De Novo Expression of the Integrin {alpha}5{beta}1 Regulates {alpha}v{beta}3-mediated Adhesion and Migration on Fibrinogen*

Daphne P. Ly, Kathleen M. Zazzali and Siobhan A. Corbett {ddagger} §

From the {ddagger} Department of Surgery, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, New Brunswick, New Jersey 08903

Received for publication, December 9, 2002 , and in revised form, April 2, 2003.
    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Recent evidence demonstrates that interactions between different integrins that are present on the cell surface can strongly influence the adhesive function of individual receptors. In this report, we show that Chinese hamster ovary cells that express the integrin {alpha}v{beta}3 in the absence of {alpha}5{beta}1 demonstrate increased adhesion and migration on fibrinogen. Furthermore, {alpha}v{beta}3-mediated adhesion to fibrinogen is not augmented by the soluble agonist, MnCl2, suggesting that {alpha}v{beta}3 exists in a higher affinity state in these cells. De novo expression of wild-type {alpha}5{beta}1 negatively regulates {alpha}v{beta}3-mediated adhesion and migration. This effect is not seen with expression of a chimeric {alpha}5{beta}1 integrin in which the cytoplasmic portion of the {alpha}5 integrin subunit is replaced by the cytoplasmic portion of the {alpha}4 integrin. In addition, it does not require ligation of {alpha}5{beta}1 by fibronectin. Cells that express a constitutively active {beta}3 integrin that contains a point mutation in the conserved membrane proximal region of the cytoplasmic tail, D723R, are resistant to the effect of {alpha}5{beta}1 expression. These data provide additional evidence of "cross-talk" between the integrins {alpha}5{beta}1 and {alpha}v{beta}3, and support the idea that {alpha}5{beta}1 regulates {alpha}v{beta}3-mediated ligand binding. This provides a relevant biological mechanism whereby variations in {alpha}5{beta}1 expression in vivo may modulate activation of {alpha}v{beta}3 to influence its adhesive function.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Integrins are transmembrane glycoproteins that are the principle mediators of cell interactions with the extracellular matrix (ECM)1 (13). They are composed of noncovalently associated {alpha} and {beta} subunits, which together determine the ligand-binding specificity of the receptor. Integrin-ECM interactions are essential for a diverse variety of important biological processes, including embryogenesis, cell survival, and wound healing (46). During wound healing, alterations in integrin expression coincide with deposition of a newly formed provisional ECM whose primary structural components include fibrinogen (FBG) and fibronectin (FN) (5, 7).

Members of the {beta}3 integrin family are the primary receptors that mediate cell interactions with FBG (810). In platelets, the interaction of {alpha}IIb{beta}3 with FBG is dependent upon agonist-stimulated signaling events that alter the ligand-binding function of the {alpha}IIb{beta}3 receptor, a process termed "integrin activation" (11). The adhesive function of {alpha}v{beta}3 also seems to be regulated by intracellular events as recent studies indicate that the basal affinity of this receptor varies both by intracellular location and among cell types (1214). Activation of {alpha}v{beta}3 results in different functional states of the receptor that influence important integrin-dependent events, including cell migration, angiogenesis, and metastatic activity (14, 15). Although rapid regulation of {alpha}v{beta}3 function may represent a common mechanism for the modulation of cell-ECM interactions, little is known about the molecular events that affect receptor affinity.

Recent evidence demonstrates that interactions between different integrins that are present on the cell surface can strongly influence the adhesive function of individual receptors. This effect, referred to as integrin "cross-talk," has been demonstrated in a number of systems (16). For example, ligation of {alpha}4{beta}1 inhibits {alpha}5{beta}1-dependent expression of metalloproteases (17). Interactions between {beta}3 integrins and {alpha}5{beta}1 have also been described. Ligation of {alpha}IIb{beta}3 by an integrin-specific FBG ligand suppresses the adhesive function of both {alpha}5{beta}1 and {alpha}2{beta}1 (16). Similarly, anti-{alpha}v{beta}3 antibodies block both {alpha}5{beta}1-mediated phagocytosis of FN-coated beads and {alpha}5{beta}1-mediated migration toward FN (18). The reverse effect also appears to be true as anti-{alpha}5{beta}1 antibodies inhibit {alpha}v{beta}3-mediated cell migration, without influencing cell adhesion (19). Interestingly, both integrins are up-regulated on migrating cells post-injury where they are important receptors for provisional ECM proteins (5, 20, 21).

In this paper, we present further evidence of cross-talk between the integrins {alpha}5{beta}1 and {alpha}v{beta}3. We report that in the absence of {alpha}5{beta}1, {alpha}v{beta}3 exists in an "activated" state demonstrating high affinity for FBG that is not augmented by soluble agonists. De novo expression of {alpha}5{beta}1, however, suppresses {alpha}v{beta}3-mediated adhesive functions. The {alpha}5{beta}1-mediated modulation of {alpha}v{beta}3 occurs through a mechanism that is dependent on the cytoplasmic tail of the {alpha} integrin subunit. Furthermore, cells that express a constitutively active {beta}3 integrin are resistant to the effects of {alpha}5{beta}1, supporting evidence that the expression of {alpha}5{beta}1 regulates {alpha}v{beta}3 affinity for its ligands. These data provide a relevant biological mechanism whereby variations in {alpha}5{beta}1 expression in vivo may modulate activation of {alpha}v{beta}3 to influence its adhesive function.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Construction of Chimeric Integrin cDNA—A 1.8-kb BamHI-XhoI fragment encoding the amino-terminal portion of the human {alpha}5 cDNA in pLJ (a gift of Dr. Jean Schwarzbauer, Princeton University, Princeton, NJ) was cloned into pcDNA 3.1(+) (pcDNA 3.1 {alpha}5-N, Invitrogen). A 2.5-kb fragment containing the carboxyl terminus of the human {alpha}5 cDNA was then ligated into a XhoI digest of pcDNA 3.1 {alpha}5-N to yield a complete human {alpha}5 cDNA in pcDNA 3.1 (X5C5).

