Because the endogenously expressed beta 1 integrin very late antigen 5 is readily activated by PMA, we investigated the role of the.
AbstractThe ability of leukocytes to self-regulate adhesion during transendothelial and extravascular migration is fundamental tothe performance of immune surveillance in complex extracellular matrices. Leukocyte adhesion is regulated through the modulationof integrin receptors such as α vβ 3. In this study, we examined the activation of α vβ 3 resulting from attachment to vitronectin or fibronectin. In K562 cells stably expressing transfected α vβ 3, adhesion to vitronectin required tyrosine phosphorylation of the β 3subunit and activation of phosphoinositide 3-kinase and protein kinase C.
In contrast, adhesion to fibronectin proceeded withoutβ 3-tyrosine phosphorylation or the activities of phosphoinositide 3-kinase or protein kinase C. Firm adhesion to both ligandsand actin stress fiber formation required both Syk and Rho activity, suggesting that each ligand employs unique signalingpathways to achieve an active integrin complex, likely merging at a common RhoGEF such as Vav. Distinct signaling by a singleintegrin species interacting with different ligands permits initiation of additional cellular processes specific to the currenttask and provides an explanation for what has been described as promiscuous ligand specificity among integrins.Integrins are heterodimeric transmembrane receptors that mediate adhesion of cells to extracellular matrix proteins and toother cells. Consequently, integrins have a pivotal role in numerous developmental, physiological, and pathological processes.The integrin family contains at least 18 α and β subunits that combine to produce 24 known heterodimer combinations with distinctcell expression patterns and overlapping ligand specificities. Binding of ligand to the integrin extracellular domain induces a conformational change that is propagated to the cytoplasmicdomain and initiates downstream signaling events. 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 tothe site of inflammation. The ability of hematopoietic cells to regulate their adhesion-dependent extravasation is mediatedin part by the integrin α vβ 3.The α vβ 3 integrin plays a vital role in the adhesion of many cell types to the extracellular matrix and to other cells.
Although itis 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 (, ). However, promiscuous may be an unfortunate term because recent studies show that differential signaling by distinct α vβ 3 ligands may in fact result in unique cellular responses. We have previously shown that when α vβ 3 is expressed in the K562 cell line, these cells exhibit differential adhesion to Vn and Fn. Α vβ 3-mediated adhesion to Vn requires tyrosine phosphorylation at Tyr-747 within the β 3cytoplasmic tail and is dependent upon PKC activation, whereas α vβ 3-mediated adhesion to Fn is constitutive, requiring neither of these events (, ). In addition, α vβ 3-mediated adhesion of LNCaP cells to osteopontin, but not Vn, activates PI3K.
Cells and MaterialsK562 cells were stably transfected with cDNA encoding wild type (Kα vβ 3), Y747F,Y759F (Kα vβ 3Y747F,Y759F) β 3 or wild type (Kα Vβ 5) β 5 together with α V and maintained as previously described (, ). The anti-β 3 monoclonal antibodies 7G2 and LIBS-1 were gifts of Eric J.
Brown and Mark H. Ginsberg, respectively. Vitronectin and fibronectinwere prepared as previously described. Purified GST-FnIII-10 and III10/RGE (RGD mutated to RGE) were gifts of Denise C. 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 Cytometryβ 3 surface expression in stably transfected K562 cells was monitored using flow cytometry. Untransfected K562 cells or Kα vβ 3 or Kα vβ 3Y747F,Y759F cells were incubated with mAb anti-β 3 AP3 in IMDM for 1 h at 4 °C. Cells were then washed in IMDM and incubated with fluorescein isothiocyanate-labeled goat anti-mouseIgG for 30 min at 4 °C. To quantify binding site equality of β 3 by 7G2 and LIBS-1, Kα vβ 3 or Kα vβ 3Y747F,Y759F cells were stimulated with or without GRGDS peptide (1 m m) or wortmannin (10 μ m) in IMDM for 20 min at room temperature. Cells were washed in IMDM and incubated with either 7G2 or LIBS-1 anti-β 3 antibodies for 1 h at 4 °C.
Cells were washed in IMDM and incubated with fluorescein isothiocyanate-labeled goat anti-mouseIgG for 30 min at 4 °C. Flow cytometry was carried out using a Coulter Epics XL flow cytometer (Coulter, Miami, FL).
