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 These findings suggest that
Trang 1Alternagin-C, a nonRGD-disintegrin, induces neutrophil migration via integrin signaling
Andre´a Mariano-Oliveira1, Ana Lu´cia J Coelho1, Cristina H B Terruggi2, Heloı´sa S Selistre-de-Arau´jo2, Christina Barja-Fidalgo1and Marta S De Freitas1
1
Departamento de Farmacologia, Instituto de Biologia, Universidade do Estado do Rio de Janeiro, Brazil;2Departamento de Cieˆncias 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 vitroand 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
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
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 asL-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)
Trang 2mammalian tissues, a disintegrin and metalloproteinase
(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 a2b1integrin [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-NH2) was a gift from M
Juliano (Department Biofı´sica, UNIFESP, SP, Brazil)
Alt-C and EAlt-CD peptide were diluted in sterile distilled water
and stored at)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,
(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)
des-cribed previously [19] For chemotaxis assays, the chemo-tactic stimuli, N-formyl-methionyl-leucyl-phenylalanine peptide (fMLP, 100 nM; Sigma) and different concentra-tions of Alt-C (0.1–1000 nM) or ECD peptide (1–1000 nM) diluted in RPMI medium, were added to the bottom wells
of the chamber Cells suspended in RPMI medium (106cellsÆmL)1) were added (50 lL) to the top wells of the Boyden chamber and allowed to migrate for 60 min at
37C in a 5% (v/v) CO2atmosphere In some experiments, neutrophils were pretreated (5 min at 37C) with Alt-C (100 nM) or ECD-peptide (0.1–1000 nM) and allowed to migrate in the Boyden chamber toward fMLP (100 nM), Alt-C (100 nM) or ECD-peptide (1000 nM) In another set
of experiments, neutrophils were preincubated with LY294002 (3 lM) or PD98059 (2 lM) (Calbiochem, San Diego, CA) for 5 min at 37C prior to the chemotaxis assay After that, cells were incubated for 60 min at 37C in
a 5% (v/v) CO2atmosphere 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· 106cellsÆmL)1) were incubated with
100 nM Alt-C for 5 min at 37C and 5% (v/v) CO2 atmosphere Cells were then cytocentrifuged at 480 g
fixed with NaCl/Picontaining 4% (v/v) paraformaldehyde and 4% (w/v) sucrose for 20 min at room temperature Cells were permeabilized in NaCl/Pi containing 0.1% Triton X-100 for 5 min, washed with NaCl/Piand incuba-ted with biotin-conjugaincuba-ted anti-phosphotyrosine Ig (1 : 50 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4C 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 mM propyl gallate and 20% (v/v) glycerol in NaCl/Pi 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
Trang 3analysed byIMAGE-PRO PLUS4.0 (Media Cybernetics) and
grey images were taken using AdobePHOTOSHOPsoftware
Preparation of nuclear extracts
Nuclear extracts were obtained as described previously [20]
Briefly, neutrophils (5· 106cellsÆmL)1) were incubated
with Alt-C (100 nM) for 1 h at 37C in a 5% (v/v) CO2
atmosphere Cells were lysed in ice-cold buffer A (10 mM
Hepes, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM
EGTA, 1 mM dithiotreitol and 0.5 mM
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; 5 min at
4C) The nuclear pellet was suspended in ice-cold buffer C
(20 mMHepes, pH 7.9, 400 mMNaCl, 1 mMEDTA, 1 mM
EGTA, 1 mM dithiotreitol, 1 mM 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; 10 min at 4C)
Immunoprecipitation
Neutrophils (5· 106cellsÆmL)1) were incubated with Alt-C
(100 nM) for 5 min at 37C in a 5% (v/v) CO2atmosphere
Cells were lysed in 50 mMTris/HCl, pH 7.4, 150 mMNaCl,
1.5 mM MgCl2, 1.5 mM EDTA, 1% (v/v) Triton X-100,
10% (v/v) glycerol, 10 lg l)1L aprotinin, 10 lg l)1L
leupeptin, 2 lgÆlL)1 pepstatin and 1 mM
phenyl-methanesulfonyl fluoride Lysates (2 lgÆlL)1) were
incuba-ted overnight at 4C 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 4C 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 mM Tris/HCl, pH 7.5, 500 mM
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 byANOVAfollowed 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 nM) 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 nM) was similiar to fMLP (100 nM), a classic chemotactic agent
We also examined the effect of alternagin-C on neutro-phil chemotaxis induced by fMLP and by itself Neutroneutro-phils were pretreated with Alt-C (100 nM) 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
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 nM), 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 nM)
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 nM) 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
Trang 4Alt-C (100 nM) and the alterations of neutrophil actin
cytoskeleton network were analyzed by TRITC
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 nM) 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 nM) 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-Erk-2 could
be observed in nuclear extracts from control cells However, incubation of neutrophils with Alt-C (100 nM) for 1 h 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 < 0.05
com-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.