The X4C4 cDNA, encoding the wild-type human {alpha}4 integrin subunit (a gift of Dr. Patricia Keely, University of Wisconsin Medical School, Madison WI), was used to create the chimeric X5C4 cDNA. Briefly, a 1.3-kb KpnI-XbaI fragment of X4C4, containing the cytoplasmic portion of the receptor was cloned into pcDNA 3.1 to generate pcDNA 3.1 C4. A 3.1-kb HindIII digest of X5C5 was used to generate the amino-terminal portion of {alpha}5 that encoded the extracellular and transmembrane regions of the receptor. This HindIII fragment was cloned into a HindIII-digested pcDNA 3.1 C4 to yield X5C4. The cytoplasmic portions of X5C5 and X5C4 were sequenced at the Robert Wood Johnson Medical School DNA Core Facility to confirm the sequence of each chimeric construct.

Cells, Cell Culture, and Transfection—The {alpha}5-negative, {beta}3-negative Chinese hamster ovary (CHO) B2 cells were a kind gift of Dr. Jean Schwarzbauer. CHO B2 {alpha}v{beta}3 (D723R) cells were a kind gift of Dr. Mark Ginsberg (Scripps Research Institute, La Jolla, CA) and were designated CHO B3(D-R). CHO B2 cells were transfected with a wild-type human {beta}3 cDNA by electroporation. Stable transfectants, designated CHO B3, were established by selection in 500 µg/ml Zeocin (Invitrogen) and were analyzed for {alpha}v{beta}3 expression as described below. CHO B3 cells were then transfected with either the X5C5 cDNA (CHO B3C5) or the chimeric X5C4 cDNA (CHO B3C4)), and selected in Zeocin and 200 µg/ml Geneticin (G418, Invitrogen). CHO B3(D-R) cells were transfected with the X5C5 cDNA (CHO B3(D-R)C5). Sterile fluorescence-activated cell sorting (FACS), performed on an EPIC Altra high speed cell sorter (Beckman-Spinco, Miami, FL), was used to establish cell populations that expressed similar levels of {alpha}v{beta}3 and {alpha}5{beta}1. CHO X5C5 ({alpha}v{beta}3–/{alpha}5{beta}1+) and X5C4 cells ({alpha}v{beta}3–/{alpha}5mut{beta}1+) were established by transfection of CHO B2 cells with the X5C5 or X5C4 cDNAs, respectively and selection as described above. Cell surface expression of both {alpha}v{beta}3 and {alpha}5{beta}1 were assessed and monitored by FACS weekly to ensure stable integrin expression. All cell lines were maintained in Dulbecco's modified Eagle's medium (Invitrogen) containing 10% fetal calf serum (HyClone Laboratories, Logan, UT), 2 mM glutamine (Invitrogen), 1% non-essential amino acids (Invitrogen), 1% sodium pyruvate (Invitrogen), 1% antibiotics/antimycotics (Invitrogen), and Zeocin or G418 as indicated.

Chimeric Integrin Expression—For additional verification of the wild-type and mutant {alpha}5 integrins, cells were grown in 10-cm tissue culture plates until confluent. Cells were washed once with ice-cold PBS and lysed. Equal amounts of total cell lysate were incubated with 2 µg of a monoclonal antibody specific for the {alpha}5 integrin extracellular domain (Chemicon International Co., Temecula, CA) at 4 °C overnight. Immunocomplexes were recovered by incubating with Protein A-Protein G-Sepharose (Pierce) for 2 h. The immunoprecipitates were analyzed by SDS-PAGE and transferred to nitrocellulose (Fisher Scientific). The membranes were blocked with 5% nonfat dry milk at 4 °C overnight. Immunodetection was performed using a polyclonal antibody specific for the {alpha}5 integrin extracellular domain (Chemicon) or monoclonal antibodies specific for the {alpha}5 integrin cytoplasmic domain or the {alpha}4 integrin cytoplasmic domain (Chemicon and Santa Cruz Biotechnology, respectively) followed by horseradish peroxidase-conjugated goat anti-rabbit ({alpha}5) or bovine anti-goat ({alpha}4) secondary antibody (Pierce). The specific proteins were detected with enhanced chemiluminescence (ECL, Pierce) according to the manufacturer's instructions and exposed to film (X-Omat, Eastman Kodak).

Determination of Cell Surface Integrin Expression—Cells were trypsinized, washed with incubation buffer (1x PBS and 2% fetal calf serum) and resuspended at a concentration of 1 x 107 cells/ml in incubation buffer. Cells were incubated on ice with monoclonal antibody specific for either {alpha}v{beta}3 (LM609; 1 µg/1 x 106 cells; Chemicon) or {alpha}5{beta}1 (VC5; 1 µg/1 x 106 cells; BD Pharmingen) for 30 min, washed once, and then incubated on ice with an Alexa Fluor 488 goat anti-mouse IgG (Molecular Probes, Eugene, OR). Following additional washes, cells were pelleted, resuspended in incubation buffer, and analyzed by FACS on the EPIC Altra cell sorter.

Preparation of FN-depleted FBG—Human FBG (Calbiochem/Novabiochem, San Diego, CA) was brought into solution at a concentration of 100 mg/15 ml according to the manufacturer's instructions. FN was depleted using gelatin-Sepharose beads (Amersham Biosciences). Briefly, 7.5 ml of FBG solution was combined with 2 ml of packed gelatin-Sepharose beads. The mixture was rotated at room temperature for 1 h. After pelleting the beads, the supernatant was transferred to a second gelatin-Sepharose containing tube and the process was repeated. Beads were then pelleted, and the supernatant containing FN-depleted FBG was collected. Protein concentration was determined using BCA protein assay reagent (Pierce) and adjusted to 5–6 mg/ml. FN-depleted FBG was then aliquoted and stored at –80 °C. FBG was analyzed by SDS-polyacrylamide gel electrophoresis followed by Coomassie stain to assess FN depletion.

