Cofilin plays a critical role in IL-8-dependent chemotaxis of neutrophilic HL-60 cells through changes in phosphorylation

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1 Cofilin plays a critical role in IL-8-dependent chemotaxis of neutrophilic HL-60 cells through changes in phosphorylation Akiko Hirayama,*, Reiko Adachi,* Saki Otani,*, Tadashi Kasahara, and Kazuhiro Suzuki*,1 *Division of Biosignaling, National Institute of Health Sciences, Tokyo, Japan; and Department of Biochemistry, Kyoritsu University of Pharmacy, Tokyo, Japan Abstract: Cofilin is a ubiquitous, actin-binding protein. Only unphosphorylated cofilin binds actin and severs or depolymerizes filamentous actin (Factin), and the inactive form of cofilin is phosphorylated at Ser 3. We reported recently that cofilin plays a regulatory role in superoxide production and phagocytosis by leukocytes, and in the present study, we investigated the role of cofilin in the chemotaxis of neutrophilic HL-60 cells. IL-8 is a potent, physiological chemokine, and it triggers a rapid, transient increase in F-actin beneath the plasma membrane and rapid dephosphorylation and subsequent rephosphorylation of cofilin. In this study, cofilin phosphorylation was found to be inhibited by S3-R peptide, which consists of a peptide corresponding to part of the phosphorylation site of cofilin and a membrane-permeable arginine polymer. When S3-R peptide was introduced into the neutrophilic cells, their chemotactic activity was enhanced, whereas a control peptide that contained an inverted sequence of the phosphorylation site of cofilin had no enhancing effect. Cofilin small interfering RNA (sirna) decreased cofilin expression by about half and inhibited chemotaxis. In IL-8-stimulated cells, unphosphorylated cofilin accumulated around F-actin, and colocalization of F-actin and phosphorylated cofilin was observed, but these changes in cofilin localization were less prominent in cofilin sirna-treated cells. The inhibitors of PI-3K wortmannin and LY inhibited the chemotaxis and suppressed IL-8-evoked dephosphorylation and rephosphorylation of cofilin. These results suggested that unphosphorylated cofilin plays a critical role in leukocyte chemotaxis and that PI-3K is involved in the control of the phosphorylation/dephosphorylation cycle of cofilin. J. Leukoc. Biol. 81: ; Key Words: chemotaxis neutrophil CXCL8 PI-3K sirna INTRODUCTION Cell migration has been studied widely, and it is considered essential in many physiological and pathological events, including the immune response, inflammation, angiogenesis, wound-healing, organ development, and tumor metastasis [1]. Among the many types of mammalian chemotactic cells, neutrophils have the ability to migrate most rapidly, and they play a crucial role in the host defense system. Defects in the migration activity of neutrophils are known to result in serious syndromes [2, 3]. A number of chemokines, a family of chemoattractant proteins that induce directional migration of cells, have been found recently to be important cytokines that act in vivo as well as in vitro. Chemokines bind to specific receptors consisting of seven-transmembrane proteins coupled to trimeric Gi proteins, and they induce the cell polarization required for directional movement. Chemokines have been classified into CXC, CC, C, and CX3C chemokines, based on differences in the numbers of amino acid residues between conserved cysteines [1, 4]. IL-8 is a typical CXC chemokine. It is the most potent CXC chemokine for neutrophils, and its physiological importance at inflammatory sites has been investigated thoroughly [5]. Reorganization of the actin cytoskeleton is the central event in cell migration [6], and the actin-binding protein cofilin plays an essential role in the control of the actin cytoskeleton through depolymerization and severing of filamentous actin [7]. Cofilin is a ubiquitous phosphoprotein in mammalian cells, and unphosphorylated cofilin is the active form that binds actin. Cofilin-specific kinases, LIM kinases (LIMKs) [8, 9] and testicular protein kinases (TESKs) [10], and cofilin-specific phosphatases, slingshot (SSH) [11] and chronophin [12], have been identified recently, and their roles have been studied in adherent cells, which