Dataare expressed as mean channel fluorescence for at least three separate experiments. Cell Adhesion Assays96-Well microtiter plates (Immulon II, Dynatech, Chantilly, VA) were coated with fibronectin (10 μg/ml), vitronectin (1 μg/ml),7G2 anti-β 3 (0.5 μg/ml), LIBS-1 anti-β 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% caseinin PBS for 30 min at room temperature. Kα vβ 3 or Kα vβ 3Y747F,Y759F cells were added at 1 × 10 5 cells/well in Hank's-buffered saline solution containing 1.0 m m Ca 2+ and Mg 2+ 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 μ m) and allowed to adhere for 1 h at 37 °C. Wells were rinsed 3 times in Hank's-buffered saline solution without Ca 2+ or Mg 2+, 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 roomtemperature. Crystal violet was dispersed in methanol and quantified by absorbance at 570 nm using an Emax microplate reader(Molecular Dynamics, Sunnyvale, CA).
Β 3 Tyrosine Phosphorylation6-well tissue culture treated plates were coated with Vn (1 μg/ml), Fn (10 μg/ml), anti-β 3 7G2 (0.5 μg/ml), or anti-β 3 LIBS-1 (0.5 μg/ml) in PBS overnight at 4 °C. Kα vβ 3 or Kα vβ 3Y747F,Y759F cells were plated at 2 × 10 6 cells/well in serum-free IMDM media containing 75 μ m sodium orthovanadate with or without PMA (10 ng/ml), bisindolylmaleimide (10 μ m), piceatannol (50 μg/ml), or wortmannin (10 μ m). The cells were lysed in PBS buffer containing 1% Nonidet P-40, sodium orthovanadate (1 m m), aprotinin (10 μg/ml), leupeptin (10 mg/ml), and phenylmethylsulfonyl fluoride (1 m m). 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-Sepharosebeads (ICN, Costa Mesa, CA) coated with anti-β 3 1A2 and anti-α V 3F12 monoclonal antibodies for 2 h at 4 °C. Samples were divided equally to determine β 3-tyrosine phosphorylation and total β 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 roomtemperature for 1 h. Membranes were then incubated with mAb 4G10 (Upstate Biotechnology) for phosphotyrosine or mAb 7G2 fortotal β 3. The membranes were then incubated with peroxidase-coupled goat anti-mouse IgG2b (Caltag Laboratories, Burlington, CA) forphosphotyrosine β 3 and peroxidase-coupled goat anti-mouse IgG (Sigma) for total β 3. Proteins were detected with enhanced chemiluminescence (ECL, Amersham Biosciences).
Fluorescent MicroscopyAcid-washed glass coverslips were coated overnight with Vn (1 μg/ml) or Fn (5 μg/ml). Kα vβ 3 or Kα vβ 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). Cellswere fixed in 3.7% formaldehyde, permeabilized in ice-cold PBS containing 0.01% Nonidet P-40, and stained with 0.7 μ m 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, SterlingHeights, MI). Rho Activity Assay6-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 (10μg/ml), 7G2 (0.5 μg/ml), or LIBS-1 (0.5 μg/ml), and Kα vβ 3 or Kα vβ 3Y747F,Y759F cells were plated at 2 × 10 6 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 μ m) and allowed to adhere for 1 h at 37 °C.
Rho activity was assayed as previously described. 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 m m), and sodium orthovanadate (1 m m). 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 domainof rhotekin (GST-RBD) (kindly provided by M. 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 determinedby loading 1/10 total volume of cell lysate on a 12% SDS-PAGE under reducing conditions. Proteins were transferred to polyvinylidenedifluoride membranes and blocked in TBST with 3% v/v bovine serum albumin at room temperature for 1 h. Membranes were thenincubated with murine mAb to RhoA (Cytoskeleton Inc., Denver, CO).
