Ligand-dependent Activation of Integrin alpha vbeta 3*

Boyd Butler, Matthew P. Williams, and Scott D. BlystoneDagger

From the Department of Cell and Developmental Biology, State University of New York Upstate Medical University, Syracuse, New York 13210

Received for publication, July 12, 2002, and in revised form, November 6, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The ability of leukocytes to self-regulate adhesion during transendothelial and extravascular migration is fundamental to the performance of immune surveillance in complex extracellular matrices. Leukocyte adhesion is regulated through the modulation of integrin receptors such as alpha vbeta 3. In this study, we examined the activation of alpha vbeta 3 resulting from attachment to vitronectin or fibronectin. In K562 cells stably expressing transfected alpha vbeta 3, adhesion to vitronectin required tyrosine phosphorylation of the beta 3 subunit and activation of phosphoinositide 3-kinase and protein kinase C. In contrast, adhesion to fibronectin proceeded without beta 3-tyrosine phosphorylation or the activities of phosphoinositide 3-kinase or protein kinase C. Firm adhesion to both ligands and actin stress fiber formation required both Syk and Rho activity, suggesting that each ligand employs unique signaling pathways to achieve an active integrin complex, likely merging at a common RhoGEF such as Vav. Distinct signaling by a single integrin species interacting with different ligands permits initiation of additional cellular processes specific to the current task and provides an explanation for what has been described as promiscuous ligand specificity among integrins.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Integrins are heterodimeric transmembrane receptors that mediate adhesion of cells to extracellular matrix proteins and to other cells. Consequently, integrins have a pivotal role in numerous developmental, physiological, and pathological processes. The integrin family contains at least 18 alpha  and beta  subunits that combine to produce 24 known heterodimer combinations with distinct cell expression patterns and overlapping ligand specificities (1). Binding of ligand to the integrin extracellular domain induces a conformational change that is propagated to the cytoplasmic domain and initiates downstream signaling events (1). The ability to self-regulate adhesion to complex extracellular matrices is of particular importance to hematopoietic cells. Hematopoietic cells circulate in a non-adhesive state until such time as they encounter pro-inflammatory or thrombotic signals, after which they arrest from the circulation by means of firm adhesion to the vascular walls and, in leukocytes, migrate to the site of inflammation. The ability of hematopoietic cells to regulate their adhesion-dependent extravasation is mediated in part by the integrin alpha vbeta 3 (2).

The alpha vbeta 3 integrin plays a vital role in the adhesion of many cell types to the extracellular matrix and to other cells. Although it is termed the vitronectin (Vn)1 receptor, it is in fact a promiscuous receptor that recognizes a number of extracellular ligands including fibronectin (Fn), tenascin, and osteopontin (1, 3). However, promiscuous may be an unfortunate term because recent studies show that differential signaling by distinct alpha vbeta 3 ligands may in fact result in unique cellular responses (4). We have previously shown that when alpha vbeta 3 is expressed in the K562 cell line, these cells exhibit differential adhesion to Vn and Fn (4). alpha vbeta 3-mediated adhesion to Vn requires tyrosine phosphorylation at Tyr-747 within the beta 3 cytoplasmic tail and is dependent upon PKC activation, whereas alpha vbeta 3-mediated adhesion to Fn is constitutive, requiring neither of these events (4, 5). In addition, alpha vbeta 3-mediated adhesion of LNCaP cells to osteopontin, but not Vn, activates PI3K. However, this signaling pathway may be cell type-specific as PC3 cells activate PI3K upon adhesion to both osteopontin and Vn (6).

In this study we sought to establish the signaling events that might necessitate beta 3-tyrosine phosphorylation in alpha vbeta 3-mediated adhesion by comparing actin cytoskeletal organization and signaling events after attachment to distinct ligands. We show that alpha vbeta 3-mediated adhesion to Fn is constitutive and independent of beta 3-tyrosine phosphorylation because cells expressing alpha vbeta 3 Y747F,Y759F (Kalpha vbeta 3Y747F,Y759F) firmly adhere to Fn, equivalent to cells expressing wild type alpha vbeta 3 (Kalpha vbeta 3), even in the absence of PKC activation. In contrast, firm adhesion to Vn requires both cellular PKC activation as well as beta 3-tyrosine phosphorylation. Coincident with cell adhesion is a reorganization of the actin cytoskeleton into stress fibers. On a Fn substrate, Kalpha vbeta 3 exhibited spontaneous stress fiber formation, whereas Kalpha vbeta 3 attached to Vn required PKC activation to achieve this cellular phenotype. Mutation of the beta 3 cytoplasmic tyrosine residues had no effect on Kalpha vbeta 3Y747F,Y759F organization of stress fibers when attached to Fn but eliminated PKC-dependent stress fiber formation upon attachment to Vn. Because Rho activity is believed to mediate actin stress fiber assembly, we examined Rho activity in Kalpha vbeta 3 and Kalpha vbeta 3Y747F,Y759F cells attached to Vn and Fn. We show that the constitutive nature of the adhesion to Fn by Kalpha vbeta 3 and Kalpha vbeta 3Y747F,Y759F is due in part to direct activation of Rho. Interestingly, whereas apparently unrelated to the tyrosine phosphorylation of beta 3, adhesion to Vn, but not Fn, results in increased PI3K activity. The PI3K inhibitor wortmannin inhibits the PKC-dependent adhesion of Kalpha vbeta 3 to Vn, whereas adhesion to Fn is unaffected by the presence of wortmannin. These results indicate that alpha vbeta 3-mediated organization of the actin cytoskeleton, a requisite event in cell adhesion, occurs via unique, ligand-dependent mechanisms.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cells and Materials-- K562 cells were stably transfected with cDNA encoding wild type (Kalpha vbeta 3), Y747F,Y759F (Kalpha vbeta 3Y747F,Y759F) beta 3 or wild type (Kalpha Vbeta 5) beta 5 together with alpha V and maintained as previously described (7, 8). The anti-beta 3 monoclonal antibodies 7G2 and LIBS-1 were gifts of Eric J. Brown and Mark H. Ginsberg, respectively. Vitronectin and fibronectin were prepared as previously described (7). Purified GST-FnIII-10 and III10/RGE (RGD mutated to RGE) were gifts of Denise C. Hocking (9). All reagents unless otherwise noted were purchased from Sigma. Anti-p85 rabbit polyclonal was purchased from Upstate Biotechnology (Lake Placid, NY). Anti-Rho mouse monoclonal antibody was purchased from Cytoskeleton Inc (Denver, CO).

