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EXTRACELLULAR MATRIX, CELL INTERACTIONS
1CIHR Group in Matrix Dynamics, University of Toronto, Toronto, Ontario; 2Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia, Canada
Submitted 21 March 2008 ; accepted in final form 22 April 2008
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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2β1-integrin-dependent process that mediates extracellular matrix remodeling by fibroblasts. We showed previously that after initial contact with collagen, activated Rac1 accelerates collagen phagocytosis but the Rac guanine nucleotide exchange factors (GEFs) that regulate Rac are not defined. We examined here the GEFs that regulate collagen phagocytosis in mouse fibroblasts. Collagen binding enhanced Rac1 activity (5–20 min) but not Cdc42 or RhoA activity. Analysis of collagen bead-associated proteins showed enrichment with Vav2, which correlated temporally with increased Rac1 activity. Knockdown of Vav2 prevented Rac activation, recruitment of Rac1 to collagen bead binding sites, and collagen bead binding, but knockdown of Sos-1 or β-Pix had no effect on Rac activation or collagen binding. Vav2 was associated with the nucleotide-free Rac1 mutant (G15ARac1) after collagen binding. Collagen bead binding promoted phosphorylation of Vav2, which temporally correlated with Rac1 activation and which required Src kinase activity. Blockage of Src activity prevented collagen bead-induced Rac activation and collagen bead binding. Collectively these data indicate that Vav2 regulates the Rac1 activity associated with the binding step of collagen phagocytosis.
2β1; integrin; Vav2; small GTPases
Cell adhesion and migration involve tightly controlled regulation of actin cytoskeletal proteins. Many of the processes involved in regulation of the actin cytoskeleton are also shared by phagocytosis, a receptor-driven process. In response to a diverse group of extracellular signals, small GTPases are activated to co-ordinate actin assembly and integrin activation (13, 24), and this has also been observed in phagocytosis (10, 11, 26). As the initial events in phagocytosis may be considered as a specialized form of cell adhesion (11), studies of actin- and integrin-dependent collagen phagocytosis and its associated regulatory systems can be facilitated by models that use loading of fibroblasts on collagen-coated latex beads (3). These models recapitulate some of the processes observed in the spreading of cells on tissue culture plates and show that alteration of integrin-mediated cell adhesion to the extracellular matrix is a critical determinant of collagen phagocytosis (2). We have shown previously that within 5 min of cell binding to collagen beads through 2β1-integrins, there is greatly accelerated bead binding and Rac activation (4). Similarly, in leukocytes, CD31 stimulates an integrin-dependent amplification of adhesion (22). However, the mechanisms that mediate this amplification and result in increased integrin affinity and collagen binding are not defined.
One of the consequences of signaling arising from integrin-mediated adhesion and cell spreading is the activation of Rac and Cdc42. These small Rho family GTPases in turn regulate extension of lamellipodia and filopodia, processes that involve complex and dynamic rearrangements of the actin cytoskeleton. The recruitment and function of Rho GTPases, specifically at sites of cell attachment to the extracellular matrix, are crucial for phagocytosis and spreading (10, 17, 24). Localized Rac1 activation at particle attachment sites triggers particle internalization, a process that requires rearrangement of the actin cytoskeleton leading to actin assembly and membrane protrusion (8). Integrin-mediated cell adhesion may affect interactions of GTP-Rac with its effectors by controlling the membrane targeting of GTP-Rac (13). Currently, the regulation of integrin affinity after initial particle attachment, which leads to Rac1 activation and accelerated cell adhesion, are not well understood.