The membranes were then incubated with peroxidase-coupledanti-mouse IgG, and proteins were detected with ECL. AKT and Phospho-AKT Immunoblotting6-Well tissue culture plates (Costar, Corning, NY) were coated as above. Kα vβ 3 or Kα vβ 3Y747F,Y759F cells were plated at 2 × 10 6 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 lysisbuffer (PBS, 1% Nonidet P-40, 10 μg/ml each of leupeptin, and aprotinin, 1 m m 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 Ser 473 and total AKT and loaded onto 10% SDS-PAGE gels under reducing conditions. Proteins were transferred to polyvinylidene difluoridemembranes and blocked in TBST with 3% v/v bovine serum albumin at room temperature for 1 h. Membranes were incubated withsheep polyclonal antibodies to AKT (0.1 μg/ml) or to phospho-AKT Ser-473 (0.5 μg/ml) (Upstate Biotechnology) for 2 h at roomtemperature. The membranes were then incubated with peroxidase-coupled anti-sheep IgG, and proteins were detected with ECL. PI3K ImmunoprecipitationKα vβ 3and Kα vβ 3Y747F,Y759F (1 × 10 7) were treated with or without Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP) (1 m m) in the presence or absence of orthovanadate (0.75 μ m) for 20 min at room temperature to induce β 3-tyrosine phosphorylation. 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 m mphenylmethylsulfonyl 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-Sepharosebeads (ICN, Costa Mesa, CA) coated with anti-β 3 1A2 and anti-α V 3F12 monoclonal antibodies for 2 h at 4 °C. Samples were divided equally to determine PI3K and total β 3. Samples were separated on 7.5% SDS-PAGE gels and transferred to polyvinylidene difluoride membranes (Millipore). The membraneswere 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 β 3. The membranes were then incubated with peroxidase-coupled goat anti-mouse IgG2b (Caltag Laboratories, Burlington, CA) forphosphotyrosine β 3 and peroxidase-coupled goat anti-mouse IgG (Sigma) for total β 3.
Proteins were detected with enhanced chemiluminescence (ECL, Amersham Biosciences). Α vβ 3-mediated Firm Adhesion to Distinct Ligands and Actin Cytoskeletal ReorganizationTo investigate the influence of the extracellular ligand on α vβ 3-mediated adhesion and actin cytoskeletal organization, we examined the ability of K562 cells, expressing wild type (Kα vβ 3) or Y747F,Y759F (Kα vβ 3Y747F,Y759F) α vβ 3, to adhere to Vn or Fn in the presence or absence of the cellular agonist PMA. Kα vβ 3 cells adhere to Vn only after cellular activation by PMA (Fig.A); however, Kα vβ 3Y747F,Y759F cells are incapable of strong adhesion to Vn even after cellular activation. As we have previously reported, bothKα vβ 3 and Kα vβ 3Y747F,Y759F cells are capable of constitutive adhesion to Fn with or without cellular PKC activation by PMA (Fig.A). To determine whether the constitutive adhesion to Fn is α vβ 3-mediated, we exposed K562 cells stably expressing α Vβ 5(Kα Vβ 5) to Vn and Fn in the presence or absence of PMA. Kα Vβ 5 cells do not adhere to Fn even in the presence of PMA nor do untransfected K562 cells (data not shown), indicating that constitutiveadhesion of Kα vβ 3 and Kα vβ 3Y747F,Y759F cells to Fn is α vβ 3-mediated. Figure 1Effect of distinct extracellular ligands on α vβ 3-mediated adhesion and actin reorganization.
A, microtiter plates (96 wells) were coated with Vn (1 μg/ml) or Fn (10 μg/ml). 1 × 10 5 Kα vβ 3 or Kα vβ 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.
Kα vβ 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). Kα vβ 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 “ExperimentalProcedures.” The bar represents 5μm. Actin cytoskeletal reorganization plays a key role in the ability of cells to adhere to the extracellular matrix (, ). Therefore, we examined the structure of the actin cytoskeleton in Kα vβ 3 and Kα vβ 3Y747F,Y759F cells attached to Vn or Fn with or without cellular PKC activation. Both Kα vβ 3 and Kα vβ 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., F–I) within 1 h of attachment to Fn, and thus, actin fiber formation on Fn is independent of both β 3 tyrosine phosphorylation and PKC activation via PMA.