Flow Cytometry-- beta 3 surface expression in stably transfected K562 cells was monitored using flow cytometry. Untransfected K562 cells or Kalpha vbeta 3 or Kalpha vbeta 3Y747F,Y759F cells were incubated with mAb anti-beta 3 AP3 in IMDM for 1 h at 4 °C. Cells were then washed in IMDM and incubated with fluorescein isothiocyanate-labeled goat anti-mouse IgG for 30 min at 4 °C. To quantify binding site equality of beta 3 by 7G2 and LIBS-1, Kalpha vbeta 3 or Kalpha vbeta 3Y747F,Y759F cells were stimulated with or without GRGDS peptide (1 mM) or wortmannin (10 µM) in IMDM for 20 min at room temperature. Cells were washed in IMDM and incubated with either 7G2 or LIBS-1 anti-beta 3 antibodies for 1 h at 4 °C. Cells were washed in IMDM and incubated with fluorescein isothiocyanate-labeled goat anti-mouse IgG for 30 min at 4 °C. Flow cytometry was carried out using a Coulter Epics XL flow cytometer (Coulter, Miami, FL). Data are expressed as mean channel fluorescence for at least three separate experiments.

Cell Adhesion Assays-- 96-Well microtiter plates (Immulon II, Dynatech, Chantilly, VA) were coated with fibronectin (10 µg/ml), vitronectin (1 µg/ml), 7G2 anti-beta 3 (0.5 µg/ml), LIBS-1 anti-beta 3 (0.5 µg/ml), or casein (50 µg/ml) in PBS overnight at 4 °C. Wells were washed twice in PBS and post-coated with 1.0% casein in PBS for 30 min at room temperature. Kalpha vbeta 3 or Kalpha vbeta 3Y747F,Y759F cells were added at 1 × 105 cells/well in Hank's-buffered saline solution containing 1.0 mM Ca2+ and Mg2+ with or without PMA (10 ng/ml) and C3 exoenzyme (10 µg/ml) after treatment with saponin (50 µg/ml) for 3 min, piceatannol (50 µg/ml), or wortmannin (10 µM) and allowed to adhere for 1 h at 37 °C. Wells were rinsed 3 times in Hank's-buffered saline solution without Ca2+ or Mg2+, and adherent cells were fixed in 3.7% formaldehyde for 1 h at 4 °C and stained with 0.05% crystal violet for 30 min at room temperature. Crystal violet was dispersed in methanol and quantified by absorbance at 570 nm using an Emax microplate reader (Molecular Dynamics, Sunnyvale, CA).

beta 3 Tyrosine Phosphorylation-- 6-well tissue culture treated plates were coated with Vn (1 µg/ml), Fn (10 µg/ml), anti-beta 3 7G2 (0.5 µg/ml), or anti-beta 3 LIBS-1 (0.5 µg/ml) in PBS overnight at 4 °C. Kalpha vbeta 3 or Kalpha vbeta 3Y747F,Y759F cells were plated at 2 × 106 cells/well in serum-free IMDM media containing 75 µM sodium orthovanadate with or without PMA (10 ng/ml), bisindolylmaleimide (10 µM), piceatannol (50 µg/ml), or wortmannin (10 µM). The cells were lysed in PBS buffer containing 1% Nonidet P-40, sodium orthovanadate (1 mM), aprotinin (10 µg/ml), leupeptin (10 mg/ml), and phenylmethylsulfonyl fluoride (1 mM). Cell debris was removed by centrifugation at 12,000 × g for 10 min at 4 °C. The lysates were precleared for 1 h at 4 °C with gelatin-Sepharose and immunoprecipitated with goat anti-mouse-Sepharose beads (ICN, Costa Mesa, CA) coated with anti-beta 3 1A2 and anti-alpha V 3F12 monoclonal antibodies for 2 h at 4 °C. Samples were divided equally to determine beta 3-tyrosine phosphorylation and total beta 3. Samples were separated on 7.5% SDS-PAGE gels and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA). The membranes were blocked in Tris-buffered saline with 0.1% Tween 20 (TBST) and 3% v/v bovine serum albumin at room temperature for 1 h. Membranes were then incubated with mAb 4G10 (Upstate Biotechnology) for phosphotyrosine or mAb 7G2 for total beta 3. The membranes were then incubated with peroxidase-coupled goat anti-mouse IgG2b (Caltag Laboratories, Burlington, CA) for phosphotyrosine beta 3 and peroxidase-coupled goat anti-mouse IgG (Sigma) for total beta 3. Proteins were detected with enhanced chemiluminescence (ECL, Amersham Biosciences).

Fluorescent Microscopy-- Acid-washed glass coverslips were coated overnight with Vn (1 µg/ml) or Fn (5 µg/ml). Kalpha vbeta 3 or Kalpha vbeta 3Y747F,Y759F cells were permitted to adhere to coverslips for 1 h at 37 °C in the presence or absence of PMA (10 ng/ml). Cells were fixed in 3.7% formaldehyde, permeabilized in ice-cold PBS containing 0.01% Nonidet P-40, and stained with 0.7 µM rhodamine-phalloidin for 15 min at 37 °C. Fluorescence was visualized on a Nikon Eclipse E800 fluorescent microscope (Nikon, Melville, NY), and images were retained digitally with a SpotCamII digital camera and software (Diagnostic Instruments, Sterling Heights, MI).