Regulation of GTPases involves transition between GDP/GTP-bound activation states that are regulated by several systems, including guanine nucleotide exchange factors (GEFs) and activating proteins (GAPs). The Vav family of GEFs activates Rho GTPases (Rho, Rac, Cdc42) by catalyzing the exchange of GDP for GTP. In mammals the Vav family of proteins consists of three known members (Vav-1,-2, and -3) that differ in their tissue distribution. Vav1 is expressed predominantly in hematopoietic cells, whereas Vav2 and Vav3 are more broadly expressed. Vav phosphorylation is functionally linked to Vav activation, and in vitro studies with purified proteins demonstrate that Vav phosphorylation is the critical regulatory event triggering Vav activation and Rac activation (12). Of the known GEFs that control the activation state of Rac, Vav2 is considered important for Fc
R but not CR3 (integrin)-mediated phagocytosis in macrophages and COS cells (23). Whereas in fibroblasts Vav2 is necessary for integrin, but not growth factor-dependent activation of Rac leading to lamellipodia formation (20), Vav2 is not apparently involved in activation of Rac in response to β1-integrin ligation (18).
We have examined here the role of Rac1 GEFs in the binding step of collagen phagocytosis. The data show that collagen bead binding promotes phosphorylation of Vav2, which temporally correlates with Rac1 activation and requires Src kinase activity.
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MATERIALS AND METHODS |
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2β1 (clone no. BMA2.1) was from Millipore. T7 antibody was from Novagen (Madison, WI), and Vav2 (clone MAC 410) antibody was from Babraham Bioscience Technology. FITC-goat anti-rat antibody was purchased from Cedarlane Laboratories (Hornby, ON). Antibody to phosphorylated Vav2 (Tyr172) was from Santa Cruz Biotechnology. Antibody to Rac (clone 23A8) was obtained from Upstate Biotechnology. Rhotekin-RBD beads and RhoA antibody were purchased from Cytoskeleton (Denver, CO). Immobilized Protein G was obtained from Pierce (Rockford, IL). Cells. NIH 3T3 cells were cultured in DMEM (GIBCO BRL) supplemented with 10% fetal calf serum and 10% antibiotics. Jurkat cells were obtained from ATCC.
Collagen bead binding. Collagen-coated latex beads (2 µm) were applied to nontissue culture dishes and dried down for attachment as described previously (3) followed by being washed with PBS. The number of beads plated per dish was adjusted to produce final bead-to-cell ratios specific for each experiment. Cells were counted electronically, and the cell concentration was adjusted before cells were plated on dishes containing collagen-coated beads. The plates were maintained at room temperature for 10 min to allow the cells to settle and attach to the collagen beads, and subsequently, the cells were washed with fresh medium at 37°C. Detached cells were removed by repeated washes. Those cells that were attached, spread and rapidly internalized the collagen beads (5).
Isolation of bead attached proteins and immunoprecipitation. In some experiments to examine the proteins that were found at the phagocytic site, collagen-coated latex beads (6 µm) were attached to 100-mm nontissue culture plastic dishes. Cell suspensions were allowed to attach to beads for 20 min. Unattached, floating cells were aspirated and replaced with media warmed to 37°C to synchronize phagocytosis. Cells were collected at discrete time points thereafter. Cells and collagen-coated latex beads were collected with a cell scraper in extraction buffer (1% Triton X-100, 150 mM NaCl, 10 mM Tris·HCl at pH 7.2, 1 mM Na3VO4, 20 µg/ml aprotonin, and 1 µg/ml Pefabloc). The samples were sonicated (2 s) and centrifuged for 5 min at 8,000 g to remove unbroken cells. After clarification, equal amounts of proteins were incubated with antibodies to T7 or Vav2 to form immunocomplexes that were captured on Sepharose-G beads (Pierce Biotechnology, Rockford, IL) for 1 h at 4°C. The samples were boiled and separated on SDS-PAGE gels. Immunoblotted samples were probed with appropriate antibodies and quantified by scanning densitometry.