In contrast, actin fiber formation on Vn is dependent upon cellularPKC activation and β 3-tyrosine phosphorylation. No actin fibers are seen in Kα vβ 3 cells on Vn in the absence of PMA stimulation (Fig.B). Stress fibers can be seen in Kα vβ 3 cells after adhesion to Vn for 1 h only in the presence of PMA (Fig.C). In addition, β 3-tyrosine phosphorylation plays a pivotal role in firm adhesion to Vn, as no stress fiber formation occurs within Kα vβ 3Y747F,Y759F cells even after stimulation with PMA (Fig., D and E). Differential Inhibition of Adhesion to Distinct LigandsTo elucidate the possible signaling mechanisms underlying the differences in α vβ 3-mediated adhesion to Vn or Fn we utilized specific inhibitors to various signaling molecules. PMA-induced firm adhesion ofKα vβ 3 cells to Vn was inhibited by pretreatment with the Rho inhibitor C3 exoenzyme (100 n m), the PI3K inhibitor wortmannin (10 μ m), and the Syk inhibitor piceatannol (50 μg/ml). These results indicate that Rho, PI3K, and Syk are involved in PMA-inducedfirm adhesion to Vn (Fig.A).
Firm adhesion of Kα vβ 3 cells to Fn was also inhibited by C3 exoenzyme and piceatannol (FigA); however, no inhibition of firm adhesion to Fn was seen with wortmannin, suggesting that PI3K activity is not required forα vβ 3-mediated firm adhesion to Fn. Inhibition with C3 exoenzyme and piceatannol reduced adhesion to both Fn and Vn to levels comparablewith adhesion to casein (50 μg/ml) (data not shown). Cells expressing Kα vβ 3Y747F,Y759F are unable to adhere to Vn even after cellular PKC activation by PMA. Kα vβ 3Y747F,Y759F cells exhibit the same inhibition pattern as Kα vβ 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.B). These data suggest that α vβ 3-mediated adhesion to Fn, already shown to be independent of β 3-tyrosine phosphorylation, is dependent upon Rho and Syk activity but not PI3K activity. In contrast α vβ 3-mediated adhesion to Vn is dependent upon PI3K activity as well as Rho and Syk. The inhibitory effect of wortmannin on Vnis not due to a decrease in β 3-tyrosine phosphorylation, because we show that in response to RGD, β 3-tyrosine phosphorylation is inhibited fully in the presence of piceatannol but only minimally in the presence of wortmanninor the PKC inhibitor bisindolylmaleimide (Fig.).
Therefore, wortmannin inhibition of adhesion to Vn is not due to an inhibition of β 3-tyrosine phosphorylation, although piceatannol inhibition of adhesion to Vn may in part be due to inhibition of β 3-tyrosine phosphorylation. However, this may be unlikely because piceatannol also inhibits Kα vβ 3 and Kα vβ 3Y747F,Y759F adhesion to Fn, where β 3 phosphorylation is not required. Figure 2Selective inhibition of α vβ 3-mediated adhesion to distinct ligands. Microtiter plates (96 wells) were coated with Vn (1 μg/ml) or Fn (5 μg/ml). 10 5Kα vβ 3 ( A) or Kα vβ 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/mlC3 exoenzyme), PI3K (10 μ m 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. Ligand-specific Activation of RhoActin cytoskeletal reorganization into stress fibers has been shown to require Rho activity in many cell types. Here we demonstrate that α vβ 3-mediated adhesion to Fn as well as stress fiber formation requires neither PKC activation nor β 3-tyrosine phosphorylation.
To determine whether the constitutive firm adhesion to Fn is mediated by Rho activation we assayedRho activity using a GST-RBD pull-down assay for active GTP-bound Rho in cells that were allowed to adhere to Vn or Fn withor without PMA (Fig.A). Elevated Rho activity was seen in both Kα vβ 3 and Kα vβ 3Y747F,Y759F cells that were allowed to adhere to Fn for 1 h even in the absence of PMA stimulation. In contrast, adhesionto Vn did not lead to an increase in Rho activity in Kα vβ 3 cells unless there was coincident cellular PKC activation and only minor increases in Rho activity were seen in Kα vβ 3Y747F,Y759F under similar conditions. To confirm that the constitutive adhesion and Rho activation on Fn is α vβ 3-mediated we show that when Kα Vβ 5 cells are exposed to Fn there is no significant Rho activation even in the presence of PMA (Fig.A). Additionally, untransfected K562 cells exhibit no Rho activity on either Vn or Fn (data not shown). We also show that whenPI3K is inhibited by the presence of wortmannin, Rho activation is inhibited in Kα vβ 3 cells adherent to Vn but not on Fn (Fig.B).