Trang 5FAK 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 chemochemo-taxis
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) or ECD (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 C and then allowed
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 < 0.05 in
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 ) for 5 min
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.
Trang 6chemotaxis 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 ) for 5 min
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.
Trang 7neutrophils [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 networkwith 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 linkbetween 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 ) for 5 min at
37 C in the presence or absence of LY294002
(3 l M ) Cell lysates were immunoprecipitated
with 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
with FAK Ig and then blotted with
anti-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 ) and then
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.
Trang 8immediatelly 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
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 Our study elucidates the mechanism
of action of Alt-C, as well as establishes a potential model for the design of new therapeutic interventions in disorders involving leukocyte dysfunctions
Acknowledgements
The authors thankDr Iolanda M Fierro (UERJ, Brazil) for the discussions and critical review of the manuscript This workwas supported by CAPES, CNPq, FAPERJ, FAPESP, SR-2/UERJ (Brazil) and IFS (Sweden).
References
1 Brown, E.J (1986) The interactions of connective tissue proteins with phagocytic cells J Leukoc Biol 39, 579–591.
2 Lawrence, M.B & Springer, T.A (1991) Leukocytes roll on a selectin at physiologic flow rates: Distinction from and prerequisite for adhesion through integrins Cell 65, 859–873.
3 Berton, G., Yan, S.R., Fugamalli, L & Lowell, C.A (1996) Neutrophil activation by adhesion: Mechanisms and pathophy-siological implications Int J Clin Laboratory Res 26, 160–177.
4 Ruoslahti, E (1991) Integrins J Clin Invest 87, 1–5.
5 Hynes, R.O (1992) Integrins: versatility, modulation, and signa-ling in cell adhesion Cell 69, 11–25.
Fig 6 Involvement of Erk-2 activityin the effect of Alt-C on human
neutrophils (A) Alt-C induces Erk-2 nuclear translocation
Neu-trophils were incubated with medium alone (Control) or with Alt-C
(100 n M ) for 1 h at 37 C in the presence or absence of LY294002
(3 l M ) or PD98059 (2 l M ) The nuclear content of Erk-2 was
deter-mined by immunoblotting using an anti-Erk-2 Ig and quantified by
densitometry WB, Western blotting Data show mean ± SD from
three independent experiments *P < 0.05 compared to cells
incubated with medium alone (Control) (B) Alt-C-induced
chemo-taxis is reduced by a MEK inhibitor Neutrophils were preincubated
for 5 min at 37 C with PD98059 (2 l M ) and then allowed to migrate
in a Boyden chamber toward Alt-C (100 n M ) Data show mean ± SD
from three independent experiments *P < 0.05 compared to control.
The # indicates that LY294002 significantly increased Alt-C-induced
Erk-2 nuclear translocation (P < 0.05).
Trang 96 Yamada, K.M (1991) Adhesive recognition sequences J Biol.
Chem 266, 12809–12812.
7 Burridge, K., Turner, C.E & Romer, L.H (1992) Tyrosine
phosphorylation of paxillin and pp125 FAK accompanies cell
adhesion to extracellular matrix: a role in cytoskeletal assembly.
J Cell Biol 119, 893–903.
8 Kornberg, L., Earp, H.S., Parsons, J.T., Schaller, M & Juliano,
R.L (1992) Cell adhesion or integrin clustering increases
phos-phorylation of a focal adhesion-associated tyrosine kinase J Biol.
Chem 267, 23439–23442.
9 Parsons, J.T (1996) Integrin-mediated signalling: regulation by
protein tyrosine kinases and small GTP-binding proteins Curr.
Opin Cell Biol 8, 146–152.
10 Lo¨fgren, R., Ng-Sikorski, J., Sjo¨lander, A & Andersson, T (1993)
b 2 -integrin engagement triggers actin polymerization and
phos-phatidylinositol triphosphate formation in non-adherent human
neutrophils J Cell Biol 123, 1597–1605.