are suitable for microscopic observation of the cytoskeleton. In activated leukocytes, cofilin is dephosphorylated and translocated to the cell membrane, where it is involved in superoxide production and phagocytosis [13 17]. Phosphorylated cofilin apparently plays a positive role in respiratory burst and phagocytosis [18], and consistent with these roles, decreasing cofilin expression with antisense oligonucleotide results in enhancement of superoxide production and 1 Correspondence: Division of Biosignaling, National Institute of Health Sciences, 18-1 Kamiyoga 1-chome, Setagaya-ku, Tokyo , Japan. ksuzuki@nihs.go.jp Received May 10, 2006; revised September 29, 2006; accepted October 30, doi: /jlb Journal of Leukocyte Biology Volume 81, March /07/ Society for Leukocyte Biology

2 phagocytosis of opsonized zymosan [19]. The roles of cofilin in chemokine-stimulated phagocytes, however, remain unknown, and no reports have described the relation between IL-8 stimulation and changes in cofilin phosphorylation. In the present study, we investigated IL-8-induced control of the actin cytoskeleton and changes in the phosphorylation state of cofilin, and we found evidence that unphosphorylated cofilin plays a positive role in the IL-8-dependent chemotaxis of leukocytes. We also obtained data suggesting that phosphorylation and dephosphorylation of cofilin are controlled downstream of PI-3K. MATERIALS AND METHODS Cells HL-60 cells were obtained from the Japanese Cancer Research Resources Bank and cultured in RPMI-1640 medium supplemented with 10% FCS. The cells were induced to differentiate into neutrophil-like cells by exposure to 1.25% DMSO and 25 ng/ml G-CSF for 6 days [20]. To maintain the quality of the cells, stored, frozen cells were thawed every 3 months and used in the experiments as described previously [13]. Reagents and antibodies Human IL-8 and G-CSF were obtained from PeproTech EC Ltd. (London, UK). Alexa Fluor 488-labeled phalloidin and Alexa Fluor 568-conjugated antimouse and antirabbit IgG antibodies F(ab )2 were purchased from Molecular Probes (Eugene, OR). CellTiter Gro was a product of Promega (Madison, WI). S3-R peptide (MASGVAVSDGVIKVFNRRRRRRRR) and control peptide (NFVKIVGDSVAVGSAMRRRRRRRR) were synthesized and purified more than 95% by Biologica Co. (Nagoya, Japan). Five cofilin sirnas (ACGCCAC- CUUUGUCAAGAU, AUGCCCUCUAUGAUGCAA, GAAGGAGGAUCUG- GUGUUU, AAAUGAUUUAUGCCAGCUC, and GCAUGAAUUGCAAG- CAAAC) and nonspecific, control small interfering RNA (sirna; NNACTC- TATCTGCACGCTGAC; GC content 52%) were obtained from Dharmacon RNA Technologies (Lafayette, CO, USA). Wortmannin and LY were from Sigma Chemical Co. (St. Louis, MO, USA). Antihuman CXCR1 antibody was from BioSource Co. (Camarillo, CA, USA). Antihuman CXCR2 antibody was from BD Biosciences (San Jose, CA, USA), and antiactin mab was from Cedar Lane Laboratories Ltd. (Hornby, Ontario, Canada). Anticofilin mab (MAB22) was a kind gift from Professor Takashi Obinata and Dr. Hiroshi Abe (Chiba University, Japan). The preparation of antiphosphorylated cofilin antibody has been described previously [18]. Analysis of CXCR1 and CXCR2 expression by flow cytometry During the differentiation culture described above, aliquots of HL-60 cells were removed and washed thoroughly with PBS and then with PBS containing 1% BSA. The washed cells were incubated with anti-cxcr1, anti-cxcr2 mab, or mouse IgG (control) for 30 min and then washed with PBS containing 1% BSA. After staining the cells with FITC-conjugated, antimouse IgG for 30 min, they were washed with PBS containing 1% BSA, and the fluorescence intensity of cells was analyzed with a flow cytometer (FACSCalibur, Becton Dicksinon. San Jose, CA, USA). The entire sample preparation procedure for flow cytometry was performed at 0 4 C. Chemotaxis of differentiated HL-60 cells Chemotaxis of neutrophilic HL-60 cells was measured in a 96-well chemotaxis chamber (Neuro Probe Inc., Gaithersburg, MD, USA) [21]. The chamber consisted of a 96-well upper chamber and a 96-well lower chamber separated by a polycarbonate filter (pore size, 2 m). The lower wells were filled with 380 l 10 ng/ml IL-8 in phenol red-free RPMI-1640 medium containing 0.5% BSA and 10 mm HEPES (ph 7.3). The cells were washed and