Control addition of C3 exoenzyme inhibits Rho activity in cells adherent to either Vn or Fn. This suggests that when α vβ 3 is bound to Fn there is a direct activation of Rho that circumvents the need for PKC activity, β 3-tyrosine phosphorylation, and PI3K activity. These results support a hypothesis in which external stimulation, β 3-tyrosine phosphorylation, and PI3K are required for Rho activation and subsequent α vβ 3-mediated adhesion to Vn.
This supports our previous work suggesting that tyrosine-phosphorylated β 3 is required for the actions of PKC during α vβ 3-mediated adhesion to Vn. To determine whether sites other than the RGD motif of Fn are responsible for the constitutive adhesionof Kα vβ 3 and Kα vβ 3Y747F,Y759F cells we employed recombinant Fn fragment III-10 and assayed for Rho activity. We find activated Rho in Kα vβ 3 cells adhered to FnIII-10 regardless of PMA activation (Fig.C). However, when Kα Vβ 5 cells are adhered to FnIII-10 there is no Rho activity even in the presence of PMA.
In addition when Kα vβ 3 and Kα Vβ 5cells are allowed to adhere to FnIII-10RGE, in which the RGD motif has been changed to RGE, there is no constitutive Rho activity(Fig.C). These results indicate that it is the RGD motif of Fn that is required for constitutive α vβ 3-mediated adhesion to Fn. Figure 4Ligand-specific Rho activation.
A, Kα vβ 3, Kα vβ 3Y747F, Y759F, or Kα Vβ 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 wasdetected by Western blotting, and 1/10 of the total lysates were probed for Rho to demonstrate equal loading. WT, wild type. B, Kα vβ 3 cells were allowed to adhere to Vn or Fn in the presence or absence of PMA (10 ng/ml), wortmannin (10 μ m), or C3 exoenzyme (10 μg/ml). Cell lysates were incubated with GST-RBD immobilized on agarose beads.
GTP-Rho was detectedby Western blotting, and 1/10 of the total cell lysates were probed for Rho to demonstrate equal loading. C, Kα vβ 3 or Kα Vβ 5cells were adhered to Vn, Fn fragment III-10, or Fn fragment III-10RGE in the presence or absence of PMA (10 ng/ml) for 1h 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 LigandsIn this study we show that inhibition of PI3K activity by wortmannin inhibits both adhesion to Vn and Rho activation. To investigatethe role of PI3K in α vβ 3-mediated adhesion to Vn and Fn, we measured phosphorylated Ser-473 AKT as an indicator of PI3K activity and also the associationof PI3K with β 3. Kα vβ 3 and Kα vβ 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 Kα vβ 3 cells adherent to Vn (Fig.A), suggesting that PI3K is active in a ligand-dependant manner only on Vn. Having demonstrated that the inhibition of PI3Kby wortmannin does not inhibit β 3-tyrosine phosphorylation (Fig. ), we investigated the role of β 3-tyrosine phosphorylation in PI3K localization.
Kα vβ 3 or Kα vβ 3Y747F,Y759F were treated with RGD to induce β 3-tyrosine phosphorylation and Western blotted with anti-p85 after β 3 immunoprecipitations, demonstrating p85 association specifically with tyrosine-phosphorylated β 3 (Fig.B). Additionally, the removal of Na 3VO 4, which reduces β 3-tyrosine phosphorylation, partially blocked association of p85 with β 3. These results indicate that PI3K is activated in a α vβ 3-ligand-dependent manner and associates with α vβ 3 in a β 3-tyrosine phosphorylation-dependent manner.
Figure 5Distinct α vβ 3ligands differentially activate PI3K. A, PI3K is activated on Vn but not Fn. Kα vβ 3 or Kα vβ 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 andtotal AKT by Western blotting. WT, wild type.
B, the P85 subunit of PI3K is only associated with phosphorylated β 3. Kα vβ 3or Kα vβ 3Y747F,Y759F cells were incubated with or without RGD peptide (1 m m) and Na 3VO 4 (100 n m), and cell lysates were immunoprecipitated with mAb 7G2 bound to Sepharose.