11 Miyamoto, S., Teramoto, H., Coso, O., Silvio, G., Burbelo, P.,
Akiyama, S.K & Yamada, K.M (1995) Integrin function:
molecular hierarchies of cytoskeletal and signaling molecules.
J Cell Biol 131, 791–805.
12 Zheng, L., Sjo¨lander, A., Eckerdal, J & Andersson, T (1996)
Antibody-induced engagement of b 2 -integrins on adherent human
neutrophils triggers activation of p21ras through tyrosine
phos-phorylation of the protooncogene product Vav Proc Natl Acad.
Sci USA 93, 8431–8436.
13 Dennis, M.S., Henzel, W.J., Pitti, R.M., Lipari, M.T., Napier,
M.A., Deisher, T.A., Bunting, S & Lazarus, R.A (1990) Platelet
glycoprotein IIb-IIIa protein antagonists from snake venoms: a
family of integrin inhibitory proteins from viper venom Proc.
Natl Acad Sci USA 87, 2471–2475.
14 Gould, R.J., Polok off, M.A., Fiedman, P.A., Huang, T.-F., Holt,
J.C., Cook, J.J & Niewiarowski, S (1990) Disintegrins: a family of
integrin inhibitory proteins from viper venom Proc Soc Exp.
Biol Medical 195, 168–171.
15 Niewiarowski, S., McLane, M.A., Kloczwiak, M & Stewart, G.J.
(1994) Disintegrins and other naturally occurring antagonists of
platelet fibrinogen receptors Sem Hematol 31, 289–300.
16 Juliano, D., Wang, Y., Marcinkiewicz, C., Rosenthal, A.L.,
Stewart, G.J & Niewiarowski, S (1996) Disintegrin interaction
with a v b 3 integrin on human umbilical vein endothelial cells:
expression of ligand-induced binding site on b 3 subunit Exp Cell
Res 225, 132–142.
17 Danen, E.H., Marcinkiewicz, C., Cornelissen, I.M., van Kraats,
A.A., Pachter, J.A., Ruiter, J., Niewiarowski, S & van Muijen,
G.N (1998) The disintegrin eristostatin interferes with integrin
alpha 4 beta 1 function and with experimental metastasis of
human melanoma cells Exp Cell Res 238, 188–196.
18 McLane, M.A., Marcinkiewicz, C., Vijay-Kumar, S.,
Wierzbicka-Patynowski, I & Niewiarowski, S (1998) Viper venom
disin-tegrins and related molecules Proc Soc Exp Biol Medical 219,
109–119.
19 Coelho, A.L.J., De Freitas, M.S., Oliveira-Carvalho, A.L.,
Moura-Neto, V., Zingali, R.B & Barja-Fidalgo, C (1999) Effects
of jarastatin, a novel snake venom disintegrin, on neutrophil
migration and actin cytoskeleton dynamics Exp Cell Res 251,
379–387.
20 Coelho, A.L.J., De Freitas, M.S., Mariano-Oliveira, A.,
Oliveira-Carvalho, A.L., Zingali, R.B & Barja-Fidalgo, C (2001)
Interaction of disintegrins with human neutrophils induces
cytoskeleton reorganization, focal adhesion kinase activation and
extracellular-regulated kinase-2 nuclear translocation, interfering
with the chemotactic function FASEB J 15, 1643–1645.
21 Souza, D.H.F., Iemma, M.R.C., Ferreira, L.L., Faria, J.P., Oliva,
M.L.V., Zingali, R.B., Niewiarowski, S., Selistre-d & e-Araujo,
H.S (2000) The disintegrin-like domain of the snake venom
metalloprotease alternagin inhibits a2b1 integrin-mediated cell adhesion Arch Biochem Biophys 384, 341–350.
22 Dooley, D.C., Simpson, J.F & Merryman, H.T (1982) Isolation
of large numbers of fully viable human neutrophils: a preparative technique using Percoll density gradient centrifugation Exp Hematol 10, 591–599.
23 Bradford, M (1976) A rapid and sensitive method for quantifi-cation of microgram quantities of protein utilizing the principle of protein-dye binding Anal Biochem 72, 248–254.
24 Sheikh, S., Gratzer, W.B., Pinder, J.C & Nash, G.B (1997) Actin polymerization regulates integrin-mediated adhesion as well as rigidity of neutrophils Bioch Biophys Res Commun 238, 910–915.
25 Chen, H.C & Guan, J.L (1994) Association of focal adhesion kinase with its potential substrate phosphatidylinositol 3-kinase Proc Natl Acad Sci USA 91, 10148–10152.
