Báo cáo khoa học: Alternagin-C, a nonRGD-disintegrin, induces neutrophil migration via integrin signaling ppt

Alternagin-C, a nonRGD-disintegrin, induces neutrophil migration via integrin signaling Andre ´ a Mariano-Oliveira 1 , Ana Lu ´ cia J. Coelho 1 , Cristina H. B. Terruggi 2 , Heloı ´ sa S. Selistre-de-Arau ´ jo 2 , Christina Barja-Fidalgo 1 and Marta S. De Freitas 1 1 Departamento de Farmacologia, Instituto de Biologia, Universidade do Estado do Rio de Janeiro, Brazil; 2 Departamento de Cie ˆncias Fisiolo ´ gicas, Universidade Federal de Sa ˜ o Carlos, Brazil Recently, a new protein containing a disintegrin domain, alternagin-C (Alt-C), was purified from Bothrops alternatus venom. Unlike other disintegrins, in Alt-C an ECD amino acid motif takes the place of the RGD sequence. Most dis- integrins contain an RGD/KGD sequence and are very potent inhibitors of platelet aggregation, as well as other cell interactions with the extracellular matrix, including tumor cell metastasis and angiogenesis. The present study investi- gated the effects of Alt-C on human neutrophil chemotaxis in vitro and the activation of integrin-mediated pathways. Alt-C showed a potent chemotactic effect for human neutrophils when compared to N-formyl-methionyl-leucyl- phenylalanine peptide (fMLP), a classic chemotactic agent. Moreover, preincubation of neutrophils with Alt-C signifi- cantly inhibited chemotaxis toward fMLP and itself. In addition, a peptide containing an ECD sequence presented a chemotactic activity and significantly inhibited chemotaxis induced by Alt-C and fMLP. A significant increase of F-actin content was observed in cells treated with Alt-C, showing that the chemotactic activity of Alt-C on neu- trophils is driven by actin cytoskeleton dynamic changes. Futhermore, this protein was able to induce an increase of phosphotyrosine content triggering focal adhesion kinase activation and its association with phosphatidylinositol 3-kinase. Alt-C was also able to induce a significant increase in extracellular signal-regulated kinase 2 1 nuclear transloca- tion. The chemotactic activity of Alt-C was partially inhi- bited by LY294002, a specific phosphatidylinositol 3-kinase inhibitor, and by PD98056, a Map kinase kinase 2 inhibitor. These findings suggest that Alt-C can trigger human neutrophil chemotaxis modulated by intracellular signals characteristic of integrin-activated pathways and that these effects could be related to the ECD motif present in disintegrin-like domain. Keywords: neutrophil; chemotaxis; integrin signaling. The recruitment of polymorphonuclear neutrophils to sites of inflammation and tissue injury requires rolling on the vessel walls and subsequent migration through the vascular endothelium. Migration involves multiple neutrophil adhe- sion receptors, such as L -selectin for rolling and integrins for adherence and locomotion [1,2]. These adhesion receptors have counter-receptors on endothelial cells and also specific ligands that are extracellular matrix (ECM) proteins [3]. Integrins are comprised of noncovalently linked a and b chains that can associate in various combinations and thus determine the ligand-binding specificities of the intact heterodimer [4,5]. On the other hand, binding of integrins to the ECM is mediated by an integrin-recognition RGD motif found on some ECM components such as fibronectin, vitronectin and fibrinogen [6]. Integrin–ligand binding and receptor clustering initiate a signaling cascade that involves receptor activation, increase in tyrosine kinase activity and protein phosphorylation, and reorganization of the actin cytoskeleton [5,7]. Focal adhesion kinase (FAK) is a cytoplasmic protein tyrosine kinase that is localized to focal adhesion sites upon clustering of integrins [7,8]. Focal adhesions contain a number of specialized cytoplasmic proteins, including talin, vinculin, a-actinin and paxillin that regulate actin cytoskeleton organization [7,9]. Focal adhe- sions also trigger various signal transduction events, inclu- ding the activation of Src-family kinases, guanine nucleotide exchange factors, Ras-family proteins, mitogen activated protein (MAP) kinases and phosphatidylinositol 3-kinase (PI3-kinase) [10–12]. A significant development in the study of integrin–ligand interactions was the discovery, originally in snake