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Originally published In Press as doi:10.1074/jbc.M310374200 on November 5, 2003

J. Biol. Chem., Vol. 279, Issue 2, 1400-1407, January 9, 2004
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The Receptor for Urokinase-type Plasminogen Activator Regulates Fibronectin Matrix Assembly in Human Skin Fibroblasts*

Elizabeth Monaghan{ddagger}, Volodia Gueorguiev, Cynthia Wilkins-Port§, and Paula J. McKeown-Longo

From the Center for Cell Biology and Cancer Research, Albany Medical College, Albany, New York 12208

Received for publication, September 18, 2003 , and in revised form, November 5, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Previous studies have indicated that the receptor for urokinase-type plasminogen activator, uPAR, can form functional complexes with integrin receptors thereby modulating integrin activity. In the present study, the role of uPAR in the regulation of {alpha}5{beta}1-dependent polymerization of the fibronectin matrix was investigated. Incubation of fibroblast monolayers with the P-25 peptide, a uPAR ligand, resulted in a 12-15-fold increase in the accumulation of exogenous fibronectin in the cell layer. The exogenous fibronectin co-localized in the extracellular matrix with endogenous cell-derived fibronectin, and its deposition into the matrix was inhibited by blocking antibodies against the {beta}1 integrin receptor. The P-25-dependent increase in fibronectin assembly was associated with a 7-8-fold increase in the expression of matrix assembly sites as well as a 37-fold increase in the rate of transfer of cell surface-bound fibronectin into a detergent-insoluble matrix. The effects of P-25 on the matrix assembly were attenuated by incubating cells with either phospholipase C or with antibodies against uPAR, confirming a role for uPAR in the P-25-dependent increase in matrix assembly. P-25-treated cells exhibited a 10-fold increase in the binding of the 120-kDa cell-binding fragment of fibronectin suggesting an increase in {alpha}5{beta}1 affinity for fibronectin. Consistent with this, treatment of cells with P-25 also resulted in a 6-10-fold increase in the binding of two different monoclonal antibodies that recognize the active conformation of the {beta}1 integrin. These results indicate that P-25 increases matrix assembly by altering the activation state of the {alpha}5{beta}1 integrin receptor and suggest that changes in integrin activation affect both the number of matrix assembly sites as well as the rate of transfer of cell-bound fibronectin into a detergent-insoluble matrix. These data provide direct evidence that uPAR and integrin receptors synergistically regulate the levels of fibronectin in the extracellular matrix.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The extracellular fibronectin matrix plays an important role in several cellular processes including adhesion, migration, differentiation, and growth (1). The assembly of fibronectin into the matrix is a stepwise process that is dependent upon cell surface integrin receptors as well as appropriate cytoskeletal organization (2, 3). In the initial step of matrix assembly, soluble fibronectin binds to adherent cells through the amino-terminal domain of fibronectin, which binds to specific saturable sites on the cell surface (4, 5). This site is often defined operationally by ligand binding assays using a radiolabeled 70-kDa amino-terminal fragment of fibronectin (4). Expression of 70-kDa binding sites is regulated by the {alpha}5{beta}1 fibronectin integrin receptor as well as cytoskeletal tension (6, 7). Fibronectin binding to the cell is followed by intermolecular homophilic binding events that contribute to fibril growth and detergent insolubility (8-11). The expression of homophilic binding sites within matrix fibronectin can also be regulated by cytoskeletal tension providing a mechanism by which cells may incorporate soluble fibronectin directly into preformed fibrils (12, 13).

In most cells, assembly of the fibronectin matrix is dependent on the {alpha}5{beta}1 integrin receptor for fibronectin. Assembly of fibronectin into the extracellular matrix requires that the integrin be in an appropriate activation state to support matrix assembly (3, 14). Whether this matrix assembly-competent state reflects increased affinity of the integrin for fibronectin and/or postreceptor occupancy events necessary to support fibrillogenesis is not clear. Recent studies have suggested that integrin function can be regulated by the receptor for the urokinase-type plasminogen activator (uPAR)1 (15, 16). uPAR is not a transmembrane receptor but is linked to the outer leaflet of the plasma membrane by a glycosylphosphatidylinositol anchor (17). Fluorescence resonance energy transfer analysis, immunolocalization, and co-immunoprecipitation have identified uPAR in complexes with several integrins including {beta}1, {beta}2, {beta}3, and {beta}5 (18-22). The presence of uPAR in these complexes alters several integrin-dependent functions including signal transduction (23-25), adhesion (26), migration (27), and growth (28, 29). Peptides with known homologies to the integrin {alpha} subunits such as P-, {alpha}3-, and M-25, have been used to modulate uPAR-integrin complexes (30, 31) and effect uPAR-dependent signaling pathways. This has led to the hypothesis that uPAR can form either cis- or trans-interactions with integrins on the surface of the same or neighboring cells, thereby affecting integrin function (32, 33). Recently, high levels of expression of uPAR in human epidermoid carcinoma cells have been shown to be associated with an increase in adhesion to fibronectin and in an increase in fibronectin matrix assembly (29). However, nothing is known about the mechanism by which uPAR may regulate the assembly of the FN matrix.