suspended in the same medium and then divided into the upper wells ( cells in 260 l per well). After incubation at 37 C for 90 min, the cells that had migrated into the lower well of the 96-well chemotaxis chamber (Neuro Probe Inc.) were counted by chemiluminescence using the luciferase-containing reagent Cell- Titer Glo (Promega) as described previously [21, 22]. IL-8-dependent migration, conversely, was confirmed visually with a chemotaxis-monitoring system (EZ-TAXIScan, Effector Cell Institute Co., Tokyo, Japan). Treatment of cells with cofilin sirna and S3-R peptide Cells (usually in 7 ml culture medium) were incubated with 40 M sirna for the 48 h between Days 4 and 6 of culture for neutrophilic differentiation with a transfection reagent (Trans IT-TKO, Mirus Co., Madison, WI, USA). After washing with the medium for the chemotaxis assay, the cells were subjected to the chemotaxis assay. For the analysis of filamentous actin (F-actin) formation and IL-8-stimulated changes in cofilin phosphorylation, the cells were washed with HBSS. To assess the effect of peptides, the neutrophilic, differentiated cells (usually cells/1 ml medium for the chemotaxis assay) were incubated with S3-R or a control peptide at various concentrations at room temperature (22 C) for 30 min. Observation of fluorescence-stained cells Neutrophilic HL-60 cells were washed with HBSS and incubated with 10 ng/ml IL-8 at 37 C for various times. The cells were fixed and stained as described previously [18] with Alexa Fluor 488-phalloidin for F-actin and rabbit antiphosphocofilin antibody and then with Alexa Fluor 568-antirabbit IgG for phosphorylated cofilin. The stained cells were examined by confocal laserscanning microscopy (LSM510, Carl Zeiss Co., Jena, Germany), and their fluorescence intensity was measured with a flow cytometer (FACSCalibur, Becton Dickinson). IL-8-induced changes in cofilin phosphorylation The cells were activated with IL-8 as described above, treated with inhibitors of proteases, kinases, and phosphatases, and solubilized with sample buffer containing SDS. SDS-PAGE and immunoblotting were performed as described previously [23], with a specific antiphosphocofilin antibody [10] and anticofilin antibody (MAB22). The staining intensity of each band was measured with an image analyzer (LAS 3000, Fuji Film Co., Tokyo, Japan). RESULTS Responses of differentiated HL-60 cells to IL-8-stimulation IL-8 has been found to be a potent chemokine for neutrophils, and two types of the IL-8 receptor (IL-8R) have been identified: CXCR1 and CXCR2. Both have high affinity for IL-8, but CXCR1 is a more selective receptor for IL-8. CXCR2 responds to several other chemokines in addition to IL-8 [5]. The IL-8- response properties of the differentiated HL-60 cells were investigated. As shown in Figure 1A, neither receptor was detected on undifferentiated HL-60 cells, but expression of both increased during differentiation. The differentiated cells displayed strong chemotactic activity toward IL-8 (Fig. 1B), and the high-dose suppression observed here is a typical phenomenon of chemotaxis. More than 90% of the differentiated cells were observed to migrate toward IL-8 with a chargedcoupled device camera-equipped chemotaxis monitor (data not shown). Moreover, as shown in Figure 1C, IL-8 triggered a rapid, transient increase in F-actin, which accumulated beneath the cell membranes that formed lamellipodia or filopodia. Actin reorganization was also a typical response of chemoattractant-stimulated cells [6]. These findings indicated that the differentiated HL-60 cells had the same characteristics with respect to IL-8-dependent chemotaxis as neutrophils. Hirayama et al. Leukocyte chemotaxis is controlled by phosphorylation of cofilin 721