venoms, of disintegrins. These peptides represent a family of cysteine- rich proteins isolated from snake venoms and are known to inhibit cell-matrix and cell–cell interactions mediated by integrins [13–15]. Most disintegrins contain an RGD/KGD sequence within an amino acid hairpin loop maintained by disulfide bridges, and are very potent inhibitors of platelet aggregation as well as cell–ECM interactions involved in tumor cell metastasis and angiogenesis [16,17]. In Correspondence to M. Sampaio De Freitas, Departamento de Farmacologia, Instituto de Biologia, Universidade do stado do Rio de Janeiro, Av. 28 de setembro 87 fds, Vila Isabel, Rio de Janeiro, 20551–030, RJ, Brazil. Fax: + 55 21 2587–6808, Tel.: + 55 21 2587–6398, E-mail: martaf@uerj.br Abbreviations: Alt-C, alternagin-C; ECM, extracellular matrix; fMLP, N-formyl-methionyl-leucyl-phenylalanine peptide; ADAM, a disintegrin and metalloproteinase; MEK, Map kinase kinase; ERK, extracellular signal-regulated kinase; MAP, mitogen activated protein; PI3-kinase, phosphatidylinositol 3-kinase. (Received 30 August 2003, revised 29 September 2003, accepted 3 October 2003) Eur. J. Biochem. 270, 4799–4808 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03867.x mammalian tissues, Ôa disintegrin and metalloproteinaseÕ (ADAM) proteins have been described to mediate import- ant roles in many pathophysiological processes, including tissue development, tumor cell adhesion and inflammatory responses [18]. ADAMs are cell membrane-anchored pro- teins that contain metalloproteinase, disintegrin-like, cys- teine-rich, epidermal growth factor-like, transmembrane and cytoplasmic domains [18]. However, the physiological role of the disintegrin and cysteine-rich domains in ADAMs is not well understood. We previously reported that jarastatin, a new RGD- disintegrin isolated from Bothrops jararaca venom, inhibited human neutrophil migration in vivo and in vitro induced by chemoattractants, and promoted actin cytoskeleton reor- ganization [19]. Interestingly, jarastatin is a potent chemo- tactic for neutrophils in vitro [19]. We also demonstrated that jarastatin and two known monomeric RGD-disinte- grins, kistrin and flavoridin, affected human neutrophil chemotaxis by triggering intracellular signaling pathways mediated by integrin activation, despite kistrin and flavo- ridin not being chemotactic to neutrophils [20]. Recently, the disintegrin-like domain of a novel metallo- proteinase (alternagin) isolated from Bothrops alternatus snake venom was purified and named alternagin-C (Alt-C) [21]. This disintegrin-like domain has an additional cysteine- rich domain, which is not found in RGD-disintegrins, and the RGD motif is replaced by an ECD sequence. Further- more, Alt-C was shown to be a potent inhibitor of collagen- induced adhesion by blockage of a 2 b 1 integrin [21]. We have evaluated the effects of Alt-C on human neutrophil chemotaxis in vitro and its ability to trigger intracellular signaling pathways mediated by integrin acti- vation. We also examined the effect of a cyclic oligopeptide corresponding to a conserved fragment containing the ECD sequence in the disintegrin-like domain. The present study demonstrates that Alt-C has a chemotactic activity on neutrophils and this effect involves actin cytoskeleton rearrangement, FAK, PI3-kinase and Erk-2 activities. Moreover, we found that ECD peptide is also a potent chemotactic and that it is able to inhibit Alt-C activity. Materials and methods Disintegrin-like domain Alternagin-C, the processed disintegrin domain of altern- agin, was isolated from Bothrops alternatus venom and purified as described previously [21]. The cyclic peptide corresponding to the disintegrin loop with the ECD sequence (CRASMSECDPAEH-NH 2 )wasagiftfromM. Juliano (Department Biofı ´ sica, UNIFESP, SP, Brazil). Alt- C and ECD peptide were diluted in sterile distilled water andstoredat)20 °C until use. Isolation of human neutrophils Human neutrophils were isolated from 0.5% (w/v) EDTA- treated peripheral venous blood of healthy volunteers, with previous agreement, 3 using a four-step discontinuous Percoll (Amersham Pharmacia Biotech, San Francisco, CA) gradi- ent [22]. Erythrocytes were removed by hypotonic lysis. Isolated neutrophils (98% purity), estimated to be > 96% viable by trypan blue exclusion, were