26 Toker, A & Cantley, L.C (1997) Signalling through the lipid products of phosphoinositide-3-OH kinase Nature 387, 673–676.
27 Klemke, R.L., Cai, S., Giannini, A.L., Gallagher, P.J., de Lane-rolle, P & Cheresh, D.A (1997) Regulation of cell motility by mitogen-activated protein kinase J Cell Biol 137, 481–492.
28 Jia, L.-G., Wang, X.-M., Shannon, J.D., Bjarnasson, J.B & Fox, J.W (1997) Function of disintegrin-like/cysteine-rich domains of atrolysin A Inhibition of platelet aggregation by recombinant protein and peptide antagonists J Biol Chem 272, 13094–13102.
29 Stanton, K.J., Frewin, M.B & Gudewicz, P.W (1999) Hetero-logous desensitization of IL-8-mediated chemotaxis in human neutrophils by a cell-binding fragment of fibronectin J Leukoc Biol 65, 515–522.
30 Williams, M.A & Solonkin, J.S (1999) Integrin-mediated sig-naling in human neutrophil functioning J Leukoc Biol 65, 725–735.
31 Schwartz, M.A (2001) Integrin signaling revisited Trends Cell Biol 11, 466–470.
32 Gaudry, M., Caon, A.C., Gilbert, C., Lille, S & Naccache, P.H (1992) Evidence for the involvement of tyrosine kinases in the locomotory responses of human neutrophils J Leukoc Biol 51, 103–108.
33 McGregor, P.E., Agrawal, D.K & Edwards, J.D (1994) Attenuation of human leukocyte adherence to endothelial cell monolayers by tyrosine kinase inhibitors Biochem Biophys Res Commun 198, 359–365.
34 Huang, M.-M., Lipfert, L., Cunningham, M., Brugge, J.S., Ginsberg, M.H & Shattil, S.J (1993) Adhesive ligand binding to integrin aIIbw3 stimulates tyrosine phosphorylation of novel protein substrates before phosphorylation of pp125 FAK J Cell Biol 122, 473–483.
35 Bhattacharya, S., Fu, C., Bhattacharya, J & Greenberg, S (1995) Soluble ligands of the avb3 integrin mediate enhanced tyrosine phosphorylation of multiple proteins in adherent bovine pulmonary artery endothelial cells J Cell Biol Chem 270, 16781–16787.
36 Bryan Smith, J., Theakston, R.D.G., Coelho, A.L.J., Barja-Fid-algo, C., Calvete, J.J & Marcinkiewicz, C (2002) Characterization
of a monomeric disintegrin, ocellatusin, present in the venom of the Nigerian carpet viper, Echis ocellatus FEBS Lett 512, 111–115.
37 Ilic, D., Furuta, Y., Kanazawa, S., Tak eda, N., Sobue, K., Nakatsuji, N., Nomura, S., Fujimoto, J., Okada, M & Yama-moto, T (1995) Reduced cell motility and enhanced focal adhe-sion contact formation in cells from FAK-deficient mice Nature
377, 539–544.
38 Feniger-Barish, R., Yron, H., Meshel, T., Matityahu, E & Ben-Baruch, A (2003) Il-8-induced migratory responses thrugh CXCR1 and CXCR2 Association with phosphorylation and
Trang 10cellular redistribution of focal adhesion kinase Biochem 42,
2874–2886.
39 Li, Z., Jiang, H., Xie, W., Zhang, Z., Smrcka, A.V & Wu, D.
(2000) Roles of PLC-beta2 and – beta3 and PI3Kgamma in
chemoattractant-mediated signal transduction Science 287,
1046–1049.
40 Hirsch, E., Katanaev, V.L., Garlanda, C., Azzolino, O., Pirola, L.,
Silengo, L., Sozzani, S., Mantovani, A., Altruda, F & Wymann,
M.P (2000) Central role for G protein-coupled phosphoinositide
3-kinase gamma in inflammation Science 287, 1049–1053.
41 Coffer, P.J., Geijsen, M., M’rabet, L., Schweizer, R.C., Maikoe, T., Raaijmakers, J.A., Lammers, J.W & Koenderman, L (1998) Comparison of the roles of mitogen-activated protein kinase kinase and phosphatidylinositol 3-kinase signal transduction in neutrophil effector function Biochem J 329, 121–130.
42 Ritter, M.R & Markland, F.S Jr (2000) Contortrostatin activates ERK2 and tyrosine phosphorylation events via distinct pathways Biochem Biophys Res Commun 274, 142–148.