Immunofluorescence, confocal microscopy, and SEM. After transfection (24 h) with different constructs, cells were plated on beads and were allowed to spread and bind to beads for 30 min. In some experiments cells were fixed with 3% formaldehyde in PBS, permeabilized with 0.2% Triton-X100, and stained with appropriate antibodies. The spatial distribution of staining around beads was determined by confocal microscopy (x40 oil immersion lens; Leica, Heidelberg, Germany). Transverse optical sections were obtained at 1-µm nominal thickness. For morphological assessments of cellular interactions with beads, cells were fixed in 4% formaldehyde, dehydrated through an ethanol series, and critical point dried in a Polaron CPD7501. Samples were mounted on aluminum stubs, and plasma was sprayed with a 5-nm thick coat of platinum in a Polaron SC 515 scanning electron microscopy coating system and examined with a Hitachi C-2500 scanning electron microscope.
Plasmids and siRNA transfections. Green fluorescence protein (GFP) Vav2 and T7 tagged Vav2 were generated by one of us (P. Marignani). The Rac1 nucleotide-free mutant G15ARac1 was kindly provided by K. Burridge (University of North Carolina). Constitutively activated GFP Rac was obtained from A. Kapus (St. Michael's Hospital Research Institute, Toronto, Canada). For small interfering RNA (siRNA) knockdown, cells (106) on tissue culture plates (100 mm) were transfected with 5–20 pmol of Smart Pool siRNA oligonucleotides specific to Vav2, Sos1, and β-PIX (Dharmacon) using Oligofectamine (Invitrogen) without antibiotics. To estimate transfection efficiency and to serve as a transfection control, pEGFPluc (Clontech) was used, and the number of fluorescent cells were counted.
Rac, Cdc42, and RhoA activation. To detect Rac, Rho, and cdc42 activity, cells were serum starved and lysed. Cell lysates were collected in lysis buffer with protease inhibitors (Upstate) and clarified by low-speed centrifugation, and supernatants were incubated with a glutathione-S-transferase (GST) fusion-protein (Upstate) corresponding to the p21-binding domain (PBD), residues 67–150 of human PAK-1; expressed in Escherichia coli and bound to glutathione agarose. The samples were washed four times with wash buffer after an hour of incubation at 4°C. Pellets were boiled in 2x Laemlli buffer. Samples were separated on 10% SDS PAGE gels, transferred to nitrocellulose paper, and probed with Rac antibody (clone no. 23A8; Upstate) or cdc42 antibody (Upstate). Similarly, Rho activation assays were performed with Rhotekin beads (Cytoskeleton) and blotted with RhoA antibody. Supernatants from samples were also run on SDS-PAGE gels and immunoblotted for total Rac, cdc42, and Rho proteins. Samples stimulated with lysophosphatidic acid (LPA) were used as a positive control to detect active Rho.
Statistical analyses. For continuous variables, means and SE were computed, and differences between groups were evaluated by Student's unpaired t-test or ANOVA for multiple comparisons with statistical significance set at P < 0.05. Post hoc comparisons were performed with Tukey's test. For all experiments, at least three independent experiments were evaluated, each performed in triplicate.
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RESULTS |
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2β1-integrin with the use of a blocking monoclonal antibody (clone no. BMA2.1; at 0.2 mg/ml) followed by incubation with fluorescent collagen-coated beads. At bead cell ratios of 4:1, there was 85% reduction in the percentage of cells with bound beads in the antibody-treated samples. Western blot analysis of bead-associated proteins showed maximal association of active Rac1 with beads between 2 and 20 min, whereas Rho and Cdc42 did not detectably associate with beads (Fig. 1B). Similarly, in cells transfected with hemagglutinin-tagged Cdc42 and GFP RhoA, there was no significant localization to collagen-coated beads (Fig. 1C, a–d) as quantified by fluorescence intensity line scans (Fig. 1C, e,f).