resuspended in RPMI- 1640 medium (Sigma Chemical Co., St. Louis, MO). Neutrophil chemotaxis assay Neutrophil chemotaxis was assayed in a 48-well Boyden chamber (Neuroprobe microchemotaxis system) using a 5 lm poly(vinyl propylene) 4 -free polycarbonate filter as des- cribed previously [19]. For chemotaxis assays, the chemo- tactic stimuli, N-formyl-methionyl-leucyl-phenylalanine peptide (fMLP, 100 n M ; Sigma) and different concentra- tions of Alt-C (0.1–1000 n M ) or ECD peptide (1–1000 n M ) diluted in RPMI medium, were added to the bottom wells of the chamber. Cells suspended in RPMI medium (10 6 cellsÆmL )1 ) were added (50 lL) to the top wells of the Boyden chamber and allowed to migrate for 60 min at 37 °Cina5%(v/v)CO 2 atmosphere. In some experiments, neutrophils were pretreated (5 min at 37 °C) with Alt-C (100 n M ) or ECD-peptide (0.1–1000 n M ) and allowed to migrate in the Boyden chamber toward fMLP (100 n M ), Alt-C (100 n M ) or ECD-peptide (1000 n M ). In another set of experiments, neutrophils were preincubated with LY294002 (3 l M ) or PD98059 (2 l M ) (Calbiochem, San Diego, CA) for 5 min at 37 °C prior to the chemotaxis assay. After that, cells were incubated for 60 min at 37 °Cin a5%(v/v)CO 2 atmosphere and the filters were removed from the chambers, fixed and stained with a Diff-Quick stain kit (Baxter Travenol Laboratories, ON, Canada). Neutrophils that had migrated through the membrane were counted under light microscopy (·100 objective) on at least five random fields. Results, expressed as the number of neutrophils per field, were representative of three different experiments performed in triplicate for each sample. Neu- trophil migration toward RPMI-1640 medium alone (ran- dom movement) was used as a negative control. Immunocytochemistry and cytochemistry assays Neutrophils (1 · 10 6 cellsÆmL )1 ) were incubated with 100 n M Alt-C for 5 min at 37 °Cand5%(v/v)CO 2 atmosphere. Cells were then cytocentrifuged at 480 g 5 and fixed with NaCl/P i containing 4% (v/v) paraformaldehyde and 4% (w/v) sucrose for 20 min at room temperature. Cells were permeabilized in NaCl/P i containing 0.1% Triton X-100 for 5 min, washed with NaCl/P i and incuba- ted with biotin-conjugated anti-phosphotyrosine Ig (1 : 50 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4 °C. Subsequently, cells were incubated with streptavidin-conjugated fluorescein isothiocyanate (1 : 50 dilution; Caltag Laboratories, Burlingame, CA) for 1 h at room temperature. To evaluate the effect of Alt-C on actin cytoskeleton network, cells were also labeled with tetra- methyl rhodamine isothiocyanate (TRITC)-labelled phal- loidin (1 : 1000 dilution; Sigma) for 2 h at room temperature. Slides were mounted using a solution of 20 m M propyl gallate and 20% (v/v) glycerol in NaCl/P i . Microscopic analysis of fluorescent images was done using an epifluorescence microscope (Olympus BX40, Tokyo, Japan) equipped with appropriate filters and using ·100 oil- immersion objetives. Image capturing was performed with a cooled-charged-coupled device camera (Photometrics, Tuc- son, AR). Fluorescence intensity from original images was 4800 A. Mariano-Oliveira et al. (Eur. J. Biochem. 270) Ó FEBS 2003 analysed by IMAGE - PRO PLUS 4.0 (Media Cybernetics) and grey images were taken using Adobe PHOTOSHOP software. Preparation of nuclear extracts Nuclear extracts were obtained as described previously [20]. Briefly, neutrophils (5 · 10 6 cellsÆmL )1 ) were incubated with Alt-C (100 n M )for1hat37°C in a 5% (v/v) CO 2 atmosphere. Cells were lysed in ice-cold buffer A (10 m M Hepes, pH 7.9, 10 m M KCl, 0.1 m M EDTA, 0.1 m M EGTA, 1 m M dithiotreitol and 0.5 m M phenylmethane- sulfonyl fluoride) and after a 15 min of incubation on ice, NP-40 was added to a final concentration of 0.5% (v/v). Nuclei were collected by centrifugation (1810 g;5minat 4 °C). The nuclear pellet was suspended in ice-cold buffer C (20 m M Hepes, pH 7.9, 400 m M NaCl, 1 m M EDTA, 1 m M EGTA, 1 m M dithiotreitol, 1 m M phenylmethanesulfonyl fluoride, 1 lgÆmL )1 pepstatin, 1 lgÆmL )1 leupeptin, 20% (v/v) glycerol) and incubated for 30 min. Nuclear proteins were collected in the supernatant after centrifugation (12 000 g;10minat4°C). Immunoprecipitation Neutrophils (5 · 10 6 cellsÆmL )1 ) were incubated with Alt-C (100 n M )for5 minat37°Cina5%(v/v)CO 