Western blotting was performed using a p85 mAb.Total β 3 was determined by Western blot of half of the β 3immunoprecipitation ( IP) probed with mAb 7G2. Adhesion to Distinct anti-β 3 Antibodies Mimics Adhesion to Vn and FnVarious anti-β 3 antibodies have been developed that recognize specific epitopes within the extracellular domain of β 3, and some of the more useful anti-β 3 antibodies developed are the ligand-induced binding site (LIBS) antibodies. These LIBS antibodies recognize epitopes that are exposed within the integrin extracellular domain upon binding to an RGDsequence within an ECM protein such as Vn or Fn. Using this we have explored the possibility that α vβ 3 may in fact exhibit adhesive properties to different anti-β 3 antibodies that mimic adhesion to distinct ECM proteins.
This would enable us to eliminate any possible contributions ofother integrins expressed within our K562 system. Using multiple assays we determined that 7G2 and LIBS-1, surprisingly bothLIBS type antibodies, mimic α vβ 3-mediated adhesion to Vn and Fn, respectively. As seen with adhesion to Vn or Fn (Fig.A), adhesion to anti-β 3 mAb 7G2 and LIBS-1 induces tyrosine phosphorylation of β 3 in Kα vβ 3 cells (Fig.B).
To further analyze 7G2 and LIBS-1, we employed adhesion assays to screen anti-β 3 antibodies as possible ligand mimetics of Vn and Fn. Interestingly, we find that both Kα vβ 3and Kα vβ 3Y747F,Y759F cells bind to LIBS-1 without the need for cellular PKC activation (Fig., A and B).
In contrast, Kα vβ 3 cells need cellular PKC activation to establish strong adhesion to 7G2, and Kα vβ 3Y747F,Y759F are unable to adhere to 7G2 even in the presence of cellular PKC activation (Fig., A and B). PKC-induced adhesion of Kα vβ 3 cells to 7G2 was inhibited by C3 exoenzyme (100 n m) and wortmannin (10 μ m). As seen with adhesion of these cells to Vn, this suggests a role for Rho and PI3K in PMA-induced adhesion of Kα vβ 3cells to 7G2. In addition, adhesion of Kα vβ 3 cells to LIBS-1 is also inhibited by C3 exoenzyme (100 n m) (Fig.A), but adhesion is unaffected by pretreatment with wortmannin (10 μ m), suggesting, as seen with adhesion to Fn, that PI3K is not required for adhesion. Fig.B shows that Kα vβ 3Y747F,Y759F cells are incapable of adhering to 7G2 even in the presence of PMA. Additionally, Kα vβ 3Y747F,Y759F cells display inhibited adhesion to LIBS-1 in the presence of C3 exoenzyme (100 n m) but not wortmannin (10 μ m), also suggesting a role for Rho, but not PI3K. Table shows that both Kα vβ 3 and Kα vβ 3Y747F,Y759F cells express β 3 equally, and both 7G2 and LIBS-1 bind equally in an RGD-dependent fashion, thus confirming that any lack of adhesion to Vnor 7G2 by Kα vβ 3Y747F,Y759F cells is not due to a lack of expression of the mutant β 3integrin.
Table also shows that PI3K is uninvolved in the conformation-dependent recognition of β 3 by either 7G2 or LIBS-1, as anti-β 3 reactivity is unchanged in the presence of wortmannin. In this study we show that Rho is constitutively active on a Fn substratebut requires a cellular agonist such as PMA and active PI3K to become active on Vn. Rho is also found to be constitutivelyactive in Kα vβ 3 or Kα vβ 3Y747F,Y759F cells adherent to LIBS-1 (Fig. However, as seen with Vn, there is an absolute requirement for cellular PKC activation before Rho activation occurs inKα vβ 3 on 7G2, and no Rho activity was detected in Kα vβ 3Y747F,Y759F cells even after PMA treatment.
Taken together these data indicate that α vβ 3-mediated adhesion to 7G2 and LIBS-1 mimic α vβ 3-mediated adhesion to Vn and Fn, respectively. This fact will enable us to further decipher signaling events downstream ofα vβ 3 adhesion to specific ligands with no input from other integrins. Figure 6Ligand and antibody induced β 3-tyrosine phosphorylation. A, Vn and Fn induce β 3-tyrosine phosphorylation ( PY).
