indicated time-points after the release of G-CSF, PD150606, ALLN, or fMLP are shown. The tracks that neutrophils moved along during the indicated periods are shown at the bottom. An arrowhead indicates the micropipette tip. The images shown are representative of three independent experiments. (B) The fluorescence images shortly after the release of the Alexa Fluor 488-conjugated F(ab')2 fragment are shown (upper panel). The relative fluorescence intensity was determined along the line from the Point A (the tip) to the Point B on the upper panel images and was plotted to show a concentration gradient of this probe (lower panel).

Calpain inhibition induces activation of MAPKs, PI-3K, and PAK
Direct stimulation of neutrophil migration by calpain inhibitors suggests that certain signaling molecules involved in neutrophil migration may be activated by exposure to calpain inhibitors. As shown in Figure 3A , stimulation of neutrophils with PD150606 resulted in significant phosphorylation of MEK1/2, ERK1/2, MKK3/6, p38, JNK, and Akt. PD150606-induced phosphorylation of these molecules was rapid, and the maximum phosphorylation was detected within 1 min after stimulation with PD150606. Increased phosphorylation of MKK3/6 and p38 in unstimulated control cells was sometimes detected when cells were incubated for >60 min, presumably because of activation of the MKK3/6-p38 pathway by stresses such as cell preparation procedures and incubation of cells in the buffer solution [16 ]. The effect of PD150606 on phosphorylation of these molecules was concentration-dependent, and the significant effect was obtained at 5–10 µM (Fig. 3B) .


Figure 3
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  		Figure 3. Calpain inhibition induces activation of the distinct signaling pathways in neutrophils. (A) Neutrophils were treated with PD150606 (10 µM) or ALLN (130 µM) for the indicated periods at 37°C. (B) Neutrophils were treated with the indicated concentrations of PD150606 or ALLN or 10 µM PD145305 for 1 min at 37°C. Immunoblotting was performed using antibodies against the phosphorylated form (P) of each protein. The equal loading of proteins onto each lane was confirmed by immunoblotting using antibody against p38. The cell lysates equivalent to 2.5 x 105 cells were loaded onto each lane. The results shown are representative of three independent experiments. (C) Neutrophils were treated with calpastatin peptide (50 µM) for the indicated periods at 37°C. For comparison, neutrophils were treated with or without ALLN (130 µM) for 5 min at 37°C. Immunoblotting was performed using antibodies against the phosphorylated and unphosphorylated forms of each protein. The results shown are representative of three independent experiments.

Similar findings were obtained when ALLN was used instead of PD150606. As shown in Figure 3A , stimulation of neutrophils with ALLN resulted in significant phosphorylation of MEK1/2, ERK1/2, MKK3/6, p38, JNK, and Akt. ALLN-induced phosphorylation of these molecules was rapid, and the maximum phosphorylation was detected within 1 min after stimulation with ALLN. The effect of ALLN on phosphorylation of these molecules was concentration-dependent, and the significant effect was obtained at 65–130 µM (Fig. 3B) . JNK phosphorylation induced by PD150606 or ALLN was sometimes weak, according to the cell preparations used.

PAK, an effector molecule of Rac and Cdc42 [17 ], was also phosphorylated in neutrophils stimulated with PD150606 or ALLN (Fig. 3B) , consistent with the previous report showing that Rac and Cdc42 are activated in neutrophils stimulated with calpain inhibitors [4 ]. By contrast, stimulation of neutrophils with PD145305 failed to induce phosphorylation of all these molecules. In addition, STAT3 was not phosphorylated in neutrophils stimulated with PD150606 or ALLN (Fig. 3B) . These findings indicate that the distinct signaling pathways are selectively activated in neutrophils exposed to calpain inhibitors (PD150606 and ALLN).