Cell Adhesion Assays—96-Well dishes were coated overnight at 4 °C with increasing concentrations of FBG, thrombospondin (TSP), or FN as indicated. Control wells were incubated with PBS. For each experiment, wells were formed in triplicate for each condition. Cell adhesion assay was performed as previously described (8). Briefly, 8 x 104 cells were added to each well, and incubated at 37 °C for 1 h. Adherent cells were fixed and stained for 25 min in a mixture of 2% EtOH, 100 mM sodium borate, and 0.5% crystal violet. Dye was then eluted with 10% acetic acid. The optical density (OD) at 570 nm was measured for each well using a 96-well microtiter plate reader (Bio-Tek Instruments, Winooski, VT). To eliminate the contribution of cell spreading to adhesion, short time course experiments were also performed. In these experiments, 24-well dishes were coated with either 50 µg/ml FBG or 10 µg/ml TSP. 2 x 105 cells were added to each well and allowed to adhere for 15 min. The wells were then washed gently to remove non-adherent cells. Adherent cells were then fixed, stained, and counted as described below. Each well was formed in duplicate. In certain experiments, cell adhesion was performed in the presence of function blocking antibodies to either {alpha}v{beta}3 (LM609) or {alpha}5{beta}1 (P1D6; 25 µg/ml; Oncogene Research Products, La Jolla, CA). For integrin activation assays, cell adhesion was performed in the presence or absence of either 100 µM MnCl2 or 200 nm PMA. To quantify cell adhesion, cells were fixed with methanol for 15 min, and stained with modified Giemsa stain (Sigma) for1hat room temperature. Cells from 3 random high power fields were counted by light microscopy and averaged. For each experiment, the mean of the samples was obtained and normalized relative to the adhesion of B3 cell. The data are expressed as the mean ± S.E. of three separate experiments. A one-way analysis of variance followed by Newman-Keul's test was used for statistical analysis of the data.

Cell Spreading Assays—24-Well tissue culture dishes were coated overnight with 50 µg/ml FBG at 4 °C. Cells in serum-free medium were placed in FBG-coated wells (2.5 x 105 cells/well) and incubated for increasing time as indicated. At each time point, cells were fixed with methanol and stained with modified Giemsa. Stained cells were viewed under high power using inverted bright-field optics. Photographic images were captured using a Spot color camera (Diagnostic Instruments, Sterling Heights, MI) connected to a MacIntosh G4 computer equipped with IPLab image analysis software.

Cell Migration Assays—Costar transwells (8.0 µm pores, 6.5 mm diameter (Fisher Scientific)) were coated on both sides or on the under-surface only with 50 µg/ml FBG or 10 µg/ml FN overnight at 4 °C, washed with PBS, and blocked with 1% bovine serum albumin prior to usage. 2.5 x 105 cells in serum-free medium with 1% bovine serum albumin were placed into the upper chamber of coated transwells and incubated under tissue culture conditions for 5 h. Non-migrated cells were removed by wiping the upper side of the membrane with a cotton swab. The transwells were washed 3 times with PBS, fixed with methanol, and stained with modified Giemsa. The transwells were then rinsed with Milli-Q water. The stained cells in three random high powered fields were counted by light microscopy. Each experiment was repeated three times. Data are expressed either as the mean cell count ± S.E. or as the percent of cells migrated relative to B3 cells. Statistical analysis of the data was performed using one-way analysis of variance followed by Newman-Keul's test.

Clot Retraction Assays—12-Well non-tissue culture dishes were blocked with 1% bovine serum albumin at 4 °C overnight. Wells were washed 3 times in PBS the next day and left to dry. Cells were detached as described above and resuspended in 25 mM Hepes-buffered saline. For integrin activation assays, cells were pretreated with 100 µM MnCl2 for 15 min. To prepare fibrin clots for retraction assays, the clotting components as listed were mixed in a volume of 1.5 ml at room temperature: FN-depleted FBG (720 µg/ml), 1 mM CaCl2, 20 µg/ml aprotinin (Sigma), 2 µg/ml human plasma coagulation factor XIII (Calbiochem), 25 mM Hepes saline, ±MnCl2, 1.0 units/ml thrombin (Sigma). Cells (3 x 106 cells/clot) were added to the clot components followed quickly by thrombin. After the addition of thrombin, the mixture was rapidly pipetted into 24-well tissue culture dishes and incubated at 37 °Cfor1hina tissue culture incubator. Serum-free medium was then added to the wells and the clots were carefully detached from the dishes using a dissecting microscope. For integrin activation assays, clot retraction was performed in the presence of 100 µM MnCl2. Clot diameter was measured under a dissecting microscope at T0 and at increasing times as indicated. Clot retraction was calculated by subtracting the diameter recorded at each time point from the starting diameter (T0). The data is the mean of six separate experiments and is expressed as clot retraction (mm) ± S.E. Statistical analysis of the data was performed using two-way analysis of variance followed by Newman-Keul's test.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Determination of Integrin Expression in CHO B2-transfected Cells—The CHO B2 cell line (22), which does not express endogenous hamster {alpha}5{beta}1, was transfected with a cDNA encoding the human {beta}3 integrin subunit. Stable transfectants expressing {alpha}v{beta}3 were selected (CHO B3) and surface expression of {alpha}v{beta}3 was determined by flow cytometry using a monoclonal antibody specific for {alpha}v{beta}3. B3 cells were then transfected with either a cDNA for the wild-type human {alpha}5 integrin subunit (X5C5) or a mutant {alpha}5 cDNA in which the cytoplasmic domain of the {alpha}5 integrin was replaced by the cytoplasmic domain of the {alpha}4 integrin (X5C4). Stable cell populations expressing {alpha}v{beta}3 and either X5C5 or X5C4 were established by FACS using an anti-{alpha}5 monoclonal antibody to establish the B3C5 and B3C4 cell lines, respectively. Fig. 1A demonstrates that B3 and B3C5 cells express similar levels of {alpha}v{beta}3. B3C5 cells, but not B3 cells, express {alpha}5{beta}1. B3C5 and B3C4 cells expressed similar levels of both {alpha}5{beta}1 and {alpha}v{beta}3 (Fig. 1A). Immunoblot analysis of B3C5 and B3C4 cell lysates with antibodies specific to either the {alpha}5 or {alpha}4 cytoplasmic tails confirm the expression of the chimeric receptor (Fig. 1B).