3 Fig. 1. IL-8-responsive characteristics of differentiated HL-60 cells. (A) Expression of IL-8Rs (CXCR1 and CXCR2) during differentiation. HL-60 cells were cultured in the presence of DMSO and G-CSF for 6 days, and expression of CXCR1 ( ) and CXCR2 ( ) was monitored daily by flow cytometry. (B) IL-8-dependent chemotaxis of terminally differentiated HL-60 cells. Random migration in the absence of IL-8 (chemokinesis) is expressed as 100%, and IL-8-induced chemotaxis is expressed as a percentage of the control value. (C) Transient IL-8-induced increase in F-actin in differentiated HL-60 cells. The cells were stimulated with IL-8 for the times indicated, then stained with fluorescence-labeled phalloidin, and analyzed by flow cytometry (left) or examined with a confocal laserscanning microscope (right). DMSO-induced HL-60 cells have been used as neutrophils in a chemotaxis experiment [24]. In our own experiment, we used not only DMSO but also G-CSF, which enhances neutrophilic differentiation in terms of superoxide production [20], expression of fmlp receptors [20], and transferring receptors [25]. In the experiments below, IL-8 10 ng/ml was used for the chemotaxis assays and for cell stimulation. IL-8-induced changes in cofilin phosphorylation Cofilin is a major actin-binding protein in leukocytes and plays an important role in respiratory bursts and phagocytosis [14, 18, 19], and its activity is controlled by phosphorylation and dephosphorylation [7]. In the unstimulated state, about half of the total cofilin in neutrophils [15] and differentiated HL-60 cells [14] is phosphorylated. We used immunoblotting with a specific antibody against phosphorylated cofilin to investigate whether IL-8 is capable of inducing changes in the state of cofilin phosphorylation. As shown in Figure 2, cofilin was dephosphorylated rapidly and subsequently, highly rephosphorylated by IL-8. The highly phosphorylated state of cofilin lasted for at least 30 min. This is the first description of the effect of IL-8 on the phosphorylation of cofilin. The marked changes in phosphorylation of cofilin may reflect its role in IL-8-stimulated chemotaxis. Effect of membrane-permeable, cofilin-related peptides on chemotaxis To assess the involvement of cofilin phosphorylation in IL-8- dependent chemotaxis, we prepared a cell membrane-permeable peptide called S3-R. The peptide was constructed from an N terminus peptide of cofilin (16 amino acids: MASGVAVS- DGVIKVFN) and a polymer of eight arginines (Fig. 3A). The peptide contains Ser 3, which is the phosphorylation site of cofilin, and the arginine polymer has the ability to permeate cell membranes. The cell membrane-permeating arginine polymer was developed by Futaki et al. [26]. For a control experiment, we prepared a peptide in which the sequence of the N terminus peptide of cofilin is reversed (Fig. 3A). After exposing the cells to the peptides for 30 min, their chemotactic activity was assayed. As shown in Figure 3B, S3-R peptide had a marked, enhancing effect at 50 g/ml and 100 g/ml, but the control peptide did not, and the phosphorylation state of cofilin 722 Journal of Leukocyte Biology Volume 81, March

4 Fig. 2. IL-8-induced change in the phosphorylation state of cofilin. The neutrophilic HL-60 cells were incubated with IL-8 (10 ng/ml) for the times indicated and subjected to immunoblotting. Phosphorylated cofilin and total cofilin were visualized with a specific antibody against phosphorylated cofilin and a mab MAB22, respectively. The degree of phosphorylation was determined by image analysis and normalized by the ratio of phosphorylated cofilin:total cofilin. The means and SD of four independent experiments are indicated. under those conditions was investigated by immunoblotting. As shown in Figure 4, the S3-R peptide had no effect on the rapid dephosphorylation of cofilin but inhibited its subsequent rephosphorylation, whereas the control peptide had no significant effect on either of the IL-8-dependent changes in cofilin phosphorylation. The S3-R peptide did not have any significant effect on migration in the absence of IL-8 at the concentratons in Figure 3B (data not shown). The target of S3-R is unclear, but it may inhibit cofilin kinases and not inhibit cofilin phosphatases. Effect of cofilin sirna on chemotaxis As a dominant-negative form of cofilin cannot be designed as a result of its conformational characteristics and as disruption of the cofilin gene results in the death of animals and cells [7, 27], we investigated the role of cofilin in chemotaxis by suppressing cofilin expression with sirnas. The cofilin content of the cells exposed to cofilin sirna decreased to 55% of its content in unexposed cells. As shown in Figure 5, the cells exposed to cofilin sirna exhibited