Rapid activation of the distinct signaling pathways by PD150606 and ALLN raises the possibility that these calpain inhibitors might exert their effects on the cell surface (outside the cells) besides their effect on calpain activity inside the cells. To explore this possibility, we used a synthetic calpastatin peptide (molecular mass, 3177.7), a 27-residue peptide encoded by Exon 1B, which would be expected to be less cell-permeable as compared with ALLN (molecular mass, 383.5) because of its higher molecular mass [18 , 19 ]. If the calpastatin peptide could exert its effect on the cell surface, it would be expected that this peptide could induce rapid activation of the distinct signaling pathways. On the other hand, if the calpastatin peptide exerted its effect inside the cells as expected, it would be expected that activation of the distinct signaling pathways would be delayed to some extent because of slow penetration of this peptide into the cells. The results shown in Figure 3C indicate that the latter is the case. Significant phosphorylation of ERK1/2, p38, and Akt was detected at 10 min after stimulation with the calpastatin peptide, and the phosphorylation level of these molecules was gradually increased over the course of 60 min, which may reflect slow penetration of this peptide into the cells. Activation of the distinct signaling pathways by the calpastatin peptide may be consistent with the previous observation that this peptide can induce human neutrophil polarization [4 ]. These findings suggest that calpain inhibitors (PD150606, ALLN, and calpastatin peptide) used here may act inside the cells and exert their effects through inhibition of the calpain activity.

Involvement of MAPKs, PI-3K, and Rac in neutrophil migration induced by calpain inhibition
Possible participation of MAPKs, PI-3K, and Rac in neutrophil migration induced by calpain inhibition was explored by using pharmacological inhibitors against MEK1/2 (U0126), p38 (SB203580), JNK (SP600125), PI-3K (LY294002), and Rac (NSC23766). As shown in Figure 4 , PD150606-induced neutrophil migration was significantly suppressed in the presence of these inhibitors when they were used alone or in combination. In particular, neutrophil migration was almost completely abolished in the presence of SP600125, U0126 + SB203580 + SP600125, or U0126 + SB203580 + LY294002. Suppression of neutrophil migration by U0126 + SB203580 was significantly greater than that by each inhibitor alone (P<0.01). These findings suggest that MEK/ERK, p38, JNK, PI-3K, and Rac are involved in neutrophil migration induced by calpain inhibition.


Figure 4
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  		Figure 4. Involvement of MAPKs, PI-3K, and Rac in neutrophil migration induced by calpain inhibition. Neutrophils were pretreated with U0126 (U; 10 µM), SB203580 (SB; 10 µM), SP600125 (SP; 10 µM), LY294002 (LY; 10 µM), and/or NSC23766 (NSC; 50 µM) for 30 min at 37°C and thereafter, exposed to PD150606 (10 µM). (A) Cell migration was monitored under a video-microscope. The migration speed was determined at 20–25 min after exposure to PD150606. The data are expressed as the mean ± SEM of three independent experiments, and at least 50 cells were analyzed for each experiment. *, PD150606-induced neutrophil migration was suppressed significantly in the presence of indicated inhibitors (P<0.01). (B) The tracks that neutrophils moved along at 20–25 min after exposure to PD150606 are shown. The images shown are representative of three independent experiments.

As shown in Figure 5 , c-Raf, an upstream molecule of MEK1/2, was also phosphorylated in neutrophils stimulated with PD150606, suggesting that the target of PD150606 is an upstream molecule of c-Raf. Interestingly, PD150606-induced phosphorylation of all molecules (PAK, c-Raf, MEK1/2, ERK1/2, MKK3/6, p38, JNK, and Akt) was suppressed by NSC23766 (Rac inhibitor), suggesting that Rac is a possible key target molecule of PD150606. U0126 (MEK1/2 inhibitor) suppressed PD150606-induced phosphorylation of MEK1/2, ERK1/2, and JNK. SB203580 (p38 inhibitor) suppressed PD150606-induced phosphorylation of p38, ERK1/2, and JNK. SP600125 (JNK inhibitor) rather enhanced PD150606-induced phosphorylation of MEK1/2, ERK1/2, JNK, and PAK, whereas SP600125 by itself induced significant phosphorylation of p38 and JNK, suggesting that SP600125 might exert some nonspecific effect on neutrophils. LY294002 (PI-3K inhibitor) suppressed PD150606-induced phosphorylation of Akt, c-Raf, MEK1/2, ERK1/2, JNK, and PAK. The combination of these inhibitors caused essentially the additive effects (Fig. 5) .