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FIG. 1.
A, determination of integrin expression in transfected CHO B2 cells. CHO B3, B3C5, B3C4, B3(D-R), and B3(D-R)C5 cells were incubated with primary antibodies specific for {alpha}v{beta}3 (LM609) or {alpha}5{beta}1 (VC5) and antibody binding was detected with fluorescein isothiocyanate-labeled secondary antibody as described under "Experimental Procedures." Cells stained with secondary antibody only were used as a negative control. In B, B3C5 (1) and B3C4 (2) cells attached to tissue culture plates were lysed and equal amounts of total cellular protein were incubated with a monoclonal antibody specific for the extracellular domain of the {alpha}5 integrin subunit. Immunocomplexes were recovered and analyzed by SDS-PAGE followed by immunoblotting with antibodies specific for the extracellular domain of {alpha}5, the cytoplasmic domains of {alpha}5 or of {alpha}4 as indicated. These experiments were repeated twice with similar results. IP, immunoprecipitated.

 

{alpha}5{beta}1 Regulates {alpha}v{beta}3-mediated Adhesive Events—FBG, a key component of the provisional ECM that is deposited in early wounds, is a ligand for {alpha}v{beta}3 (21). To determine the effect of {alpha}5{beta}1 on {alpha}v{beta}3-mediated cell adhesion to FBG, CHO cells expressing {alpha}v{beta}3 alone (B3), {alpha}5{beta}1 alone (X5C5), or both {alpha}v{beta}3 and {alpha}5{beta}1 (B3C5) were examined. Cells transfected with an empty vector served as a negative control (P3). B3 cells adhere to FBG in a concentration dependent fashion (Fig. 2A). B3 cell adhesion to FBG is completely inhibited by LM609, a function-blocking antibody specific for {alpha}v{beta}3 (data not shown). Surprisingly, B3C5 cells that express both {alpha}v{beta}3 and {alpha}5{beta}1 demonstrate significantly decreased adhesion to FBG when compared with B3 cells despite expression of comparable levels of the {alpha}v{beta}3 integrin (p = 0.0002 at 100 µg/ml FBG). Control cells and cells expressing {alpha}5{beta}1 alone (X5C5) do not adhere to FBG. B3, B3C5, and X5C5 cell adhesion to FN was also examined. No discernible difference was measured, supporting previous work that demonstrates that individually both {alpha}v{beta}3 and {alpha}5{beta}1 support adhesion to FN (data not shown).



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FIG. 2.
{alpha}v{beta}3-mediated adhesion is modulated by the integrin {alpha}5{beta}1. CHO cells in serum-free medium expressing either {alpha}v{beta}3 alone (B3; stippled box) or both {alpha}v{beta}3 and {alpha}5{beta}1 (B3C5; gray box) were plated on increasing concentrations of fibrinogen (A) or thrombospondin (C) as indicated and allowed to adhere for 60 min. Alternately, cells were plated on 50 µg/ml FBG (B) or 10 µg/ml TSP (D) and allowed to adhere for 15 min. Cells transfected with empty vector served as a negative control (P3; white box). In E and F, B3C5 cells were preincubated with the anti-{alpha}5{beta}1 monoclonal antibody (cross-hatched box) prior to attachment to 50 µg/ml FBG for either 15 (E) or 60 min (F). Cell adhesion was determined either by staining cells in triplicate wells with crystal violet and elution of the dye (A and C) or by staining cells in duplicate wells with Giemsa and counting the number of cells from 3 random high power fields (B, D, E, and F). The results obtained by both methods are similar. All experiments were repeated in triplicate. The data are expressed as the mean ± S.E.

 

To determine whether the effect of {alpha}5{beta}1 on {alpha}v{beta}3-mediated adhesion was specific for FBG, adhesion of B3 and B3C5 cells to TSP was also examined. B3C5 cell adhesion to TSP was decreased by 50% when compared with B3 cells, an effect comparable with that seen for FBG (Fig. 2C). Taken together, these data suggest that co-expression of {alpha}5{beta}1 with {alpha}v{beta}3 negatively regulates {alpha}v{beta}3 interaction with its ligands.

At 1 h after plating on FBG, both B3 and B3C5 cells are partially spread (Fig. 3A). To eliminate the contribution of cell spreading to cell adhesion, short time course adhesion assays were also performed. At 15 min following adhesion to FBG, B3 cells remain round. By 30 min, however, a significant number of cells are partially spread (data not shown). Therefore, adhesion assays were performed at the 15-min time point. B3C5 cells demonstrate a significant decrease in cell adhesion to both FBG and TSP at 15 min (Fig. 2, B and D). This data confirms that co-expression of {alpha}5{beta}1 with {alpha}v{beta}3, significantly inhibits {alpha}v{beta}3-mediated cell adhesion to its ligands, independent of cell spreading.



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FIG. 3.
The integrin {alpha}5{beta}1 inhibits {alpha}v{beta}3-mediated spreading and migration on fibrinogen. A, B3 (top panels) or B3C5 (bottom panels) cells in serum-free medium were plated on FBG-coated dishes (50 µg/ml) and incubated for increasing times as indicated. Attached cells were fixed, stained with modified Giemsa, and allowed to dry. Stained cells were viewed using inverted bright-field optics. Photographic images were captured using a Spot color camera connected to a MacIntosh G4 computer equipped with IPLab image analysis software. In B, B3 or B3C5 cells in serum-free medium were plated on the upper surface of a transwell filter coated on both sides with either FBG (50 µg/ml) or FN (10 µg/ml). After 5 h, the cells attached to the lower surface of the filter were fixed, stained with modified Giemsa, and allowed to dry. Cell number was quantified microscopically from three random high power fields. Results are presented as the mean ± S.E. of at least three separate experiments.