Rho Activity Assay-- 6-well tissue-coated plates were coated with Vn (1 µg/ml), Fn (10 µg/ml), recombinant Fn III-10 (10 µg/ml), Fn III-10RGE (10 µg/ml), 7G2 (0.5 µg/ml), or LIBS-1 (0.5 µg/ml), and Kalpha vbeta 3 or Kalpha vbeta 3Y747F,Y759F cells were plated at 2 × 106 cells/well in the presence or absence of PMA (10 ng/ml), C3 exoenzyme (10 µg/ml) after treatment with saponin (50 µg/ml) for 3 min, piceatannol (50 µg/ml), or wortmannin (10 µM) and allowed to adhere for 1 h at 37 °C. Rho activity was assayed as previously described (10). Briefly, cells were lysed in 0.5 ml of ice-cold PBS buffer containing 1% Nonidet P-40, leupeptin (10 µg/ml), aprotinin (10 µg/ml), phenylmethylsulfonyl fluoride (1 mM), and sodium orthovanadate (1 mM). Cell lysates were then clarified by centrifugation at 12,000 × g for 15 min at 4 °C. GTP-bound Rho was affinity-purified from lysates using a GST fusion construct of the RhoA binding domain of rhotekin (GST-RBD) (kindly provided by M. A. Schwartz, Scripps Institute). Lysates were incubated with GST-RBD (~25 µg) beads for 1 h at 4 °C. GTP-bound RhoA was separated using 12% SDS-PAGE gels under reducing conditions. Total Rho was determined by loading 1/10 total volume of cell lysate on a 12% SDS-PAGE under reducing conditions. Proteins were transferred to polyvinylidene difluoride membranes and blocked in TBST with 3% v/v bovine serum albumin at room temperature for 1 h. Membranes were then incubated with murine mAb to RhoA (Cytoskeleton Inc., Denver, CO). The membranes were then incubated with peroxidase-coupled anti-mouse IgG, and proteins were detected with ECL.

AKT and Phospho-AKT Immunoblotting-- 6-Well tissue culture plates (Costar, Corning, NY) were coated as above. Kalpha vbeta 3 or Kalpha vbeta 3Y747F,Y759F cells were plated at 2 × 106 cells/well in the presence or absence of PMA (10 ng/ml) for 1 h at 37 °C. Cells were lysed in 0.5 ml of ice-cold PBS lysis buffer (PBS, 1% Nonidet P-40, 10 µg/ml each of leupeptin, and aprotinin, 1 mM phenylmethylsulfonyl fluoride), and lysates were cleared by centrifugation at 12,000 × g for 15 min at 4 °C. Lysates were divided evenly for phospho-AKT Ser473 and total AKT and loaded onto 10% SDS-PAGE gels under reducing conditions. Proteins were transferred to polyvinylidene difluoride membranes and blocked in TBST with 3% v/v bovine serum albumin at room temperature for 1 h. Membranes were incubated with sheep polyclonal antibodies to AKT (0.1 µg/ml) or to phospho-AKT Ser-473 (0.5 µg/ml) (Upstate Biotechnology) for 2 h at room temperature. The membranes were then incubated with peroxidase-coupled anti-sheep IgG, and proteins were detected with ECL.

PI3K Immunoprecipitation-- Kalpha vbeta 3 and Kalpha vbeta 3Y747F,Y759F (1 × 107) were treated with or without Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP) (1 mM) in the presence or absence of orthovanadate (0.75 µM) for 20 min at room temperature to induce beta 3-tyrosine phosphorylation (11). Cells were then lysed in 0.5 ml of ice-cold PBS lysis buffer (PBS, 1% Nonidet P-40, 10 µg/ml each of leupeptin and aprotinin, 1 mM phenylmethylsulfonyl fluoride). Cell debris was removed by centrifugation at 12,000 × g for 10 min at 4 °C. The lysates were precleared for 1 h at 4 °C with gelatin-Sepharose and immunoprecipitated with goat anti-mouse-Sepharose beads (ICN, Costa Mesa, CA) coated with anti-beta 3 1A2 and anti-alpha V 3F12 monoclonal antibodies for 2 h at 4 °C. Samples were divided equally to determine PI3K and total beta 3. Samples were separated on 7.5% SDS-PAGE gels and transferred to polyvinylidene difluoride membranes (Millipore). The membranes were blocked in Tris-buffered saline with 0.1% Tween 20 (TBST) and 3% v/v bovine serum albumin at room temperature for 1 h. Membranes were then incubated with anti-p85 rabbit polyclonal (Upstate Biotechnology) or mAb 7G2 for total beta 3. The membranes were then incubated with peroxidase-coupled goat anti-mouse IgG2b (Caltag Laboratories, Burlington, CA) for phosphotyrosine beta 3 and peroxidase-coupled goat anti-mouse IgG (Sigma) for total beta 3. Proteins were detected with enhanced chemiluminescence (ECL, Amersham Biosciences).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

alpha vbeta 3-mediated Firm Adhesion to Distinct Ligands and Actin Cytoskeletal Reorganization-- To investigate the influence of the extracellular ligand on alpha vbeta 3-mediated adhesion and actin cytoskeletal organization, we examined the ability of K562 cells, expressing wild type (Kalpha vbeta 3) or Y747F,Y759F (Kalpha vbeta 3Y747F,Y759F) alpha vbeta 3, to adhere to Vn or Fn in the presence or absence of the cellular agonist PMA. Kalpha vbeta 3 cells adhere to Vn only after cellular activation by PMA (Fig. 1A); however, Kalpha vbeta 3Y747F,Y759F cells are incapable of strong adhesion to Vn even after cellular activation. As we have previously reported, both Kalpha vbeta 3 and Kalpha vbeta 3Y747F,Y759F cells are capable of constitutive adhesion to Fn with or without cellular PKC activation by PMA (Fig. 1A) (4). To determine whether the constitutive adhesion to Fn is alpha vbeta 3-mediated, we exposed K562 cells stably expressing alpha Vbeta 5 (Kalpha Vbeta 5) to Vn and Fn in the presence or absence of PMA. Kalpha Vbeta 5 cells do not adhere to Fn even in the presence of PMA nor do untransfected K562 cells (data not shown), indicating that constitutive adhesion of Kalpha vbeta 3 and Kalpha vbeta 3Y747F,Y759F cells to Fn is alpha vbeta 3-mediated.