little chemotactic activity, whereas cells exposed to control sirna displayed chemotaxis comparable with that of the unexposed cells. The data shown in Figures 3 5 suggest that unphosphorylated, active cofilin plays an important, positive role in IL-8- dependent chemotaxis. More specifically, although IL-8 induced rapid dephosphorylation and subsequent rephosphorylation of cofilin, interception of rephosphorylation resulted in augmentation of IL-8-induced chemotaxis, and down-regula- Fig. 3. Effect of S3-R peptide on IL-8-dependent chemotaxis. (A) Amino acid sequences of S3-R and control peptides. (B) Concentration-dependent effect of the peptides on IL-8-induced chemotaxis. The cells were treated with the peptides for 30 min at room temperature, and their chemotactic activity was assayed with 10 ng/ml IL-8. The means and SD are indicated. **, Significantly different (t test, P 0.01) from the value in the untreated, control cells. Hirayama et al. Leukocyte chemotaxis is controlled by phosphorylation of cofilin 723

5 Fig. 4. Inhibition of cofilin phosphorylation by the S3-R peptide. Neutrophilic HL-60 cells were treated with the peptides the same as in Figure 3 and stimulated with IL-8 for the times indicated. Phosphorylated cofilin and total cofilin were detected and determined as in Figure 2. One of the typical results of immunoblotting is shown, and means of four independent experiments are plotted in the graph. The SD were within 17% of the means. Untreated cells (e); control peptide-treated cells ( ); S3-R peptide-treated cells ( ). tion of cofilin expression by introduction of sirna abrogated chemotaxis. IL-8-induced changes in the intracellular localization of cofilin and actin F-actin formation and active F-actin and G-actin turnover are known to occur at the leading edge of moving cells [28], and as the major actin-binding protein cofilin seemed to play an important role in chemotaxis, we investigated the intracellular localization of cofilin in relation to the F-actin forming in chemotactic cells and chemotaxis-suppressed cells, which had been exposed to cofilin sirna. As shown in Figure 6, IL-8 induced rapid accumulation of cofilin just around the F-actin, which formed 30 s after IL-8 stimulation in the regions of the membrane protrusions in the nontransfected cells and in the control, sirna-treated cells. Some of the phosphorylated cofilin that stained with a specific antibody was colocalized with the F-actin that had formed, whereas hardly any colocalization Fig. 5. Effect of cofilin sirna on IL-8- dependent chemotaxis. The cells were treated with cofilin sirna or control sirna for 48 h, and cellular cofilin and actin were detected by immunoblotting (left). Their chemotactic activity was assayed, and the IL-8-independent, migrating activity of untreated cells is expressed as 100%. The means and SD of triplicate data are shown. Similar results were obtained in five independent experiments. 724 Journal of Leukocyte Biology Volume 81, March

6 Fig. 6. Intracellular localization of cofilin and F-actin. Neutrophilic, differentiated HL-60 cells treated with cofilin sirna or control sirna were stimulated with IL-8 (10 ng/ml) at 37 C for the times indicated and then stained with fluorescein-labeled phalloidin and anticofilin antibody (left) or antiphosphorylated cofilin antibody (right). F-actin appears green, and cofilin appears red. The colocalized area of F-actin and cofilin appear as the merged color yellow, and several examples are indicated by arrowheads. was observed in the cells exposed to cofilin sirna. Phosphorylated cofilin may play an important role in the IL-8-stimulated cells by supporting the F-actin that formed. Effect of PI-3K inhibitors on chemotaxis and cofilin phosphorylation It has been shown recently that the intracellular gradient of phosphorylated phosphatidylinositols in polarized chemotactic cells is important and that their kinases (PI-3Ks) are involved in the chemotaxis of various cells, including Dictiostellium discoydium [29] and mammalian cells (reviewed in ref. [30]). We investigated the effect of PI-3K inhibitors on the chemotaxis of neutrophilic, differentiated HL-60 cells. As shown in Figure 7, wortmannin and LY clearly suppressed IL- 8-dependent chemotaxis