Figure 5
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  		Figure 5. Effects of various inhibitors on PD150606-induced phosphorylation of signaling molecules. Neutrophils were pretreated with U0126 (10 µM), SB203580 (10 µM), SP600125 (10 µM), LY294002 (10 µM), and/or NSC23766 (50 µM) for 30 min at 37°C and thereafter, exposed to PD150606 (10 µM) for 1 min at 37°C. Immunoblotting was performed using antibodies against the phosphorylated form of each protein. The equal loading of proteins onto each lane was confirmed by immunoblotting using antibody against p38. The cell lysates equivalent to 2.5 x 105 cells were loaded onto each lane. The results shown are representative of three independent experiments.

Involvement of PTX-sensitive G protein in neutrophil migration induced by calpain inhibition
As shown in Figure 6 , IL-8- and fMLP-induced neutrophil migration and polarization were markedly suppressed by pretreatment of cells with PTX (250 ng/ml) for 4 h at 37°C. PD150606- and ALLN-induced neutrophil migration and polarization were also markedly suppressed by pretreatment of cells with PTX, in contrast to the previous report [4 ]. Under the same conditions, phosphorylation of p38, ERK1/2, JNK, PAK, or Akt induced by stimulation with IL-8, fMLP, PD150606, or ALLN, but not with GM-CSF, was markedly suppressed by pretreatment of cells with PTX. These findings indicate that a PTX-sensitive G protein is involved in calpain inhibition-mediated neutrophil migration and activation of the distinct signaling pathways.


Figure 6
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  		Figure 6. Involvement of PTX-sensitive G protein in neutrophil migration induced by calpain inhibition. Neutrophils (5x106 cells/ml) were pretreated with PTX (250 ng/ml) for 4 h at 37°C and thereafter, exposed to IL-8 (100 ng/ml), fMLP (10 nM), PD150606 (10 µM), ALLN (130 µM), or GM-CSF (5 ng/ml). (A) The images were obtained at 15 min after exposure to IL-8 and at 40 min after exposure to PD150606. The images shown are representative of three independent experiments. (B) Cell migration was monitored under a video-microscope. The migration speed was determined at 10–15 min after exposure to IL-8 or fMLP and at 35–40 min after exposure to PD150606 or ALLN. The data are expressed as the mean ± SEM of three independent experiments, and at least 20 cells were analyzed for each experiment. *, Significantly suppressed by pretreatment of cells with PTX (P<0.01). (C) Neutrophils were treated with PD150606 or ALLN for 1 min, IL-8 or fMLP for 3 min, and GM-CSF for 5 min at 37°C, respectively. Immunoblotting was performed using antibodies against the phosphorylated and unphosphorylated forms of each protein. The results shown are representative of three independent experiments.

Differentiated, but not undifferentiated, HL-60 cells respond to calpain inhibition
As shown in Figure 7 , the phosphorylation level of p38, ERK1/2, JNK, and Akt in the resting state of differentiated, neutrophil-like HL-60 cells was reduced as compared with that of undifferentiated HL-60 cells. Differentiated, but not undifferentiated, HL-60 cells showed increased phosphorylation of p38, ERK1/2, JNK, and Akt in response to stimulation with PD150606, ALLN,or fMLP. Consistent with activation of the distinct signaling pathways, differentiated but not undifferentiated HL-60 cells displayed chemotaxis in response to PD150606, ALLN, or fMLP (data not shown for undifferentiated HL-60 cells) (Fig. 8 ). These findings indicate that the cell responsiveness to calpain inhibition develops during differentiation into mature neutrophils and that calpain inhibitors exert the specific effects on mature neutrophils.


Figure 7
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  		Figure 7. Calpain inhibition induces phosphorylation of MAPKs and Akt in differentiated, but not undifferentiated, HL-60 cells. Undifferentiated and differentiated HL-60 cells were exposed to PD150606 (50 µM), ALLN (130 µM), or fMLP (1 µM) for 1 min at 37°C. Immunoblotting was performed using antibodies against the phosphorylated and unphosphorylated forms of each protein. The cell lysates equivalent to 2.5 x 105 cells were loaded onto each lane. The results shown are representative of three independent experiments.