 

The cell adhesion experiments were performed in the absence of exogenous FN. However, CHO B2 cells do produce a small amount of cellular FN. Therefore, the inhibitory effect of {alpha}5{beta}1 on {alpha}v{beta}3-mediated adhesion could require the ligation of {alpha}5{beta}1 by endogenous FN. To test this hypothesis, B3C5 cells were allowed to adhere to FBG in the presence of the {alpha}5{beta}1 function-blocking antibody. As demonstrated in Fig. 2, E and F, incubation of B3C5 cells with P1D6 did not significantly affect {alpha}v{beta}3-mediated adhesion to FBG at either the 15- or 60-min time points (p = 0.24). This suggests that the inhibitory effect of {alpha}5{beta}1 on {alpha}v{beta}3 does not require the binding of FN to {alpha}5{beta}1.

Cell spreading follows cell attachment to adhesive substrates. Whereas B3C5 cell adhesion to FBG is significantly decreased, some cells do adhere. When B3C5 cell spreading on FBG was examined, however, cells show decreased spreading at all time points when compared with B3 cells (Fig. 3A).

{alpha}5{beta}1 Blocks {alpha}v{beta}3-mediated Migration on FBG—Integrin {alpha}v{beta}3 has an important role in cell migration, particularly during wound healing. Therefore, we sought to determine the effect of {alpha}5{beta}1 expression on {alpha}v{beta}3-mediated cell migration on FBG. Random migration on FBG was determined using a Boyden chamber assay in which transwells were coated on both sides with FBG. As demonstrated in Fig. 3B, B3 cells migrate efficiently on FBG. Expression of integrin {alpha}5{beta}1, however, inhibited greater than 90% of {alpha}v{beta}3-mediated cell migration (p = 0.0001). To determine whether this reflected a global decrease in migratory function of B3C5 cells, cell migration on FN was also examined. When B3 and B3C5 cell migration on FN were compared, no discernible difference was measured (Fig. 3B). Cells expressing {alpha}5{beta}1 alone do not migrate on FBG (Fig. 4A). However, these cells can migrate efficiently on FN (Fig. 4B). These data indicate that expression of the integrin {alpha}5{beta}1 suppresses {alpha}v{beta}3-mediated cell migration



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FIG. 4.
The inhibition of {alpha}v{beta}3-mediated migration by {alpha}5{beta}1 is regulated by the cytoplasmic domain of the {alpha}5 integrin subunit. A, CHO cells in serum-free medium expressing {alpha}v{beta}3 alone (B3), {alpha}5{beta}1 alone (X5C5), or both {alpha}v{beta}3 and wild type {alpha}5{beta}1 (B3C5) or mutant {alpha}5{beta}1 integrin receptors (B3C4) as indicated were plated on the upper surface of a transwell filter coated on both sides with FBG (50 µg/ml). In B, X5C5 or X5C4 cells were plated on the upper surface of a transwell filter coated on both sides with fibronectin (5 µg/ml). Cells transfected with empty vector served as a negative control (P3). After 5 h, cell migration was quantified by performing microscopic cell counts as described in the legend to Fig. 3.

 

Inhibition of {alpha}v{beta}3 by {alpha}5{beta}1 Is Dependent on the Cytoplasmic Tail of the {alpha}5 Integrin Subunit—One possible explanation for the effect of {alpha}5{beta}1 expression on {alpha}v{beta}3-mediated adhesive events may be that the presence of {alpha}5{beta}1 sterically hinders the interaction of {alpha}v{beta}3 with its ligand. To test this hypothesis, B3 cells were transfected with a chimeric {alpha}5 integrin cDNA, in which the cytoplasmic domain of the {alpha}5 integrin was replaced by the cytoplasmic domain of the {alpha}4 integrin. A stable cell line expressing the mutant {alpha}5 was established as described above (B3C4).

B3C5 and B3C4 cells express similar levels of cell surface {alpha}5{beta}1 (Fig. 1A). As previously demonstrated, the expression of wild-type {alpha}5{beta}1 significantly inhibits {alpha}v{beta}3-mediated migration on FBG (Fig. 4A). When the B3C4 cells expressing the chimeric {alpha}5 integrin are examined, however, their migration on FBG is significantly greater than the B3C5 cells and is comparable with the untransfected B3 cells. To confirm that the chimeric {alpha}5 integrin is functional, CHO B2 cells were transfected with wild-type (X5C5) or mutant (X5C4) {alpha}5{beta}1 cDNA and stable cell lines were established by FACS. There was no difference between the X5C5 and X5C4 cell migrations on FN (Fig. 4B). Taken together, these data suggest that {alpha}5{beta}1 acts in an "inside-out" fashion to negatively regulate {alpha}v{beta}3-mediated migration and that this effect is dependent on the {alpha}5 integrin cytoplasmic domain.

Mn2+ Promotes B3C5 Cell Adhesion to FBG—Intracellular signaling events can modulate integrin ligand binding affinity, a process termed activation or inside-out signaling (23, 24). This process has been well described for members of the {beta}3 integrin family (12, 25). We hypothesized that co-expression of the integrin {alpha}5{beta}1 leads to an alteration in the affinity state of {alpha}v{beta}3 resulting in decreased ligand binding. To test this hypothesis, we used Mn2+, a divalent cation known to stimulate integrin activity and interactions with ligand (26). B3, B3C5, or X5C5 cells were allowed to adhere to FBG in the presence or absence of 100 µM Mn2+ for 1 h (Fig. 5A). Treatment with Mn2+ does not significantly improve B3 cell adhesion to FBG, suggesting that {alpha}v{beta}3 exists in a higher affinity state in these cells. When Mn2+ is added to B3C5 cells, however, there is a significant increase in adhesion to FBG to a level that is comparable with the B3 cells (p = 0.41). X5C5 cells show no increased affinity for FBG in the presence of MnCl2. Similar results were obtained when adhesion was performed at 15 min (data not shown). Interestingly, enhanced adhesion of B3C5 cells to FBG is not seen in the presence of PMA (Fig. 5B). These data demonstrate that {alpha}v{beta}3 exists in a low affinity state when co-expressed with {alpha}5{beta}1. Furthermore, the decreased {alpha}v{beta}3-mediated ligand binding can be rescued by the addition of MnCl2 but not by PMA.