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Role of Vav2 in collagen binding. Although many of the GEFs share homologous common domains that promote their interactions with small GTPase-binding proteins, the distribution and biological activity of GEFs varies depending on cell type. Therefore, we examined protein expression levels of different GEFs in total cell lysates and proteins isolated from collagen beads after 5 min of incubation with NIH 3T3 fibroblasts (Fig. 2 A,a). We found Vav2, Sos1, and β-Pix in total cell lysates and abundant Vav2 and Sos1 in samples from collagen bead-associated proteins. β-Pix was barely detectable in collagen bead-associated proteins. Consistent with its restricted distribution to hematopoietic cells, there was no detectable Vav1. We did not detect Vav3, and C3G was only just detectable. Since these findings could be dependent on antibody reactivity, we assessed protein expression for all of these GEFs in Swiss 3T3 cells, human embryonic kidney (HEK) cells, Cos cells, and Jurkat cells. These cells were chosen on the basis of their known expression of the expected proteins. Immunoblots of whole cell lysates prepared from these cells indicated that our failure to detect Vav1 and Vav3 was not because of detection problems because these proteins were found in Jurkat and HEK cells, and C3G was readily detected in the Swiss 3T3 cells (Fig. 2, A,b).
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We reduced the expression of Vav2, Sos1, and β-Pix by siRNA and studied collagen bead binding. Knockdown of Vav2 resulted in a 50% reduction of collagen bead binding compared with controls (P < 0.01). Cells transfected with Sos1, β-PIX, or nontargeted siRNA showed no significant difference compared with controls (Fig. 2E; P > 0.2).
Vav2 targets Rac1. Since Western blot analysis showed a temporal correlation between the relative abundance of Rac1 and Vav2 in bead-associated proteins after the initiation of bead binding, we examined their localization in more detail. Cells cotransfected with GFP Rac1 and wild-type Vav2-T7 were incubated with collagen beads for 5 min. We observed marked colocalization of the two proteins (Fig. 3A, a–d). We then studied the role of Vav2 on the recruitment of active Rac in cells cotransfected with siRNA and YFP-PBD (to detect active Rac). In cells treated with siRNA for Vav2, there was no evidence for recruitment of active Rac1 to collagen bead binding sites (Fig. 3B, a–c). Although YFP-PBD could potentially bind to cdc42 (6), since cdc42 was not associated with collagen beads (Fig. 1B), we assume that our results reflect predominantly, activated Rac1. Optical sectioning by confocal microscopy showed the presence of YFP-PBD and Vav2 around beads (Fig. 3B, d–f).
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The Rac1 nucleotide-free mutant G15ARac1 can bind active GEFs with high affinity (15, 16). We examined binding of Vav2, β-PIX, and Sos1 to G15ARac1 beads in lysates prepared from cells that had been treated with collagen beads, BSA-coated beads, or no beads. We found that collagen bead binding enhanced the association of Vav2 with G15ARac1 after 5 min of bead incubation, whereas BSA-coated beads elicited no response (Fig. 3D). We found no evidence of β-PIX or Sos1 binding to G15ARac1 before or after collagen bead incubation with cells.
Src-mediated Vav2 tyrosine phosphorylation in response to collagen binding. Tyrosine phosphorylation mediates both activation and downmodulation of the biological activity of Vav proteins (19), and integrin-mediated binding can induce tyrosine phosphorylation of various GEFs, including Vav2 (21). Accordingly, we examined tyrosine-phosphorylated proteins in response to collagen binding and observed a gradual increase in tyrosine phosphorylation of proteins with molecular mass of 72–130 kDa in total cell lysates (Fig. 4 A,a). We determined whether endogenous Vav2 was one of these phosphorylated proteins. After incubation with collagen beads, Vav2 was immunoprecipitated, and the immunoprecipitates were immunoblotted with an antibody that recognizes phosphorylated Y172 (19). Phosphorylation of Tyr172 in Vav2 relieves an intramolecular inhibition, thus resulting in enhanced Vav2 activity by exposing the Dbl homology domain (1). We observed increased phosphorylation of Y172 particularly at 2 and 5 min after incubation with collagen beads (Fig. 4 A,b). A similar experimental design was used for cells transfected with wild-type Vav2 tagged with T7. Vav2 was immunoprecipitated from lysates using agarose beads conjugated with T7 antibody and analyzed for tyrosine phosphorylation by immunoblotting with PY20. Tyrosine-phosphorylated Vav2 was maximal at 5 min after exposure to collagen beads. There was no tyrosine phosphorylation of Vav2 in response to cells binding BSA-coated beads at 5 min (Fig. 4 A,c).