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Fig. 1.   Effect of distinct extracellular ligands on alpha vbeta 3-mediated adhesion and actin reorganization. A, microtiter plates (96 wells) were coated with Vn (1 µg/ml) or Fn (10 µg/ml). 1 × 105 Kalpha vbeta 3 or Kalpha vbeta 3Y747F,Y759F cells were allowed to adhere for 1 h at 37 °C in the absence (gray bars) or presence (black bars) of PMA and quantified as described under "Experimental Procedures." Data bars represent the mean absorbance ±S.D. from triplicate wells from three separate experiments. WT, wild type. B-I, fluorescent microscopy. Kalpha vbeta 3 cells were allowed to adhere to Vn in the presence (C) or absence (B) of PMA (10 ng/ml) or Fn in the presence (G) or absence (F) of PMA (10 ng/ml). Kalpha vbeta 3Y747F,Y759F cells were allowed to adhere to Vn in the presence (E) or absence (D) of PMA (10 ng/ml) or Fn in the presence (I) or absence (H) of PMA (10 ng/ml). Cells were then stained with rhodamine phalloidin to reveal actin organization as described under "Experimental Procedures." The bar represents 5µm.

Actin cytoskeletal reorganization plays a key role in the ability of cells to adhere to the extracellular matrix (13, 14). Therefore, we examined the structure of the actin cytoskeleton in Kalpha vbeta 3 and Kalpha vbeta 3Y747F,Y759F cells attached to Vn or Fn with or without cellular PKC activation. Both Kalpha vbeta 3 and Kalpha vbeta 3Y747F,Y759F cells, when allowed to adhere to Fn for 1 h, display a spread phenotype independent of PKC activation via PMA. Fluorescent microscopy of these cells reveals the formation of actin fibers (Fig. 1, F-I) within 1 h of attachment to Fn, and thus, actin fiber formation on Fn is independent of both beta 3 tyrosine phosphorylation and PKC activation via PMA. In contrast, actin fiber formation on Vn is dependent upon cellular PKC activation and beta 3-tyrosine phosphorylation. No actin fibers are seen in Kalpha vbeta 3 cells on Vn in the absence of PMA stimulation (Fig. 1B). Stress fibers can be seen in Kalpha vbeta 3 cells after adhesion to Vn for 1 h only in the presence of PMA (Fig. 1C). In addition, beta 3-tyrosine phosphorylation plays a pivotal role in firm adhesion to Vn, as no stress fiber formation occurs within Kalpha vbeta 3Y747F,Y759F cells even after stimulation with PMA (Fig. 1, D and E).

Differential Inhibition of Adhesion to Distinct Ligands-- To elucidate the possible signaling mechanisms underlying the differences in alpha vbeta 3-mediated adhesion to Vn or Fn we utilized specific inhibitors to various signaling molecules. PMA-induced firm adhesion of Kalpha vbeta 3 cells to Vn was inhibited by pretreatment with the Rho inhibitor C3 exoenzyme (100 nM), the PI3K inhibitor wortmannin (10 µM), and the Syk inhibitor piceatannol (50 µg/ml). These results indicate that Rho, PI3K, and Syk are involved in PMA-induced firm adhesion to Vn (Fig. 2A). Firm adhesion of Kalpha vbeta 3 cells to Fn was also inhibited by C3 exoenzyme and piceatannol (Fig 2A); however, no inhibition of firm adhesion to Fn was seen with wortmannin, suggesting that PI3K activity is not required for alpha vbeta 3-mediated firm adhesion to Fn. Inhibition with C3 exoenzyme and piceatannol reduced adhesion to both Fn and Vn to levels comparable with adhesion to casein (50 µg/ml) (data not shown). Cells expressing Kalpha vbeta 3Y747F,Y759F are unable to adhere to Vn even after cellular PKC activation by PMA. Kalpha vbeta 3Y747F,Y759F cells exhibit the same inhibition pattern as Kalpha vbeta 3 cells on Fn in that there is complete inhibition of adhesion after pretreatment with C3 exoenzyme and piceatannol; however, we see no inhibition of adhesion with wortmannin (Fig. 2B). These data suggest that alpha vbeta 3-mediated adhesion to Fn, already shown to be independent of beta 3-tyrosine phosphorylation, is dependent upon Rho and Syk activity but not PI3K activity. In contrast alpha vbeta 3-mediated adhesion to Vn is dependent upon PI3K activity as well as Rho and Syk. The inhibitory effect of wortmannin on Vn is not due to a decrease in beta 3-tyrosine phosphorylation, because we show that in response to RGD, beta 3-tyrosine phosphorylation is inhibited fully in the presence of piceatannol but only minimally in the presence of wortmannin or the PKC inhibitor bisindolylmaleimide (Fig. 3). Therefore, wortmannin inhibition of adhesion to Vn is not due to an inhibition of beta 3-tyrosine phosphorylation, although piceatannol inhibition of adhesion to Vn may in part be due to inhibition of beta 3-tyrosine phosphorylation. However, this may be unlikely because piceatannol also inhibits Kalpha vbeta 3 and Kalpha vbeta 3Y747F,Y759F adhesion to Fn, where beta 3 phosphorylation is not required.


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Fig. 2.   Selective inhibition of alpha vbeta 3-mediated adhesion to distinct ligands. Microtiter plates (96 wells) were coated with Vn (1 µg/ml) or Fn (5 µg/ml). 105 Kalpha vbeta 3 (A) or Kalpha vbeta 3Y747F,Y759F (B) cells were allowed to adhere for 1 h at 37 °C in the absence or presence of PMA and specific inhibitors for Rho (10 µg/ml C3 exoenzyme), PI3K (10 µM wortmannin), or Syk (50 µg/ml piceatannol) and quantified as described under "Experimental Procedures." Data bars represent the mean absorbance ±S.D. from triplicate wells from three separate experiments. Unst., unstimulated, treated with vehicle only.