Kα vβ 3 or Kα vβ 3Y747F,Y759F cells were either kept in suspension and incubated with PMA (10 ng/ml) or RGD (1 m m) peptide or allowed to adhere to Vn (1 μg/ml) or Fn (10 μg/ml). Cell lysates were immunoprecipitated with mAb 7G2 bound toSepharose and Western-blotted with anti-phosphotyrosine 4G10. Total β 3was determined by Western blot of half of the β 3immunoprecipitation probed with mAb 7G2. WT, wild type. B, LIBS-1 and 7G2 binding to β 3 induce tyrosine phosphorylation. Kα vβ 3 and Kα vβ 3Y747F,Y759F were either kept in suspension and incubated with PMA (10 ng/ml) or allowed to adhere to mAb 7G2 (7G2) or mAbLIBS-1 (LIBS-1), and cell lysates were immunoprecipitated with mAb 7G2 bound to Sepharose and Western-blotted with anti-phosphotyrosine4G10.
Total β 3 was determined by Western blot of half of the β 3 immunoprecipitation probed with mAb 7G2. Figure 7Selective inhibition of α vβ 3-mediated adhesion to anti-β 3 antibodies. Microtiter plates (96 wells) were coated with mAb 7G2 (0.5 μg/ml) or mAb LIBS-1 (0.5 μg/ml). 10 5 Kα vβ 3( A) or Kα vβ 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 μ m wortmannin) and quantified as described under “Experimental Procedures.” Data bars represent the mean absorbance ±S.D. From triplicate wells from three separate experiments. DISCUSSIONThe ability of integrins, including α vβ 3, to adhere to multiple ECM proteins has prompted the use of the term “promiscuous.” However, this term may be inaccuratebecause we and others show that α vβ 3-mediated adhesion to distinct ligands is differentially regulated (, ). Hematopoietic cells and leukocytes in particular are able to regulate their adhesive properties, a requirement for appropriatevascular egress of leukocytes and subsequent extravascular immune surveillance.
Integrin activation can be defined as the events necessary to permit firm adhesion, including a possible enhancement ofintegrin affinity, avidity, and integrin-cytoskeletal interactions. A requirement for β 3-tyrosine phosphorylation during α vβ 3 activation has been established; however, the mechanism and the circumstances under which this modification contributes tothe overall activation state of α vβ 3 have not been have not been completely established (, ).In this study we show that α vβ 3-mediated firm adhesion to distinct ECM ligands initiates unique signaling pathways and distinct cellular phenotypes. We showthat α vβ 3-mediated firm adhesion to Vn requires both cellular PKC activity and β 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 phosphorylationof β 3.
PI3K activity is up-regulated after adhesion to Vn, but not Fn, and PI3K translocation to α vβ 3 complexes occurs only with tyrosine-phosphorylated β 3. In contrast, α vβ 3-mediated firm adhesion to Fn results in or in fact may be a result of constitutive Rho activation that requires no cellularPKC activation, β 3-tyrosine phosphorylation, or PI3K activity. These data clearly outline distinct signaling pathways that are involved in α vβ 3 activation by and subsequent adhesion to distinct ligands. Parallel results using ligand mimetic anti-β 3 mAbs validate the single integrin-multiple pathway conclusion. The unique behavior of the anti-β 3mAb 7G2 indicates that the signaling pathway utilized by α vβ 3 adhesion to Vn defines the leukocyte response integrin, a β 3-related integrin of neutrophils originally characterized using this mAb.
These mAb data support our hypothesis that β 3-tyrosine phosphorylation and the mechanisms dependent upon this modification are likely to be a hematopoietic cell-specificevent. If this conclusion is correct, a role exists for CD47 co-characterized with leukocyte response integrin in α vβ 3 function that has not yet been determined.
The role for CD47 in α vβ 3-mediated adhesion to Vn is perhaps related to the recruitment of a greater complexity of signaling molecules than is requiredfor α vβ 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 abilityof specific integrins to adhere to distinct ligands in a regulated manner may be a fundamental part of the regulated adhesionrequired for hematopoietic cell function.