2 atmosphere. Cells were lysed in 50 m M Tris/HCl, pH 7.4, 150 m M NaCl, 1.5 m M MgCl 2 ,1.5m M EDTA, 1% (v/v) Triton X-100, 10% (v/v) glycerol, 10 lg l )1 L aprotinin, 10 lg l )1 L leupeptin, 2 lgÆlL )1 pepstatin and 1 m M phenyl- methanesulfonyl fluoride. Lysates (2 lgÆlL )1 ) were incuba- ted overnight at 4 °C with anti-FAK Ig (1 : 200; Santa Cruz Biotechnology). Then, protein A/G-agarose (20 lLÆmg protein )1 ; Santa Cruz Biotechnology) was added and the samples were incubated at 4 °C under rotation for 2 h. The content of phosphorylated FAK and PI3-kinase associated with FAK was analyzed by Western blotting as subse- quently described. Immunoblotting analysis The total protein content in the cell extracts was determined by the Bradford method [23]. Cellular proteins (30 lg) were subjected to 12% (w/v) SDS/PAGE, trans- ferred to poly(vinylidene difluoride) filters (PVDF Hybond-P, Amersham Pharmacia Biotech) and blocked with Tween/TBS [20 m M Tris/HCl, pH 7.5, 500 m M NaCl, 0.1% (v/v) Tween-20] containing 1% (w/v) bovine serum albumin. Primary antibodies used in Western analysis were anti-actin (diluted 1 : 500; Santa Cruz Biotechnology); anti-phosphotyrosine (diluted 1 : 200; Santa Cruz Biotechnology); anti-FAK (diluted 1 : 1000); anti-(PI3-kinase p85 subunit) (diluted 1 : 1000; Santa Cruz Biotechnology) or anti-Erk-2 (1 : 1000; Santa Cruz Biotechnology). The poly(vinylidene difluoride) filters were next washed three times with Tween/TBS, followed by 1 h incubation with the appropriate secondary antibody conjugated to biotin (Santa Cruz Biotechnology). Then, the filters were incubated with streptavidin-conjugated horseradish peroxidase (diluted 1 : 1000; Caltag Laborat- ories). Immunoreactive proteins were visualized by 3,3¢- diaminobenzidine (Sigma) staining. The bands were quantified by densitometry, using SCION IMAGE SOFTWARE (Scion Co, Frederick, MD, USA). Statistical analysis Statistical significance was assessed by ANOVA followed by Bonferroni’s t-test, and P < 0.05 was taken as statistically significant. Results Effect of Alt-C on human neutrophil chemotaxis To evaluate the effect of Alt-C as a direct chemotatic stimulus for human neutrophils in vitro, the cells were allowed to migrate toward different concentrations of Alt-C (0.1–1000 n M ). Figure 1A shows that Alt-C induced signi- ficant chemotaxis of neutrophils in a concentration-depend- ent manner. The chemotatic activity of Alt-C (100 n M )was similiar to fMLP (100 n M ), a classic chemotactic agent. We also examined the effect of alternagin-C on neutro- phil chemotaxis induced by fMLP and by itself. Neutrophils were pretreated with Alt-C (100 n M ) for 5 min, and the cells were allowed to migrate toward fMLP or Alt-C in a Boyden chamber (directional cell movement). In the presence of Alt-C, the chemotactic response of neutrophils to fMLP was significantly inhibited, and we observed the same effect in response to Alt-C (Fig. 1B). In addition, pretreatment of neutrophils with Alt-C did not affect the random cell movement. Effect of ECD-peptide on human neutrophil chemotaxis To better understand the mechanism of action of 6 Alt-C, the activity of a synthetic peptide containing the ECD sequence was examined in chemotaxis assays. This peptide was synthesized based on the disintegrin-like domain and cyclized by a disulfide bond between the two cysteines. Cells were allowed to migrate toward different concentra- tions of ECD-peptide (1–1000 n M ), placed in the bottom wells of the Boyden chamber. The ECD-peptide showed a chemotactic effect only at the highest dose used (Fig. 2A; 1000 n M ). The inhibition of a chemotactic effect by prior exposure to structurally related and unrelated chemotactic factors has been already described for known neutrophil activators [19,20]. In this regard, the effect of ECD-peptide on neutrophil chemotaxis induced in vitro by Alt-C or fMLP was also investigated. In the presence of ECD-peptide, neutrophil chemotaxis in response to Alt-C (100 n M )was significantly inhibited at all studied doses (Fig. 2B). When the random cell movement was analysed in the presence of ECD-peptide, no alterations were observed (Fig. 2C). However, the chemotactic response of neutrophils to fMLP was completely blocked by ECD-peptide (Fig. 2C). Alt-C-induced rearrangement of neutrophil actin network It has been reported that some neutrophil activities, such as chemotaxis and phagocytosis, are mediated by cytoskeletal actin polymerization [24]. Neutrophils were incubated with Ó FEBS 2003 Effects of alternagin-C on neutrophil functions (Eur. J. Biochem. 