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Fig. 3.   Selective inhibition of ligand induced beta 3-tyrosine phosphorylation (PY). Shown is a Western blot using anti-phosphotyrosine mAb from beta 3 immunoprecipitates from1 × 106 Kalpha vbeta 3 cells treated with or without RGD (1 mM) and specific inhibitors for PKC (10 µM bisindolylmaleimide), PI3K (10 µM wortmannin), or Syk (50 µg/ml piceatannol). Total beta 3 was determined by Western blot of half the beta 3 immunoprecipitation probed with mAb 7G2. Control cells are left untreated in suspension.

Ligand-specific Activation of Rho-- Actin cytoskeletal reorganization into stress fibers has been shown to require Rho activity in many cell types (14-18). Here we demonstrate that alpha vbeta 3-mediated adhesion to Fn as well as stress fiber formation requires neither PKC activation nor beta 3-tyrosine phosphorylation. To determine whether the constitutive firm adhesion to Fn is mediated by Rho activation we assayed Rho activity using a GST-RBD pull-down assay for active GTP-bound Rho in cells that were allowed to adhere to Vn or Fn with or without PMA (Fig. 4A). Elevated Rho activity was seen in both Kalpha vbeta 3 and Kalpha vbeta 3Y747F,Y759F cells that were allowed to adhere to Fn for 1 h even in the absence of PMA stimulation. In contrast, adhesion to Vn did not lead to an increase in Rho activity in Kalpha vbeta 3 cells unless there was coincident cellular PKC activation and only minor increases in Rho activity were seen in Kalpha vbeta 3Y747F,Y759F under similar conditions. To confirm that the constitutive adhesion and Rho activation on Fn is alpha vbeta 3-mediated we show that when Kalpha Vbeta 5 cells are exposed to Fn there is no significant Rho activation even in the presence of PMA (Fig. 4A). Additionally, untransfected K562 cells exhibit no Rho activity on either Vn or Fn (data not shown). We also show that when PI3K is inhibited by the presence of wortmannin, Rho activation is inhibited in Kalpha vbeta 3 cells adherent to Vn but not on Fn (Fig. 4B). Control addition of C3 exoenzyme inhibits Rho activity in cells adherent to either Vn or Fn. This suggests that when alpha vbeta 3 is bound to Fn there is a direct activation of Rho that circumvents the need for PKC activity, beta 3-tyrosine phosphorylation, and PI3K activity. These results support a hypothesis in which external stimulation, beta 3-tyrosine phosphorylation, and PI3K are required for Rho activation and subsequent alpha vbeta 3-mediated adhesion to Vn. This supports our previous work suggesting that tyrosine-phosphorylated beta 3 is required for the actions of PKC during alpha vbeta 3-mediated adhesion to Vn. To determine whether sites other than the RGD motif of Fn are responsible for the constitutive adhesion of Kalpha vbeta 3 and Kalpha vbeta 3Y747F,Y759F cells we employed recombinant Fn fragment III-10 and assayed for Rho activity. We find activated Rho in Kalpha vbeta 3 cells adhered to FnIII-10 regardless of PMA activation (Fig. 4C). However, when Kalpha Vbeta 5 cells are adhered to FnIII-10 there is no Rho activity even in the presence of PMA. In addition when Kalpha vbeta 3 and Kalpha Vbeta 5 cells are allowed to adhere to FnIII-10RGE, in which the RGD motif has been changed to RGE, there is no constitutive Rho activity (Fig. 4C). These results indicate that it is the RGD motif of Fn that is required for constitutive alpha vbeta 3-mediated adhesion to Fn.


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Fig. 4.   Ligand-specific Rho activation. A, Kalpha vbeta 3, Kalpha vbeta 3Y747F, Y759F, or Kalpha Vbeta 5 cells were plated on 6-well plates coated with either Vn (1 µg/ml) or Fn (10 µg/ml), treated with or without PMA (10 ng/ml), and allowed to adhere for 1 h at 37 °C. Cell lysates were incubated with GST-RBD immobilized on agarose beads. GTP-Rho was detected by Western blotting, and 1/10 of the total lysates were probed for Rho to demonstrate equal loading. WT, wild type. B, Kalpha vbeta 3 cells were allowed to adhere to Vn or Fn in the presence or absence of PMA (10 ng/ml), wortmannin (10 µM), or C3 exoenzyme (10 µg/ml). Cell lysates were incubated with GST-RBD immobilized on agarose beads. GTP-Rho was detected by Western blotting, and 1/10 of the total cell lysates were probed for Rho to demonstrate equal loading. C, Kalpha vbeta 3 or Kalpha Vbeta 5 cells were adhered to Vn, Fn fragment III-10, or Fn fragment III-10RGE in the presence or absence of PMA (10 ng/ml) for 1 h at 37 °C. Cell lysates were incubated with GST-RBD immobilized on agarose beads. GTP-Rho was detected by Western blotting, and 1/10 of the total lysates were probed for Rho to demonstrate equal loading.