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We assessed whether blockade of Src kinase activity would interfere with activation of Rac1. Fibroblasts were pretreated with PP2 or PP3 and then incubated with collagen beads. Samples of bead-associated proteins isolated after 5 min showed inhibition of Rac activation in PP2-treated samples but not in PP3-treated samples (Fig. 4E). These data indicated that collagen bead binding induces tyrosine phosphorylation of Vav2 on Tyr172, which is mediated by Src. Therefore Src kinase activity and Vav2 are likely required for Rac activation in response to collagen binding.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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2β1-integrins (5). We previously observed that the initial interaction of collagen with 2β1-integrins activates Rac1 and greatly enhances collagen binding over time (2, 4), but how specific exchange factor signaling molecules orchestrate the initial interaction of collagen binding leading to acceleration of Rac1 activity is not defined. In this study we have shown that the guanidine exchange factor Vav2 mediates Rac activation at collagen bead binding sites. We used the nucleotide-free Rac mutant G15ARac1 (15) to determine whether Vav2 interacts with active Rac1 after collagen binding. These experiments clearly indicated that Vav2 interacts with active Rac1 and is required for enhanced Rac1 activity around bound collagen beads. Furthermore, functional experiments showed that knockdown of Vav2 inhibited collagen binding over time and also inhibited enhanced Rac1 activation around the beads. These data are in contrast to an earlier report in which Vav2 activated Rac1 downstream of growth factor receptors but not after binding to β1-integrins (18). Thus while early steps in phagocytosis are appropriately modeled by adhesive cellular processes (11), adhesion is likely to be dependent on the type of cells that are examined. This may be particularly true for the binding step of phagocytosis in fibroblasts where small and spatially discrete activation sites are required for particle binding.
Role of Vav2 and Rac. Our results with siRNA knockdown of Rac1 showed that enhancement of collagen bead binding required Rac1, whereas collagen binding did not affect Cdc42 or RhoA activation. Whereas these data do not rule out possible roles for Cdc42 or Rho in collagen internalization, the internalization of collagen is not detectable at early time points after collagen ligation (2). We also found that following bead binding, Rac1 activity increased. This is in agreement with previous studies showing that integrin-mediated cell adhesion promotes activation of Rac (9, 24). In contrast, FcR-mediated phagocytosis in macrophages is initiated by recruitment of activated Rac at particle attachment sites (7). Collectively our data show that Vav2 plays a crucial role in the initial binding step of collagen phagocytosis. Although other GEFs (i.e., Sos1 and β-PIX) were detected in the samples isolated from collagen bead binding sites, which raised the possibility of their involvement in Rac1 activation, knockdown of these exchange factors by siRNA demonstrated no effect on activation of Rac1 and enhanced collagen binding.
Role of Src kinase activity. We found that tyrosine phosphorylation of Vav2 occurs specifically in response to collagen binding and that active Vav2 is required for collagen (but not BSA)-induced activation of Rac1. Notably, tyrosine phosphorylation of Vav2 is sufficient to catalyze GDP-GTP exchange on Rac1 (12) and Src is an effector of Vav2 phosphorylation (25). We found that inhibition of Src kinase activity with PP2 prevented Vav2 phosphorylation and Rac1 activity and greatly reduced collagen bead binding. Furthermore, our experiments with Vav2 knockdown demonstrated greatly reduced localization of Rac1 to collagen beads, suggesting that Vav2 is required for Rac1 targeting to collagen beads. These results are consistent with previous findings showing that in fibroblasts, Vav2 is necessary for fibronectin-dependent activation of Rac, leading to lamellipodia extension and cell spreading (20). Furthermore, Chinese hamster ovary and Jurkat cells transfected with Vav2 show β1-integrin mediated cell adhesion to fibronectin triggered by phosphorylation of Vav2 (27). Collectively, our data indicate a central role for Vav2 in collagen phagocytosis and Rac activation in this process.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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