270) 4801 Alt-C (100 n M ) and the alterations of neutrophil actin cytoskeleton network were analyzed by TRITC 7 –phalloidin staining. Figure 3 shows that Alt-C was able to induce a marked increase in the F-actin content in these cells (Fig. 3B) when compared with nonstimulated cells (Fig. 3A). When the fluorescence intensity was measured, Alt-C induced 77% more actin polymerization in compar- ison to the control (nontreated cells: 30.10 ± 4.35; treated cells: 53.41 ± 6.02; in arbitrary units, P < 0.05) (Fig. 3C). This suggests that the motile activities of neutrophils induced by Alt-C are driven by actin cytoskeleton dynamic rearrangement. Involvement of tyrosine kinase pathways in neutrophil activation by Alt-C We evaluated the involvement of tyrosine kinase pathways in the neutrophil activation induced by Alt-C. The altera- tions in protein tyrosine phosphorylation were analyzed by immunocytochemistry. As shown in Fig. 4B, Alt-C (100 n M ) was able to increase the content of phosphotyro- sine when compared with nonstimulated neutrophils (Fig. 4A). The immunoreactivity to phosphotyrosine was 80% greater in cells treated with Alt-C when compared to the control (nontreated cells: 51.56 ± 8.16; treated cells: 93.00 ± 10.05; in arbitrary units, P < 0.05) (Fig. 4C). Alt-C induced FAK and PI3-kinase activation Focal adhesion contacts are comprised by integrins, cyto- skeletal proteins and FAK association followed by FAK autophosphorylation and activation [7]. By immunopreci- pitation of FAK, we determined its activation by the increase in the content of phosphotyrosine. As shown in Fig. 5A, incubation of neutrophils with Alt-C (100 n M )was able to increase FAK phosphorylation. The tyrosine phosphorylation of FAK generates docking sites for several proteins containing Src homology 2 (SH2) domains, as the p85 regulatory subunit of PI3-kinase [25]. PI3-kinase activation can modulate some cellular responses such as cell migration [26]. Activation of PI3-kinase was evaluated by its p85 subunit association with FAK in cells treated with Alt-C. Figure 5B shows that Alt-C induced an increase in the PI3-kinase association with FAK. Confirming the involvement of the PI3-kinase pathway in the chemotactic effect of Alt-C on human neutrophil chemotaxis, a specific PI3-kinase inhibitor, LY294002, completely blocked Alt-C-induced chemotaxis (Fig. 5C). In addition to the determination of FAK and PI3-kinase activation in response to Alt-C-induced chemotaxis, we analyzed the effect of PI3-kinase inhibitor on FAK phosphorylation and FAK-associated PI3-kinase upon stimulation with Alt-C. As shown in Fig. 5A,B, the levels of FAK phosphorylation and PI3-kinase association with FAK in LY294002-treated cells were similar to those in control cells. Alt-C-induced activation of Erk-2 It has been reported that some disintegrins are able to increase the activation and translocation of Erk-2 from the cytoplasm to the nucleus [20]. Here, we investigated whether Alt-C would be able to induce nuclear translocation of Erk- 2 in human neutrophils. Figure 6A shows that Erk-2 could be observed in nuclear extracts from control cells. However, incubation of neutrophils with Alt-C (100 n M )for1h induced a significant increase on Erk-2 nuclear translocation when compared to nontreated cells (Fig. 6A). Fig. 1. Effect of Alt-C on human neutrophil chemotaxis. (A) Alt-C induces chemotaxis in a dose-dependent manner. The cells were allowed to migrate in a Boyden chamber toward medium alone (RPMI, control, j), fMLP (100 n M , d) or Alt-C (m). Data shows mean ± SD from three independent experiments. *P <0.05com- pared to control. (B) Alt-C inhibits fMLP- and Alt-C-induced chemo- taxis. Cells were incubated in presence or absence of Alt-C (100 n M ) for 5 min at 37 °C. Neutrophil chemotaxis was evaluated in a Boyden chamber toward medium alone (RPMI), fMLP (100 n M ) or Alt-C (100 n M ). Data shows mean ± SD from three independent experi- ments. *P < 0.05 compared to control. 4802 A. Mariano-Oliveira et al. (Eur. J. Biochem. 