Many reports have shown distinct phenotypes resulting from different integrins recognizinga single substrate. Here we show that distinct phenotypes can also result from a single integrin recognizing different substrates. This wouldallow hematopoietic cells to use distinct ligands for discrete steps in the processes of arresting from the vasculature, transendothelialmigration, and migration within the ECM to the site of inflammation. Although α vβ 3-mediated adhesion to Fn or Vn can both ultimately result in firm adhesion, the activation of distinct signaling pathwayswould allow for the initiation of additional cellular functions specific to an immediate cellular challenge such as transendothelialmigration.The ability of cells to firmly adhere to multiple ECM proteins requires regulated integrin ligation and initiation of specificsubsequent signaling pathways that ultimately lead to an actin cytoskeletal reorganization that supports firm adhesion, andintegrin activation is a critical step in this process. Integrin activation can be defined as the process by which integrinligation leads to actin cytoskeletal reorganization via Rho activity and consequent firm adhesion. By this definition we canutilize active GTP-bound Rho as a biochemical marker of an activated integrin complex. Here we show that distinct ligandscan initiate two modes of integrin activation that lead to Rho activation and firm adhesion employing different signalingcomponents downstream of integrin ligation.
Integrin activation on Vn is highly regulated and requires β 3 tyrosine phosphorylation, cellular PKC activity, PI3K activity, and Syk activity, which ultimately lead to Rho activationand stress fiber formation. In contrast, adhesion to Fn does not require β 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 β 3, it is unlikely that this is the mechanism whereby piceatannol inhibits α vβ 3-mediated adhesion to Fn, because this event is independent of β 3-tyrosine phosphorylation and yet inhibited by piceatannol. In contrast to another report, we find that wortmannin has noeffect on RGD-induced β 3-tyrosine phosphorylation, and therefore, its inhibitory effects are likely due to inhibition of the increase in PI3K activityand/or the translocation of PI3K to the integrin complex.
Furthermore, we do not find an effect of wortmannin on the RGD-induced conformational change of α vβ 3. This correlates with our previous reports showing the requirement for tyrosine phosphorylation of β 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 suggestthat these signaling pathways converge at the level of a RhoGEF. One potential scenario involves PKC-dependent activationof Src causing Pyk2 activation and translocation to the integrin complex. This simplistic path provides neither a role forPI3K activity or its translocation to the integrin unless it functions to recruit the RhoGEF or Syk.
It is also possible thatPI3K is required for the activation of Src. The requirement of Syk activity in both pathways probably implicates Vav as theRhoGEF responsible for Rho activation. In cells adherent to Fn or LIBS-1, Rho activation may result from direct α vβ 3 association with and auto-activation of Syk as previously described. This determination will require additional reagents specific for active Vav. Of equal interest is the ability of Fn ligationof α vβ 3 to up-regulate Rho without the need for β 3-tyrosine phosphorylation, PI3K activity, or PKC activity.
It has been reported that β 3-tyrosine phosphorylation can be a negative regulator of Fn adhesion; thus, it seems likely that the recruitment of a α vβ 3 binding partner in a β 3-tyrosine phosphorylation-dependent manner is responsible for the initiation of these distinct signaling pathways. The natureof the proximal binding partner is most likely that of an adapter molecule, although we cannot rule out that PI3K may binddirectly to tyrosine-phosphorylated β 3 integrins. Identification of this proximal binding partner will permit further discrimination of the signaling events initiatedby α vβ 3 after attachment to Vn or Fn.
Furthermore, the Rho activation, firm adhesion, and stress fiber formation seen in Kα vβ 3Y747F,Y759F cells adherent to fibronectin argues that these cytoplasmic tyrosine mutations selectively target signaling pathwaysand 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 anadditional level of ligand discrimination and integrin regulation. Whether this is unique to hematopoietic cell types remainsto be determined; however, these cell types are the most in need of self-regulated adhesion.
Related phenomena such as tumormetastasis may utilize similar signaling pathways. The characterization of ligand-mimetic mAb, which also initiate uniquesignaling, 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 ArthritisFoundation and NIAID, National Institutes of Health Grant 40602.The costs of publication of this article were defrayed inpart 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.‡ To whom correspondence should be addressed: Cell and Developmental Biology, SUNY Upstate Medical University, 750 East AdamsSt, Syracuse, NY. Tel.: 315-464-8512; Fax: 315-464-8535; E-mail: [email protected], JBC Papers in Press, November 22, 2002, DOI 10.1074/jbc.M206997200.
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