PI3K Activation on Distinct Ligands-- In this study we show that inhibition of PI3K activity by wortmannin inhibits both adhesion to Vn and Rho activation. To investigate the role of PI3K in alpha vbeta 3-mediated adhesion to Vn and Fn, we measured phosphorylated Ser-473 AKT as an indicator of PI3K activity and also the association of PI3K with beta 3 (6). Kalpha vbeta 3 and Kalpha vbeta 3Y747F,Y759F were adhered to Vn or Fn, and phospho-Ser-473 levels were detected using a phospho-specific AKT Ser-473 antibody. Phospho-Ser-473 can only be detected in Kalpha vbeta 3 cells adherent to Vn (Fig. 5A), suggesting that PI3K is active in a ligand-dependant manner only on Vn. Having demonstrated that the inhibition of PI3K by wortmannin does not inhibit beta 3-tyrosine phosphorylation (Fig. 3), we investigated the role of beta 3-tyrosine phosphorylation in PI3K localization. Kalpha vbeta 3 or Kalpha vbeta 3Y747F,Y759F were treated with RGD to induce beta 3-tyrosine phosphorylation and Western blotted with anti-p85 after beta 3 immunoprecipitations, demonstrating p85 association specifically with tyrosine-phosphorylated beta 3 (Fig. 5B). Additionally, the removal of Na3VO4, which reduces beta 3-tyrosine phosphorylation, partially blocked association of p85 with beta 3. These results indicate that PI3K is activated in a alpha vbeta 3-ligand-dependent manner and associates with alpha vbeta 3 in a beta 3-tyrosine phosphorylation-dependent manner.


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Fig. 5.   Distinct alpha vbeta 3 ligands differentially activate PI3K. A, PI3K is activated on Vn but not Fn. Kalpha vbeta 3 or Kalpha vbeta 3Y747F,Y759F cells were plated on 6-well plates coated with either Vn (1 µg/ml) or Fn (10 µg/ml), treated with or without PMA (10 ng/ml), and allowed to adhere for 1 h at 37 °C. Cell lysates were analyzed for the presence of phosphorylated AKT and total AKT by Western blotting. WT, wild type. B, the P85 subunit of PI3K is only associated with phosphorylated beta 3. Kalpha vbeta 3 or Kalpha vbeta 3Y747F,Y759F cells were incubated with or without RGD peptide (1 mM) and Na3VO4 (100 nM), and cell lysates were immunoprecipitated with mAb 7G2 bound to Sepharose. Western blotting was performed using a p85 mAb. Total beta 3 was determined by Western blot of half of the beta 3 immunoprecipitation (IP) probed with mAb 7G2.

Adhesion to Distinct anti-beta 3 Antibodies Mimics Adhesion to Vn and Fn-- Various anti-beta 3 antibodies have been developed that recognize specific epitopes within the extracellular domain of beta 3, and some of the more useful anti-beta 3 antibodies developed are the ligand-induced binding site (LIBS) antibodies (19). These LIBS antibodies recognize epitopes that are exposed within the integrin extracellular domain upon binding to an RGD sequence within an ECM protein such as Vn or Fn. Using this we have explored the possibility that alpha vbeta 3 may in fact exhibit adhesive properties to different anti-beta 3 antibodies that mimic adhesion to distinct ECM proteins. This would enable us to eliminate any possible contributions of other integrins expressed within our K562 system. Using multiple assays we determined that 7G2 and LIBS-1, surprisingly both LIBS type antibodies, mimic alpha vbeta 3-mediated adhesion to Vn and Fn, respectively. As seen with adhesion to Vn or Fn (Fig. 6A), adhesion to anti-beta 3 mAb 7G2 and LIBS-1 induces tyrosine phosphorylation of beta 3 in Kalpha vbeta 3 cells (Fig. 6B). To further analyze 7G2 and LIBS-1, we employed adhesion assays to screen anti-beta 3 antibodies as possible ligand mimetics of Vn and Fn. Interestingly, we find that both Kalpha vbeta 3 and Kalpha vbeta 3Y747F,Y759F cells bind to LIBS-1 without the need for cellular PKC activation (Fig. 7, A and B). In contrast, Kalpha vbeta 3 cells need cellular PKC activation to establish strong adhesion to 7G2, and Kalpha vbeta 3Y747F,Y759F are unable to adhere to 7G2 even in the presence of cellular PKC activation (Fig. 7, A and B). PKC-induced adhesion of Kalpha vbeta 3 cells to 7G2 was inhibited by C3 exoenzyme (100 nM) and wortmannin (10 µM). As seen with adhesion of these cells to Vn, this suggests a role for Rho and PI3K in PMA-induced adhesion of Kalpha vbeta 3 cells to 7G2. In addition, adhesion of Kalpha vbeta 3 cells to LIBS-1 is also inhibited by C3 exoenzyme (100 nM) (Fig. 7A), but adhesion is unaffected by pretreatment with wortmannin (10 µM), suggesting, as seen with adhesion to Fn, that PI3K is not required for adhesion. Fig. 7B shows that Kalpha vbeta 3Y747F,Y759F cells are incapable of adhering to 7G2 even in the presence of PMA. Additionally, Kalpha vbeta 3Y747F,Y759F cells display inhibited adhesion to LIBS-1 in the presence of C3 exoenzyme (100 nM) but not wortmannin (10 µM), also suggesting a role for Rho, but not PI3K. Table I shows that both Kalpha vbeta 3 and Kalpha vbeta 3Y747F,Y759F cells express beta 3 equally, and both 7G2 and LIBS-1 bind equally in an RGD-dependent fashion, thus confirming that any lack of adhesion to Vn or 7G2 by Kalpha vbeta 3Y747F,Y759F cells is not due to a lack of expression of the mutant beta 3 integrin. Table I also shows that PI3K is uninvolved in the conformation-dependent recognition of beta 3 by either 7G2 or LIBS-1, as anti-beta 3 reactivity is unchanged in the presence of wortmannin. In this study we show that Rho is constitutively active on a Fn substrate but requires a cellular agonist such as PMA and active PI3K to become active on Vn. Rho is also found to be constitutively active in Kalpha vbeta 3 or Kalpha vbeta 3Y747F,Y759F cells adherent to LIBS-1 (Fig. 8). However, as seen with Vn, there is an absolute requirement for cellular PKC activation before Rho activation occurs in Kalpha vbeta 3 on 7G2, and no Rho activity was detected in Kalpha vbeta 3Y747F,Y759F cells even after PMA treatment. Taken together these data indicate that alpha vbeta 3-mediated adhesion to 7G2 and LIBS-1 mimic alpha vbeta 3-mediated adhesion to Vn and Fn, respectively. This fact will enable us to further decipher signaling events downstream of alpha vbeta 3 adhesion to specific ligands with no input from other integrins.