270) Ó FEBS 2003 FAK activation mediates the Ras/Raf/MAP kinase kinase (MEK) signal transduction cascade leading to Erk-2 activation and regulating cell motility [27]. To investigate the involvement of the Ras/Raf/MAP kinase pathway in the effects of Alt-C on neutrophil chemotaxis, cells were preincubated with PD98059, a MEK inhibitor, and then allowed to migrate toward Alt-C. Figure 6B demonstrates that chemotaxis induced by Alt-C was significantly inhibited by PD 98059 (24% inhibition). As shown in Fig. 6A, preincubation of neutrophils with PD98059 abolished Erk-2 nuclear translocation. To provide further insight into the regulation of Alt-C-induced Erk-2 activation, we determined whether PI3-kinase activity is a prerequisite for Erk-2 activation. Figure 6A shows that nuclear translocation of Erk-2 in response to Alt-C was significantly increased by 38% in neutrophils by exposure to PI3-kinase inhibitor. Discussion Cell adhesion to the ECM is primarily mediated by the binding of cell surface integrins to the RGD motif found on ECM proteins [6]. Disintegrins mostly express an RGD sequence at an integrin-binding loop. The type and position of the amino acids flanking the RGD motif determine the selectivity of disintegrin interaction with integrin [15,18]. The understanding of the precise mechanism of action and structure of disintegrins will provide information about adhesive ligands and their integrin receptors. We previously reported that RGD-disintegrins interfered with neutrophil chemotaxis induced by chemoattractants [19,20]. It has been postulated that disintegrins are approximately 1000-fold more potent than linear RGD-containing peptides, being determined by the conformation of the RGD amino acid sequence within their structures [13–15]. In the present study we have investigated the effects of a disintegrin-like protein, Alt-C, on neutrophil activation and function. Alt-C has been described to inhibit collagen-induced adhesion of cells expressing a2b1 integrin in a dose-dependent manner [21]. Alt-C also has a cysteine-rich disulfide-bonding pattern and the primary structure containing an ECD sequence presents homology with the disintegrins [21]. The results demonstra- ted that Alt-C strongly induced human neutrophil chemo- taxis in vitro. Furthermore, this protein inhibited chemotaxis of neutrophils induced by fMLP and by itself. We exam- ined the effect of a synthetic ECD-peptide on neutrophil Fig. 2. Effect of ECD-peptide on Alt-C- induced human neutrophil chemotaxis. (A) ECD-peptide induces chemotaxis. The cells were allowed to migrate in a Boyden chamber toward medium alone (RPMI, control, j), fMLP (100 n M , d)orECD(r). Data shows mean ± SD from three independent experi- ments. *P < 0.05 in comparison with control. (B) ECD-peptide inhibits Alt-C-induced che- motaxis. Neutrophils were preincubated in presence or absence of ECD-peptide (0.1– 1000 n M ) for 5 min at 37 °Candthenallowed to migrate in a Boyden chamber toward medium alone (RPMI, control, j) or Alt-C (100 n M , m). Data shows mean ± SD from three independent experiments. *P <0.05in comparison with Alt-C-incuced chemotaxis. (C) ECD-peptide inhibits fMLP-induced chemotaxis. Cells were incubated in absence or presence of ECD-peptide (1000 n M )for5min at 37 °C. Neutrophil chemotaxis was evalu- ated in a Boyden chamber toward medium alone (RPMI) or fMLP (100 n M ). Data shows means ± SD from three independent experi- ments. *P < 0.05 compared to random movement. Ó FEBS 2003 Effects of alternagin-C on neutrophil functions (Eur. J. Biochem. 270) 4803 chemotaxis in vitro. The peptide induced neutrophil chemo- taxis and also had the ability of inhibiting Alt-C-induced chemotaxis. These data strongly suggest that the chemotac- tic effect of Alt-C appears to be mediated by ECD sequence conformation. It has been demonstrated that synthetic peptides having the sequence RSECD inhibit collagen- induced platelet aggregation [28]. Thus, our results indicate that Alt-C may interact with adhesive receptors on the neutrophil surface inducing cell activation and leading to desensitization of the receptor to other chemotactic stimuli after prior stimulation. These findings are in agreement with previous studies describing that neutrophil migration can be inhibited or desensitized to a given chemoattractant by prior exposure to the same agonist (homologous