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FIG. 5.
Activation of {alpha}v{beta}3 with MnCl2 increases B3C5 cell adhesion to fibrinogen. B3, B3C5, or X5C5 cells in serum-free Dulbecco's modified Eagle's medium were plated on 50 µg/ml FBG in the presence or absence of either 100 µM MnCl2 (A) or 200 nM PMA (B). Cells were incubated at 37 °C and 5% CO2 for 1 h. The wells were rinsed and attached cells were fixed and stained with Giemsa. Cells from 3 random high power fields were counted. Results are presented as the mean ± S.E. of at least three separate experiments performed in triplicate.

 

{alpha}5{beta}1 Regulates {alpha}v{beta}3-mediated Retraction of Fibrin Matrices—The retraction of fibrin matrices by cells that express the integrin {alpha}v{beta}3 has been well described (8, 27). Therefore, we sought to determine whether expression of {alpha}5{beta}1 could inhibit {alpha}v{beta}3-mediated fibrin clot retraction. B3 cells retract fibrin clots. As demonstrated in Fig. 6, the extent of B3 cell retraction of fibrin matrices was significantly greater at all time points when compared with B3C5 cells (p = 0.002 at 7 h). Furthermore, Mn2+ treatment increases B3C5 cell retraction of fibrin matrices. These data indicate that co-expression of {alpha}5{beta}1 broadly inhibits a variety of cell adhesive events that are dependent on {alpha}v{beta}3.



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FIG. 6.
The integrin {alpha}5{beta}1 inhibits {alpha}v{beta}3-mediated retraction on fibrin matrices. Equal numbers of B3 or B3C5 cells were added to fibrin matrix components followed by thrombin to allow clot formation. The mixture was rapidly pipetted into albumin-coated tissue culture dishes and incubated at 37 °C for 30 min. Serum-free Dulbecco's modified Eagle's medium was then added to each well. In some experiments, 100 µM MnCl2 was added to the B3C5 cells. Clots were then detached from the dishes and allowed to contract. The diameter of each clot was recorded at increasing times as indicated. The data are expressed as the mean retraction (mm) ± S.E. of six separate experiments.

 

Constitutive Activation of {alpha}v{beta}3 Maintains {alpha}v{beta}3-mediated Ligand Binding Despite {alpha}5{beta}1 Expression—Our data suggest that {alpha}5{beta}1 expression may convert {alpha}v{beta}3 to a less active state. We reasoned that a constitutively active {alpha}v{beta}3 integrin would be resistant to negative regulation by {alpha}5{beta}1. To test this hypothesis, we used CHO B2 cells expressing an {alpha}v{beta}3 integrin containing a point mutation in the conserved membrane-proximal region of the {beta}3 cytoplasmic tail, D723R. This mutation results in constitutive activation of the {beta}3 subunit (CHO B3(D-R)) (28). When dimerized with the {alpha}v subunit, the resulting {alpha}v{beta}3 integrin demonstrates increased ligand binding affinity.

CHO B3(D-R) surface expression of the mutant {alpha}v{beta}3 was determined by flow cytometry to ensure that receptor expression was comparable with the wild type B3 cells (Fig. 1A). B3(D-R) cells were then transfected with a cDNA for the wild-type human {alpha}5 integrin subunit. Stable cell populations expressing {alpha}v{beta}3(D-R) and {alpha}5{beta}1 were established by FACS to establish the B3(D-R)C5 cell line. Fig. 1A demonstrates that B3C5 and B3(D-R)C5 cells express similar levels of both {alpha}v{beta}3 and {alpha}5{beta}1.

B3 and B3(D-R) cell adhesion to FBG is comparable and is significantly greater than that seen for B3C5 cells (p = 0.0001 and p = 0.013, respectively; Fig. 7A). Interestingly, B3(D-R)C5 cells also adhere well to FBG indicating that the {alpha}v{beta}3 receptor in these cells maintains a high affinity state despite the expression of {alpha}5{beta}1.



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FIG. 7.
Integrin {alpha}v{beta}3 activation renders resistance to the effect of {alpha}5{beta}1 expression. CHO cells in serum-free medium expressing wild-type {alpha}v{beta}3 alone (B3), the constitutively activated mutant {alpha}v{beta}3(D723R) alone (B3(D-R)), or both the mutant {alpha}v{beta}3(D-R) and {alpha}5{beta}1 (B3(D-R)C5) were plated on 50 µg/ml FBG or 10 µg/ml FN. Cells were incubated at 37 °C and 5% CO2 for 1 h. The wells were rinsed and attached cells were fixed and stained with Giemsa. Cells from 3 random high power fields were counted. Results are presented as the mean ± S.E. of at least three separate experiments performed in triplicate. B3, B3(D-R), B3C5, or B3(D-R)C5 cells in serum-free medium were plated on the upper surface of a transwell filter coated on the under surface only (B) with either FBG (50 µg/ml) or fibronectin (5 µg/ml). After 5 h, the cells attached to the lower surface of the filter were fixed, stained with modified Giemsa, and allowed to dry. Cell number was quantified microscopically from three random high power fields at x100. Results are presented as the mean ± S.E. of at least three separate experiments.