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Fig. 6.   Ligand and antibody induced beta 3-tyrosine phosphorylation. A, Vn and Fn induce beta 3-tyrosine phosphorylation (PY). Kalpha vbeta 3 or Kalpha vbeta 3Y747F,Y759F cells were either kept in suspension and incubated with PMA (10 ng/ml) or RGD (1 mM) peptide or allowed to adhere to Vn (1 µg/ml) or Fn (10 µg/ml). Cell lysates were immunoprecipitated with mAb 7G2 bound to Sepharose and Western-blotted with anti-phosphotyrosine 4G10. Total beta 3 was determined by Western blot of half of the beta 3 immunoprecipitation probed with mAb 7G2. WT, wild type. B, LIBS-1 and 7G2 binding to beta 3 induce tyrosine phosphorylation. Kalpha vbeta 3 and Kalpha vbeta 3Y747F,Y759F were either kept in suspension and incubated with PMA (10 ng/ml) or allowed to adhere to mAb 7G2 (7G2) or mAb LIBS-1 (LIBS-1), and cell lysates were immunoprecipitated with mAb 7G2 bound to Sepharose and Western-blotted with anti-phosphotyrosine 4G10. Total beta 3 was determined by Western blot of half of the beta 3 immunoprecipitation probed with mAb 7G2.


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Fig. 7.   Selective inhibition of alpha vbeta 3-mediated adhesion to anti-beta 3 antibodies. Microtiter plates (96 wells) were coated with mAb 7G2 (0.5 µg/ml) or mAb LIBS-1 (0.5 µg/ml). 105 Kalpha vbeta 3 (A) or Kalpha vbeta 3Y747F,Y759F (B) cells were allowed to adhere for 1 h at 37 °C in the absence or presence of PMA (10 ng/ml) and specific inhibitors of Rho (10 µg/ml C3 exoenzyme) or PI3K (10 µM wortmannin) and quantified as described under "Experimental Procedures." Data bars represent the mean absorbance ±S.D. from triplicate wells from three separate experiments.

                              
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Table I
PI3K inhibition does not affect beta 3 conformational change
Kalpha vbeta 3, Kalpha vbeta 3Y747F,Y759F, or untransfected K562 cells were incubated with either 7G2 (0.5 µg/ml) or LIBS-1 (0.5 µg/ml) in the absence (control) or presence of RGD (1 mM) or wortmannin (Wn) (10 µM). Antibody binding was quantified as described under "Experimental Procedures." Data are expressed as mean channel fluorescence for three separate experiments.


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Fig. 8.   Rho becomes activated on LIBS type anti-beta 3 antibodies. Kalpha vbeta 3 or Kalpha vbeta 3Y747F,Y759F were plated on 6-well tissue culture plates coated with either mAb 7G2 (0.5 µg/ml) or mAb LIBS-1 (0.5 µg/ml), treated with or without PMA (10 ng/ml), and allowed to adhere for 1 h at 37 °C. Cell lysates were incubated with GST-RBD immobilized on agarose beads. GTP-Rho was detected by Western blotting, and 1/10 of the total lysates were probed for Rho to demonstrate equal loading. WT, wild type.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The ability of integrins, including alpha vbeta 3, to adhere to multiple ECM proteins has prompted the use of the term "promiscuous." However, this term may be inaccurate because we and others show that alpha vbeta 3-mediated adhesion to distinct ligands is differentially regulated (4, 5). Hematopoietic cells and leukocytes in particular are able to regulate their adhesive properties, a requirement for appropriate vascular egress of leukocytes and subsequent extravascular immune surveillance (2). Integrin activation can be defined as the events necessary to permit firm adhesion, including a possible enhancement of integrin affinity, avidity, and integrin-cytoskeletal interactions. A requirement for beta 3-tyrosine phosphorylation during alpha vbeta 3 activation has been established; however, the mechanism and the circumstances under which this modification contributes to the overall activation state of alpha vbeta 3 have not been have not been completely established (5, 12, 20, 21).

In this study we show that alpha vbeta 3-mediated firm adhesion to distinct ECM ligands initiates unique signaling pathways and distinct cellular phenotypes. We show that alpha vbeta 3-mediated firm adhesion to Vn requires both cellular PKC activity and beta 3-tyrosine phosphorylation. Additionally, we show that firm adhesion to Vn also requires both PI3K and Syk activities. Furthermore, Rho activity is only up-regulated in cells adherent to Vn when there is coincident PKC and PI3K activities and tyrosine phosphorylation of beta 3. PI3K activity is up-regulated after adhesion to Vn, but not Fn, and PI3K translocation to alpha vbeta 3 complexes occurs only with tyrosine-phosphorylated beta 3. In contrast, alpha vbeta 3-mediated firm adhesion to Fn results in or in fact may be a result of constitutive Rho activation that requires no cellular PKC activation, beta 3-tyrosine phosphorylation, or PI3K activity. These data clearly outline distinct signaling pathways that are involved in alpha vbeta 3 activation by and subsequent adhesion to distinct ligands. Parallel results using ligand mimetic anti-beta 3 mAbs validate the single integrin-multiple pathway conclusion. The unique behavior of the anti-beta 3 mAb 7G2 indicates that the signaling pathway utilized by alpha vbeta 3 adhesion to Vn defines the leukocyte response integrin, a beta 3-related integrin of neutrophils originally characterized using this mAb. These mAb data support our hypothesis that beta 3-tyrosine phosphorylation and the mechanisms dependent upon this modification are likely to be a hematopoietic cell-specific event. If this conclusion is correct, a role exists for CD47 co-characterized with leukocyte response integrin in alpha vbeta 3 function that has not yet been determined. The role for CD47 in alpha vbeta 3-mediated adhesion to Vn is perhaps related to the recruitment of a greater complexity of signaling molecules than is required for alpha vbeta 3-mediated adhesion to Fn or its mimetic mAb LIBS-1.