Figure 8
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  		Figure 8. Calpain inhibition induces chemotaxis in differentiated HL-60 cells. PD150606 (2.5 mM), ALLN (6.5 mM), or fMLP (1 µM) was loaded into a sterile injection capillary, Femtotip II. A gradient was formed by slow release of the stimuli from the tip into the medium by using an Eppendorf microinjector. The images at the indicated time-points after the release of PD150606, ALLN, or fMLP are shown. The tracks that HL-60 cells moved along during the indicated periods are shown at the bottom. Each arrow in each series indicates the same cell. An arrowhead indicates the micropipette tip. The images shown are representative of three independent experiments.


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DISCUSSION
 
The present experiments show that calpain inhibition promotes PTX-sensitive neutrophil migration and induces rapid activation of distinct signaling molecules, including c-Raf, MEK1/2, ERK1/2, MKK3/6, p38, JNK, Akt, and PAK. The studies with pharmacological inhibitors suggest that these molecules are involved in neutrophil migration induced by calpain inhibition. The results also suggest that calpain, which is constitutively active in resting neutrophils, exerts a negative regulation on activation of the distinct signaling pathways as well as cell migration in resting neutrophils. Calpain-mediated negative regulation of the distinct signaling pathways and cell migration is specific for mature neutrophils, as the cell responsiveness to calpain inhibition develops during differentiation of HL-60 cells into mature neutrophils.

Lokuta et al. [4 ] have demonstrated that active calpain I is expressed predominantly and constitutively in resting neutrophils, and the specific inhibitor for calpain I but not calpain II induces neutrophil polarization and migration. These findings suggest that constitutively active calpain I in resting neutrophils may function as a negative regulator on neutrophil polarization and migration. We confirmed this observation and extended the study to show that neutrophil migration induced by calpain inhibition was chemotaxis but not random migration. In addition, we found that the distinct signaling pathways were rapidly activated upon exposure to calpain inhibitors, suggesting that constitutively active calpain in resting neutrophils may also function as a negative regulator on activation of the distinct signaling pathways. Human peripheral blood lymphocytes did not show increased phosphorylation of these signaling molecules nor cell migration in response to calpain inhibitors (data not shown). Negative regulation of the distinct signaling pathways by calpain thus appears to be specific for mature neutrophils. This regulatory system may develop during differentiation into mature neutrophils, as differentiated but not undifferentiated HL-60 cells show increased phosphorylation of distinct signaling molecules as well as cell migration in response to calpain inhibitors. Calpain-mediated suppression of the distinct signaling pathways in resting neutrophils may, at least in part, contribute to suppression of neutrophil polarization and migration as well as maintenance of a spherical phenotype in resting cells.

The studies with pharmacological inhibitors suggest that calpain inhibition-mediated neutrophil migration is dependent on activation of MEK/ERK, p38, JNK, and PI-3K/Akt. Possible involvement of these molecules in neutrophil migration has been shown in several systems, although the roles of these molecules may be dependent on the agonists used [8 , 9 , 14 , 20 21 22 ]. Our previous studies show that MEK/ERK, p38, and PI-3K/Akt are involved in actin reorganization in human neutrophils stimulated with G-CSF, GM-CSF, TNF-{alpha}, or TLR agonists [8 , 9 , 14 , 21 ]. MEK/ERK is involved in G-CSF-induced neutrophil random migration and is associated with G-CSF-induced redistribution of F-actin and phosphorylated myosin light chain [8 , 14 ]. Coordinated action of MEK/ERK and p38 is involved in neutrophil random migration induced by TLR2 and TLR4 agonists [9 ]. Activation of p38 and MAPK-activated protein kinase-2, a downstream kinase of p38, plays a role in fMLP-induced chemotaxis [22 ]. Although possible involvement of JNK has not been reported in neutrophil migration, the role of JNK in cell migration has been demonstrated in several other systems, including hepatocyte growth factor-induced migration of human brain endothelial cells and IL-1β-induced migration of rat cardiac fibroblasts [23 , 24 ].