desensitization) or to unrelated chemotactic factors (heterologous desensi- tization) [20,29]. Different chemotactic and phagocytic stimuli generate dynamic alterations in the actin cytoskeleton network in neutrophils [30]. Integrins induce assembly of actin filaments and high-order structures, such as focal adhesions, in response to extracellular stimuli and during cell adhesion and migration [reviewed in 31]. We previously showed that RGD-disintegrins are able to induce the integrin activation and rearrangement of the actin cytoskeleton in human Fig. 3. Alt-C increases actin cytoskeleton polymerization on human neutrophils. Neutrophils were incubated with Alt-C (100 n M )for5min at 37 °C and actin filaments were stained with TRITC-phalloidin for 2 h at room temperature. (A) Control and (B) Alt-C incubated neu- trophils labelled with TRITC-phalloidin. (C) Fluorescence intensity with mean ± SD from three independent experiments. *P <0.05 compared to control values. Fig. 4. Involvement of tyrosine kinase activity in the effect of Alt-C on human neutrophils. Neutrophils were incubated with Alt-C (100 n M ) for 5 min at 37 °C, permeabilized with 0.1% (v/v) Triton X-100, incubated with anti-phosphotyrosine Ig conjugated to biotin followed by streptavidin-conjugated fluorescein isothiocyanate. (A) Control and (B) Alt-C incubated FITC-labelled neutrophils. (C) The measure of fluorescence intensity with mean ± SD from three independent experiments. *P < 0.05 compared to control values. 4804 A. Mariano-Oliveira et al. (Eur. J. Biochem. 270) Ó FEBS 2003 neutrophils [19,20]. Because Alt-C causes remarkable neu- trophil chemotaxis, we examined whether Alt-C also produces changes in cytoskeletal F-actin. Treatment of neutrophils with Alt-C induced profound alterations in the actin network with an increase of F-actin content, suggest- ing that the Alt-C effect on neutrophils could involve integrin-mediated pathways. Neutrophil functional responses that require cytoskeletal reorganization, such as adhesion to the endothelium and ECM, cell migration and phagocytosis, result in the activation of protein tyrosine kinases [31–33]. Interaction between integrins and ligands leads to a profound increase in tyrosine phosphorylation of several cellular proteins. It is well established that simple dimerization of integrins is sufficient to initiate tyrosine phosphorylation events [11]. This has been accomplished with crosslinked anti-integrin Igs, multimeric integrin ligands [34,35] as well as disinte- grins, potent inducers of conformational changes in both subunits of integrins [36]. Results reported here show that Alt-C induces an increase in tyrosine kinase activity and tyrosine phosphorylation. One of the initial events in integrin-mediated signaling is the activation of FAK, resulting in its autophosphoryla- tion [7]. This is supported by the findings that distinct disintegrins binding to integrin stimulate FAK activity [20] and that activated FAK might mediate signal transduction in a manner similar to that of integrins. According to this, the present study demonstrated that Alt-C was able to induce an increase in phosphotyrosine content of FAK and that FAK phosphorylation may be directly involved in the activation of the migratory process in response to Alt-C. A recent report showed that FAK phosphorylation is directly required for neutrophil chemotaxis by using a dominant negative mutant of FAK [37]. Interestingly, it also has been described that FAK-deficient cells exhibit an elevated number of focal adhesions accompanied by a decrease rate of cell migration [38]. Furthermore, FAK, as a nonreceptor tyrosine kinase that associates with the cytoplasmic domain of integrins at focal adhesions, might be critical for cytoskeleton reorganization [7,9]. Earlier studies have demonstrated that two cytoskeletal proteins, paxillin and tensin, are substrates for FAK, which could account for a role of FAK in actin cytoskeleton assembly and disassembly [7,9]. In the present study we provide evidence of a link between FAK activation and rearrangement of the actin cytoskeleton in neutrophils Fig. 5. Involvement of FAK and PI3-kinase in the effect of Alt-C on human neutrophils. (A) Alt-C induces FAK activation. FAK phos- phorylation was determined in neutrophils incubated with Alt-C (100 n M )for5minat 