at PI-3K-inhibitable concentrations [31], and the effect of the PI-3K inhibitors on the phosphorylation states of cofilin was then investigated. In the untreated, control cells, cofilin was dephosphorylated rapidly and subsequently rephosphorylated, as described above (Fig. 2), whereas the rapid dephosphorylation and subsequent rephosphorylation of cofilin were inhibited in the wortmannin- or LY treated cells (Fig. 8). These results suggested that the IL-8- evoked changes in phosphorylation of cofilin are crucial for cell motility and that PI-3K lies upstream of the phosphatases and kinases of cofilin. neutrophilic cells, suggesting that control of cofilin phosphorylation plays an essential role in IL-8-dependent chemotaxis, as the phosphorylation state changed markedly during IL-8 stimulation; a cell-permeating peptide S3-R, which inhibits cofilin phosphorylation, enhanced IL-8-dependent chemotaxis; DISCUSSION In this paper, we first reported that cofilin is dephosphorylated rapidly and subsequently rephosphorylated in IL-8-stimulated Fig. 7. PI-3K-inhibitor suppression of IL-8-induced chemotaxis. The differentiated HL-60 cells were treated with wortmannin or LY at the concentrations indicated for 30 min, and their chemotactic activity was assayed by using 10 ng/ml IL-8 as the chemoattractant. Hirayama et al. Leukocyte chemotaxis is controlled by phosphorylation of cofilin 725

7 Fig. 8. Effect of PI-3K inhibitors on IL-8-induced changes in cofilin phosphorylation. The cells were treated with 10 nm wortmannin ( ), 10 M LY (e), or vehicle ( ) at 37 C for 30 min. Phosphorylated cofilin and total cofilin were detected by immunoblotting, and the degree of phosphorylation was determined as in Figure 2. The graph shows the means of three independent experiments. The SD were less than 18% of the means. the decrease in level of cofilin after exposure to sirna resulted in suppression of IL-8-induced chemotaxis; F-actin formed rapidly beneath the protruding membranes in IL-8-stimulated cells, and some of the cofilin was translocated close to the F-actin; and PI-3K inhibitors abolished the IL-8-triggered changes in cofilin phosphorylation and IL-8-dependent chemotaxis. Cofilin is known to bind actin and depolymerize and/or sever F-actin [7]. The rapidly dephosphorylated cofilin in IL-8-stimulated cells may be involved in disruption of pre-existing, old F-actin in unstimulated cells. Conversely, Ghosh et al. [32] have reported that cofilin promotes actin polymerization in directionally moving cells. Dephosphorylated cofilin may also be involved in rapid F-actin formation. In fact, S3-R peptide treatment resulted in enhancement of chemotaxis and inhibition of cofilin phosphorylation. The results indicating that unphosphorylated, active cofilin participates positively in the chemotaxis seemed consistent with the fact that unphosphorylated cofilin is involved in membrane protrusion in epidermal growth factor-treated cells [33 35]. We also observed heavy phosphorylation of cofilin in chemotactic cells after rapid dephosphorylation. Nishita et al. [36] observed that stimulation with the chemoattractant stromal cell-derived factor 1 (SDF-1) caused cofilin phosphorylation in Jurkat T cells. Phosphorylation of cofilin may result in the release of actin bound to unphosphorylated cofilin and/or in stabilization of F-actin. It can be concluded that cyclical phosphorylation/dephosphorylation of cofilin was stimulated in the chemotactic cells and that F-actin/G-actin turnover was promoted as a result. Actually, we observed that IL-8 induced translocation of total cofilin to the membrane region, the same as in opsonized, zymosan-stimulated cells [14]. Some of the phosphorylated cofilin that was visualized specifically with phospho-specific antibody colocalized with F-actin, especially beneath the protruding membranes. The precise physiological significance of the slightly different localization of phoshorylated cofilin and total cofilin is unclear, but F-actin may be supported or stabilized by phosphorylated cofilin. We have reported observing that cytosolic LIMK1 translocated to the cell membrane region upon stimulation with opsonized zymosan in macrophage-like U937 cells [18], and Soosairajah et al. [37] found recently