Calpain inhibition caused phosphorylation of PAK (an effector molecule of Rac and Cdc42), and NSC23766 (Rac inhibitor) suppressed calpain inhibition-mediated phosphorylation of PAK, c-Raf, MEK1/2, ERK1/2, MKK3/6, p38, JNK, and Akt, as well as cell migration. These findings suggest that calpain negatively regulates activation of Rac, and calpain inhibition results in rapid activation of Rac, leading to activation of the Rac-dependent downstream signaling pathways, i.e., the c-Raf-MEK1/2-ERK1/2, MKK3/6-p38, JNK, and PI-3K-Akt pathways. In this regard, it is of interest that calpain I is active in adherent bovine aortic endothelial cells, and calpain may positively regulate Rac- and Rho-mediated cytoskeletal reorganization by acting at sites upstream of Rac1 and RhoA [25 ]. Using a murine model, Li et al. [26 ] have demonstrated that upon activation of G-coupled receptors, Gβ{gamma} binds PAK1 and via interaction with PAK-interacting exchange factor, activates Cdc42, which in turn leads to activation of PAK1. Thus, it appears that PAK1 is not only an effector for Cdc42, but it also functions as a scaffold protein required for Cdc42 activation. This pathway may play a role in directional sensing and persistent polarized migration of neutrophils [26 ]. In neutrophils, calpain inhibition results in activation of Rac and Cdc42 [4 ] as well as PAK, suggesting that calpain might negatively regulate the interaction of Rac/Cdc42 and PAK. It has been reported that the c-Raf-MEK-ERK and MKK3/6-p38 pathways can be activated by PAK [27 , 28 ], and expression of constitutively active Rac results in activation of JNK and p38 in certain situations [29 ]. These findings support the notion that Rac/PAK activation can lead to activation of ERK, p38, and JNK. In this study, we used antibody against Ser338-phosphorylated c-Raf, indicating that c-Raf was phosphorylated at Ser-338 on calpain inhibition. It has been reported that c-Raf is phosphorylated at Ser-338 by PAK [27 ]. These findings support the notion that in neutrophils, calpain inhibition causes Rac/PAK activation, leading to phosphorylation of c-Raf at Ser-338. It has also been shown that there is a positive-feedback loop between Rac and PI-3K [30 , 31 ], which may explain the similar suppressive effects of NSC23766 and LY294002 on phosphorylation of signaling molecules in PD150606-stimulated neutrophils (Fig. 5) .

The studies with PTX indicate that a PTX-sensitive G protein is involved in calpain inhibition-mediated neutrophil migration and activation of the distinct signaling pathways (PAK, ERK1/2, p38, JNK, and PI-3K/Akt) and suggest that the PTX-sensitive G protein and Rac/PAK axis may be a possible key target of calpain inhibitors. The mechanisms by which calpain inhibition induces activation of a PTX-sensitive G protein remain to be determined. One possibility is that constitutively active calpain may negatively regulate activation of a PTX-sensitive G protein. Another possibility is that the calpain-calpain inhibitor complexes might activate a PTX-sensitive G protein in association with or without cytoplasmic domain of G protein-coupled receptors.

Another important function of constitutively active calpain may be associated with regulation of neutrophil apoptosis. X-linked inhibitor of apoptosis, the efficient inhibitor of caspase-3, -7, and -9 [32 ], is cleaved by calpain, leading to acceleration of spontaneous neutrophil apoptosis. In fact, calpain inhibitors (PD150606, ALLN, or calpeptin) can delay spontaneous and TNF-{alpha}-induced neutrophil apoptosis [5 , 6 ]. The results presented here suggest that calpain inhibitors might delay neutrophil apoptosis, not only by inhibiting calpain activity but also by activating prosurvival molecules such as MEK/ERK and PI-3K/Akt, both of which are known to be involved in delayed neutrophil apoptosis under certain situations [33 , 34 ]. However, the latter possibility appears to be unlikely, as we observed that MEK and PI-3K inhibitors did not affect calpain inhibition-mediated delay of spontaneous neutrophil apoptosis (unpublished).

The present experiments show a unique role of calpain in resting neutrophils; i.e., calpain may negatively regulate activation of the distinct signaling pathways and cell migration in resting neutrophils. This regulatory system may develop during differentiation into mature neutrophils. The calpain-mediated regulatory system for activation of the distinct signaling pathways and cell migration and apoptosis may play an important role in the functions and the fate of mature neutrophils, highly motile and terminally differentiated cells.


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ACKNOWLEDGEMENTS
 
This work was supported by a Grant-in-Aid for Scientific Research, Japan. We thank Dr. F. Hato for helpful discussion.

Received September 30, 2007; revised March 9, 2008; accepted March 28, 2008.


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