37 °C in the presence or absence of LY294002 (3 l M ). Cell lysates were immunoprecipitated with anti-FAK Ig and blotted with either anti- phosphotyrosine or anti-FAK Igs. IP, immu- noprecipitation; WB, Western blotting. Blots were analyzed by densitometry and the con- tent of phosphorylated FAK was expressed in densitometric units. Data show mean ± SD from three independent experiments. *P < 0.05 compared to cells incubated with medium alone (Control) (B) Alt-C increases FAK- associated PI3-kinase. Cell lysates of neu- trophils incubated with Alt-C (100 n M )for 5 min at 37 °C in the presence or absence of LY294002 (3 l M ) were immunoprecipitated withanti-FAKIgandthenblottedwithanti- FAK or anti-PI3-kinase p85 subunit Igs. IP, immunoprecipitation; WB, Western blotting. Blots were analyzed by densitometry and the content of PI3-kinase associated to FAK was expressed in densitometric units. Data show mean ± SD from three independent experi- ments. *P < 0.05 compared to cells incubated with medium alone (Control). (C) Alt-C- induced chemotaxis is reduced by a PI3-kinase inhibitor. Neutrophils were preincubated for 5 min at 37 °C with LY294002 (3 l M )andthen allowed to migrate in a Boyden chamber to- ward Alt-C (100 n M ). Data show mean ± SD from three independent experiments. *P < 0.05 compared to control. Ó FEBS 2003 Effects of alternagin-C on neutrophil functions (Eur. J. Biochem. 270) 4805 immediatelly following stimulation with Alt-C, an ECD- disintegrin. FAK is also considered a focal adhesion docking protein that recruits PI3-kinase and other signaling molecules to form a multimolecular complex, altering their activities [10,12,25]. In Alt-C-stimulated neutrophils, PI3-kinase was found to be associated with FAK. Therefore, it is reasonable to postulate that this association promotes PI3-kinase activation, which correlates with a variety of cellular responses to external stimuli including chemotaxis, which was completely blocked by a PI3-kinase inhibitor. These results are in agreement with previous studies showing that neutrophils lacking PI3-kinase failed to orient toward different chemotactic stimuli [39,40]. Thus Alt-C-induced neutrophil chemotaxis could be driven by PI3-kinase activation, which associates with autophosphorylated FAK through their SH2 domains. FAK activation may also trigger the Ras signal trans- duction cascade [11]. Downstream signal molecules such as Erk-2 have also been implicated in the regulation of the neutrophil effector functions [41]. Our study revealed that Alt-C can induce Erk-2 activation, as observed by its translocation to the nucleus. Activation of Erk-2 is often associated with enhanced myosin light chain kinase 9 activity and increased migration [27]. The effect of Alt-C on neutrophil chemotaxis was partially reversed by PD98059, a MEK inhibitor, supporting a role for Erk-2 in Alt-C- induced neutrophil migration. These findings suggest that activation of Erk-2 induced by Alt-C may function as a positive regulator of migration. Recently, some paradoxical findings have reported the effects of different RGD-disin- tegrins on cell migration supporting the role for Erk-2 as a positive or negative effector [20,42]. In addition to Alt-C- induced Erk-2 activation that accounts for its positive effect on neutrophil chemotaxis, other cellular responses may be related to this pathway. Along this line, our results also demonstrated that PI3-kinase inhibition is accompanied by an increase of Erk-2 nuclear translocation suggesting a modulatory role of PI3-kinase signaling pathway on Erk-2 activity. Studies on the expression of cytokines and chemo- kines and on neutrophil apoptosis are under investigation. The present study provides evidence that Alt-C, a disintegrin-like protein presenting an ECD motif, interacts with neutrophils promoting integrin-mediated signaling and inducing chemotaxis. 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Res. Commun. 274, 142–148. 4808 A. Mariano-Oliveira et al. (Eur. J. Biochem. 270) Ó FEBS 2003 . Alternagin-C, a nonRGD-disintegrin, induces neutrophil migration via integrin signaling Andre ´ a Mariano-Oliveira 1 , Ana Lu ´ cia J. Coelho 1 ,. focal adhesion kinase activation and its association with phosphatidylinositol 3-kinase. Alt-C was also able to induce a significant increase in extracellular