that LIMK1, SSH phosphatase, actin, scaffolding protein , and p21-activated kinase 4 form a complex that controls cofilin activity. Cofilin-phosphorylation-controlling machinery, including the enzymes and signal-tranduction proteins, may be organized beneath the IL-8-induced, protruding membranes. A LIMK-inhibitable peptide (S3) was developed originally by Aizawa et al. [38] and has been applied to nerve cells [38] and Jurkat human T cells [36]. The peptide was constructed from an N terminus sequence of cofilin, which contains the Ser 3 phosphorylation site and a penetratin sequence derived from antennapedia of Drosophila as the part that permeates cell membranes [39]. We tried applying the peptide to the neutrophilic cells, but the penetratin peptide itself was found to have stimulating activity on the neutrophilic, differentiated HL-60 cells, probably as the cell membrane of the neutrophilic phagocytes is sensitive to such exogenous substances. We then replaced the penetratin part of the sequence of the S3 peptide with an arginine polymer, and we called the resulting peptide S3-R. The arginine polymer was developed by Futaki et al. [26] and even at 4 C, permeates the cell membranes via a nonendocytic process, but it has no stimulating activity on neutrophilic cells. Peptide S3-R, which inhibited phosphorylation of 726 Journal of Leukocyte Biology Volume 81, March

8 cofilin, probably through inhibition of LIMK, will be useful for investigating the cellular signaling of LIMK/cofilin in various other types of cells by specific inhibition of LIMKs. The PI-3K inhibitors wortmannin and LY displayed strong inhibitory activity against IL-8-dependent chemotaxis. They reduced the dephosphorylation and subsequent rephosphorylation of cofilin, suggesting that PI-3K acts upstream of cofilin phosphatases (SSH and/or chronophin) and cofilin kinases (LIMKs and/or TESKs). There are several types of PI-3K, and Sadhu et al. [40] suggested that the type of PI-3K may be involved in neutrophil chemotaxis based on the finding that IC87114, a synthetic, selective inhibitor of PI-3K, suppresses fmlp-dependent, directional movement by neutrophils. Nishita et al. [41] found that insulin-dependent activation of SSH is mediated by PI-3K in human breast cancer cell line MCF7. Wang et al. [42] reported recently that SSH1 is activated by calcium signaling-dependent calcineurin. We, conversely, observed that IL-8 induces a typical calcium response in neutrophilic HL-60 cells, which is not inhibited by PI-3K inhibitors (data not shown). It therefore seemed unlikely that the IL-8-induced dephosphorylation of cofilin is mediated by calcineurin. Recently, Nishita et al. [43] reported that LIMK1 is involved in initial lamellipodium formation and that SSH1 is crucially involved in directional migration by SDF-1-stimulated Jurkat cells. IL-8-dependent, rapid dephosphorylation may be required to initiate chemotaxis, as PI-3K inhibitors suppressed dephosphorylation and rephosphorylation of cofilin and inhibited chemotaxis, whereas the S3-R peptide did not inhibit the rapid dephosphorylation and enhanced the chemotaxis. Dephosphorylated, active cofilin may be involved in remodeling old cytoskeleton that exists in unstimulated cells. By suppressing the rephosphorylation of cofilin, the S3-R peptide may release phosphorylation-dependent regulation of chemotaxis. Local and time-dependent, intracellular events in polarized cells moving toward chemoattractants should be clarified to understand the precise signaling mechanisms underlying the control of cofilin phosphorylation. ACKNOWLEDGMENTS This study was supported by research grants from Ministry of Environment and from the Ministry of Health, Labor and Welfare of Japan. REFERENCES 1. Rossi, D., Zlotnik, A. (2000) The biology of chemokines and their receptors. Annu. Rev. Immunol. 18, Kretschmer, R.R., Collado, M.L. (1980) Chemotaxis. Infection 8 (Suppl. 3), S299 S Svanborg, C., Bergsten, G., Fischer, H., Frendeus, B., Godaly, G., Gustafsson, E., Hang, L., Hedlund, M., Karpman, D., Lundstedt, A. C., Samuelsson, M., Samuelsson, P., Svensson, M., Wullt, B. (2001) The innate host response protects and damages the infected urinary tract. Ann. 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