 

When cell migration is examined, B3 cell migration on FBG is significantly higher than that observed for both B3(D-R) and B3(D-R)C5, neither of which migrate efficiently on FBG-coated surfaces (data not shown). We therefore examined haptotactic cell migration toward both FBG- and FN-coated surfaces. As demonstrated in Fig. 7B, B3 haptotactic cell migration toward FBG is significantly greater than that observed for B3C5 cells (p = 0.0003) and remains greater than that seen for both B3(D-R) and B3(D-R)C5 cells. Furthermore, both B3(D-R) and B3(D-R)C5 cell migration toward FBG is also significantly greater than observed with B3C5 cells (both at p = 0.0003). Importantly, when B3(D-R) and B3(D-R)C5 cell migrations are compared, no significant difference is observed (p = 0.62). Migration of all four cell lines toward FN is comparable (Fig. 7C). These data demonstrate that the constitutively active {alpha}v{beta}3 is resistant to the regulatory effect of {alpha}5{beta}1 and provides further support to the idea that {alpha}5{beta}1 expression acts as an inside-out suppressor of {alpha}v{beta}3-mediated ligand binding.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In this article, we present further evidence of cross-talk between integrins {alpha}5{beta}1 and {alpha}v{beta}3. We demonstrate that 1) de novo expression of {alpha}5{beta}1 alters {alpha}v{beta}3-mediated interaction with its ligands resulting in reduced adhesion, migration, and clot retraction; 2) the effect of {alpha}5{beta}1 on {alpha}v{beta}3 does not require ligation of {alpha}5{beta}1 by FN; 3) the suppression of {alpha}v{beta}3 by {alpha}5{beta}1 is dependent on the cytoplasmic domain of the {alpha} integrin subunit; 4) activation of {alpha}v{beta}3 by MnCl2 or by a point mutation in the {beta}3 integrin subunit confers resistance to the effect of {alpha}5{beta}1. Taken together, these data suggest that {alpha}5{beta}1 can down-regulate the ligand binding affinity of {alpha}v{beta}3 and provide a relevant biological mechanism whereby variations in {alpha}5{beta}1 expression in vivo may modulate activation of {alpha}v{beta}3 to influence its adhesive function.

A general feature of integrins is that their adhesive function may be modified by extracellular or intracellular events to produce distinct functions (11, 29). For example, ligand-binding by the integrin {alpha}IIb{beta}3 is regulated by soluble agonists that effect a conformational change in the integrin and allow it to bind soluble FBG to facilitate blood clotting (9, 11). Differences in the activation state of {alpha}v{beta}3 have also been described. Studies have shown that activation of {alpha}v{beta}3 can be induced by model agonists, including the phorbol ester, PMA and the divalent cation MnCl2, to regulate {alpha}v{beta}3-mediated adhesion and migration on specific ligands (12, 14, 25). More recently, work has demonstrated that activation of {alpha}v{beta}3 promotes breast cancer cell arrest during blood flow, contributing significantly to the metastatic phenotype (14). Furthermore, in migrating cells, high affinity {alpha}v{beta}3 is recruited to the leading edge of lamellopodia and appears to be required for directed cell movement (13). In this paper, we present evidence that the default status of {alpha}v{beta}3 may be one of constitutive high affinity and that the expression of the integrin {alpha}5{beta}1 in this system can suppress {alpha}v{beta}3 function. This supports previous work suggesting that inside-out signaling events in cells that express {alpha}v{beta}3 may serve to suppress constitutive activity (30).

Relatively little is known about the factors that suppress integrin function, but they are likely to involve the modulation of the same events that influence integrin activation. Integrin activation results as a consequence of two potential mechanisms, that may act separately or in concert. The first involves an alteration in integrin affinity that occurs because of a change in receptor conformation as detected by epitope-specific or ligand-mimetic antibodies (23, 31, 32). The second involves an alteration in integrin avidity or clustering. Alterations in integrin avidity are likely to reflect changes in the tethering of the integrin to the cytoskeleton and/or the diffusion of the integrin in the plane of the membrane (3335). These complex events facilitate integrin clustering and support multivalent interactions in response to ligand binding. Our data demonstrate that de novo expression of {alpha}5{beta}1 strongly influences the ligand binding function of {alpha}v{beta}3 as measured by cell adhesion and migration assays, but does not distinguish whether this reflects a conformational change in the integrin or an alteration in integrin clustering. Two lines of evidence, however, support the idea that the suppression of {alpha}v{beta}3 function may occur as a consequence of a conformational change in the receptor. First, MnCl2 is a divalent cation that can convert {alpha}v{beta}3 to a high affinity conformation by a direct effect on the integrin extracellular domain (12, 25, 26, 36). B3C5 cell adhesion to FBG in the presence of MnCl2 is significantly increased and is comparable with that of B3 cells. However, MnCl2 has little effect on B3 cell adhesion to FBG suggesting that {alpha}v{beta}3 exists in a moderate to high affinity state in these cells. Second, previous work has shown that expression of {beta}3(D-R) results in a functionally activated integrin that supports tumor cell arrest during blood flow and that binds an activation-specific antibody (14, 32). This point mutation in the {beta}3 cytoplasmic tail is believed to produce a conformational change in the intracellular domain of the receptor that is propagated to the extracellular domain to modulate ligand binding affinity (28). In our studies, cells that express the constitutively active {alpha}v{beta}3(D-R) integrin adhere to FBG in a manner that is comparable with B3 cells. Furthermore, B3(D-R)C5 cells maintain a high affinity for FBG despite expression of {alpha}5{beta}1. Taken together, our data suggest that {alpha}5{beta}1-mediated inside-out signaling can alter the conformation of {alpha}v{beta}3 to negatively regulate ligand binding affinity.