The ECM is composed of multiple proteins that serve as ligands for integrins expressed on a variety of cells. The ability of specific integrins to adhere to distinct ligands in a regulated manner may be a fundamental part of the regulated adhesion required for hematopoietic cell function. Many reports have shown distinct phenotypes resulting from different integrins recognizing a single substrate (1). Here we show that distinct phenotypes can also result from a single integrin recognizing different substrates. This would allow hematopoietic cells to use distinct ligands for discrete steps in the processes of arresting from the vasculature, transendothelial migration, and migration within the ECM to the site of inflammation. Although alpha vbeta 3-mediated adhesion to Fn or Vn can both ultimately result in firm adhesion, the activation of distinct signaling pathways would allow for the initiation of additional cellular functions specific to an immediate cellular challenge such as transendothelial migration.

The ability of cells to firmly adhere to multiple ECM proteins requires regulated integrin ligation and initiation of specific subsequent signaling pathways that ultimately lead to an actin cytoskeletal reorganization that supports firm adhesion, and integrin activation is a critical step in this process. Integrin activation can be defined as the process by which integrin ligation leads to actin cytoskeletal reorganization via Rho activity and consequent firm adhesion. By this definition we can utilize active GTP-bound Rho as a biochemical marker of an activated integrin complex. Here we show that distinct ligands can initiate two modes of integrin activation that lead to Rho activation and firm adhesion employing different signaling components downstream of integrin ligation. Integrin activation on Vn is highly regulated and requires beta 3 tyrosine phosphorylation, cellular PKC activity, PI3K activity, and Syk activity, which ultimately lead to Rho activation and stress fiber formation. In contrast, adhesion to Fn does not require beta 3-tyrosine phosphorylation, cellular PKC activity, or PI3K activity; however, firm adhesion to both ligands requires Rho activity. Although the Syk inhibitor piceatannol blocks RGD-induced phosphorylation of beta 3, it is unlikely that this is the mechanism whereby piceatannol inhibits alpha vbeta 3-mediated adhesion to Fn, because this event is independent of beta 3-tyrosine phosphorylation and yet inhibited by piceatannol. In contrast to another report, we find that wortmannin has no effect on RGD-induced beta 3-tyrosine phosphorylation, and therefore, its inhibitory effects are likely due to inhibition of the increase in PI3K activity and/or the translocation of PI3K to the integrin complex (22). Furthermore, we do not find an effect of wortmannin on the RGD-induced conformational change of alpha vbeta 3 (23). This correlates with our previous reports showing the requirement for tyrosine phosphorylation of beta 3 for Vn adhesion, shown here to be PI3K-dependent, is unrelated to the conformation change in the receptor after ligand binding.

Although the need for ligand-dependent pathways of cytoskeletal reorganization remains to be determined, our data suggest that these signaling pathways converge at the level of a RhoGEF. One potential scenario involves PKC-dependent activation of Src causing Pyk2 activation and translocation to the integrin complex. This simplistic path provides neither a role for PI3K activity or its translocation to the integrin unless it functions to recruit the RhoGEF or Syk. It is also possible that PI3K is required for the activation of Src. The requirement of Syk activity in both pathways probably implicates Vav as the RhoGEF responsible for Rho activation. In cells adherent to Fn or LIBS-1, Rho activation may result from direct alpha vbeta 3 association with and auto-activation of Syk as previously described (24). This determination will require additional reagents specific for active Vav. Of equal interest is the ability of Fn ligation of alpha vbeta 3 to up-regulate Rho without the need for beta 3-tyrosine phosphorylation, PI3K activity, or PKC activity. It has been reported that beta 3-tyrosine phosphorylation can be a negative regulator of Fn adhesion; thus, it seems likely that the recruitment of a alpha vbeta 3 binding partner in a beta 3-tyrosine phosphorylation-dependent manner is responsible for the initiation of these distinct signaling pathways. The nature of the proximal binding partner is most likely that of an adapter molecule, although we cannot rule out that PI3K may bind directly to tyrosine-phosphorylated beta 3 integrins. Identification of this proximal binding partner will permit further discrimination of the signaling events initiated by alpha vbeta 3 after attachment to Vn or Fn. Furthermore, the Rho activation, firm adhesion, and stress fiber formation seen in Kalpha vbeta 3Y747F,Y759F cells adherent to fibronectin argues that these cytoplasmic tyrosine mutations selectively target signaling pathways and do not grossly disturb the functional structure of the receptor.

Our description of distinct signaling pathways resulting from different ligand recognition by the same integrin provides an additional level of ligand discrimination and integrin regulation. Whether this is unique to hematopoietic cell types remains to be determined; however, these cell types are the most in need of self-regulated adhesion. Related phenomena such as tumor metastasis may utilize similar signaling pathways. The characterization of ligand-mimetic mAb, which also initiate unique signaling, will advance future study of this integrin behavior in cell types with complex integrin expression patterns.

    FOOTNOTES

* This work was supported in part by grants from the American Heart Association New York State Affiliate and the Arthritis Foundation and NIAID, National Institutes of Health Grant 40602.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.

Dagger To whom correspondence should be addressed: Cell and Developmental Biology, SUNY Upstate Medical University, 750 East Adams St, Syracuse, NY. Tel.: 315-464-8512; Fax: 315-464-8535; E-mail: Blystons@mail.upstate.edu.

Published, JBC Papers in Press, November 22, 2002, DOI 10.1074/jbc.M206997200

    ABBREVIATIONS

The abbreviations used are: Vn, vitronectin; Fn, fibronectin; PKC, protein kinase C; PI3K, phosphoinositide 3-kinase; GST, glutathione S-transferase; mAb, monoclonal antibody; IMDM, Iscove's modified Dulbecco's medium; LIBS, ligand-induced binding site; PBS, phosphate-buffered saline; PMA, phorbol 12-myristate 13-acetate; RBD, RhoA binding domain.

    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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