The integrin {alpha} subunit cytoplasmic domain plays an important role in the translation of ligand binding information to cellular events. For example, the integrin {alpha}2{beta}1 supports cell adhesion to collagen and the contraction of collagen gels. The exchange of the {alpha}2 cytoplasmic domain with the {alpha}5 or {alpha}4 sequences altered {alpha}2 integrin-mediated contraction of collagen gels but not adhesion to collagen (37). In these experiments, the {alpha}5 cytoplasmic domain had a similar capacity to {alpha}2 to contract collagen gels whereas the function of the {alpha}2/{alpha}4 chimera was significantly reduced (37). In this work, we have shown that cells expressing {alpha}v{beta}3 and a chimeric {alpha}5/{alpha}4 integrin retain their ability to migrate on FBG when compared with cells expressing {alpha}v{beta}3 and the wild-type {alpha}5 integrin. However, our recent work has determined that cell migration on FBG is not rescued by expression of a chimeric {alpha}5/{alpha}2 integrin.2 These data support the idea that {alpha}5- and {alpha}2-mediated signaling events are similar. Furthermore, the data suggest that the ability of the {alpha}4 cytoplasmic domain to rescue {alpha}v{beta}3-mediated adhesion may depend upon the distinctive function of {alpha}4{beta}1 with respect to these integrins.

The integrin {alpha}4{beta}1 is expressed on a variety of cell types, including leukocytes. In previous experiments in which the integrin {alpha}4 cytoplasmic domain has been joined to other integrin subunits, {alpha}4 supports increased cell migration, but not cell spreading or contractility (37). Recently, a molecular mechanism for the effects of the {alpha}4 cytoplasmic domain on cell adhesion and migration has been determined. The integrin {alpha}4 directly and tightly binds paxillin, a multidomain adaptor protein that plays an important role in the regulation of cell migration (38, 39). Inhibition of paxillin-{alpha}4 binding with a peptide fragment of paxillin blocks {alpha}4-dependent cellular responses (40). Additionally, the interaction between {alpha}4 and paxillin can be modulated by the phosphorylation of the {alpha}4 integrin subunit or by a phosphorylation-mimicking point mutation in {alpha}4 that blocks paxillin binding and facilitates cell spreading (41).

In our experiments, both the X5C5 and X5C4 cells migrate well on FN-coated transwells. However, X5C4 cells display both decreased adhesion and spreading on FN.2 This data suggests that the chimeric X5C4 receptor is also regulated by an interaction with paxillin and that this interaction may regulate its ability to modulate {alpha}v{beta}3-mediated signaling events.

Our data demonstrates that the effect of {alpha}5{beta}1 on {alpha}v{beta}3 ligand binding affinity does not require FN binding to {alpha}5{beta}1. This suggests that unligated {alpha}5{beta}1 can mediate distinct intracellular signaling events and may be required to negatively regulate the affinity state of {alpha}v{beta}3. Recent work supports this concept establishing that unligated integrins can have significant biological function (19, 42). For example, overexpression of unligated integrin {alpha}5{beta}1 has been associated with reduced tumor cell growth in vitro and in vivo (42, 43). Furthermore, expression of both unligated {alpha}5{beta}1 and {alpha}v{beta}3 integrins are sufficient to initiate cell death among adherent cells (42). This "integrin-mediated cell death" can be induced by the cytoplasmic domain of both the {beta}1 and {beta}3 integrins resulting in the recruitment of caspase-8 to the cell membrane and its subsequent activation (42). Unligated integrins appear to have the capacity to cluster on the cell surface, however, these integrins are not associated with FAK or tethered by the cytoskeleton. These untethered integrin clusters may serve to recruit signaling molecules other than caspase-8 that can modulate inside-out signaling events that influence integrin activation.

One such target for unligated integrins may be the protein kinase A (PKA) signaling pathway. Ligation of integrin {alpha}5{beta}1 by FN suppresses PKA activity in cells adherent to vitronectin (19). Blockade of FN-{alpha}5{beta}1 interactions with an anti-{alpha}5{beta}1 antibody increases PKA activity and decreases {alpha}v{beta}3-mediated migration on vitronectin. This effect could be replicated by treatment with PKA agonists such as forskolin or dibutyryl cAMP (19). In contrast to the data presented in this report, treatment with PKA agonists did not influence {alpha}v{beta}3-mediated adhesion to vitronectin, suggesting that PKA does not modulate integrin ligand binding activity in these cells. These differences may reflect a cell type-specific response to de novo {alpha}5{beta}1 integrin expression. Furthermore, the effects of unligated {alpha}5{beta}1 may be more readily apparent in cells that secrete relatively low levels of endogenous, cellular FN such as those used for these experiments.3

In summary, we report that in the absence of {alpha}5{beta}1, {alpha}v{beta}3 exists in an activated state demonstrating high affinity for FBG that is not augmented by soluble agonists. De novo expression of {alpha}5{beta}1, however, suppresses {alpha}v{beta}3-mediated adhesive functions through a mechanism that is dependent on the cytoplasmic tail of the {alpha} integrin subunit and independent of ligand binding. Furthermore, cells that express a constitutively active {beta}3 integrin are resistant to the effects of {alpha}5{beta}1 expression. These data provide a relevant biological mechanism whereby variations in {alpha}5{beta}1 expression in vivo may modulate activation of {alpha}v{beta}3 to influence its adhesive function


    FOOTNOTES
 
* This work was supported by National Institutes of Health Grant GM-061847-01 (to S. A. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ To whom correspondence should be addressed. Tel.: 732-235-7348; Fax: 732-235-7079; E-mail: corbetsi{at}umdnj.edu.

1 The abbreviations used are: ECM, extracellular matrix; CHO, Chinese hamster ovary; FBG, fibrinogen; FN, fibronectin; TSP, thrombospondin; PMA, phorbol 12-myristate 13-acetate; PKA, protein kinase A; PBS, phosphate-buffered saline; FACS, fluorescence-activated cell sorter. Back

2 D. Ly and S. A. Corbett, unpublished observations. Back

3 S. A. Corbett, unpublished results. Back


    ACKNOWLEDGMENTS
 
We thank Ramsey Foty for helpful discussions and George P. Tuszynski (MCP-Hahneman) for the gift of thrombospondin.



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 ABSTRACT
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