In the current study, we demonstrate that the addition of the P-25 peptide ligand for uPAR can up-regulate the polymerization of fibronectin in human dermal fibroblasts. In the presence of P-25, the rate of incorporation of cell surface-bound fibronectin into the detergent-insoluble matrix was increased 37-fold. P-25-treated cells exhibit a 7-8-fold increase in the expression of matrix assembly sites and in the level of activated {alpha}5{beta}1 integrins. These results suggest that uPAR and {alpha}5{beta}1 receptors coordinately and synergistically regulate the assembly of the fibronectin matrix.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents and Antibodies—Unless otherwise stated, all chemicals were purchased from Sigma. Peptides P-25, sequence AESTYHHLSLGYMYTLN, and S-25, sequence NYHYLESSMTALYTLGH, were synthesized by Cell Essentials (Boston, MA). R2 antibody to human uPAR was a gift from Drs. Liliana Ossowski and Julio Aguirre Ghiso (Mt. Sinai School of Medicine, New York). The {beta}1 function-blocking antibody, clone P5D2, and the HUTS-4 antibody against the active conformation of the {beta}1 integrin were purchased from Chemicon (Temecula, CA). Monoclonal antibody 9EG7, which recognizes the activated form of the {beta}1 integrin, was purchased from Pharmingen. Monoclonal antibody IST-9 was obtained from Abcam (Cambridge, England). Secondary antibodies goat anti-mouse HRP and goat anti-rat HRP were purchased from Bio-Rad and Santa Cruz Biotechnology (Santa Cruz, CA), respectively. Alexa Fluor 594-labeled goat anti-mouse antibody was obtained from Molecular Probes (Eugene, OR).

Cell Culture—Human foreskin fibroblasts (A1-F) were a gift from Dr. Lynn Allen-Hoffmann (University of Wisconsin, Madison, WI). A1-F cells were grown in Dulbecco's modified Eagle's medium (DMEM, Invitrogen) supplemented with 10% fetal bovine serum (FBS, Hyclone Laboratories, Logan, UT). Cells were used at passage 6-12 and unless otherwise noted experiments were performed on monolayer cultures.

Purification and Derivatization of Proteins—Human plasma fibronectin was purified from a fibronectin- and fibrinogen-rich by-product of Factor VIII production by ion exchange chromatography on DEAE-cellulose (Amersham Biosciences) as described previously (34) and further purified by affinity chromatography with gelatin-agarose and heparin-agarose (35). The 70-kDa amino-terminal fragment of fibronectin was generated by limited digestion of intact fibronectin with cathepsin D followed by gelatin affinity chromatography as described previously (34). The 120-kDa cell-binding fragment was generated through a chymotrypsin digestion of plasma fibronectin and isolated from the unbound fractions of sequential gelatin- and heparin-Sepharose affinity columns as described previously (36). Vitronectin was purified from human serum by heparin-Sepharose (Amersham Biosciences) affinity chromatography according to the methods of Yatohgo et al. (37).

Purified plasma fibronectin (400 µg), the 70-kDa fibronectin (100 µg), or the 120-kDa fragment (100 µg) was iodinated with 1 mCi of Na125I (PerkinElmer Life Sciences) as described previously (38). Iodinated proteins were mixed with bovine albumin, 1 mg/ml, dialyzed against Tris-buffered saline, and frozen at -80 °C until used. Fibronectin (1 mg/ml) was derivatized with Alexa Fluor 488 according to the manufacturer's protocol (Molecular Probes).

Matrix Incorporation Assays—125I-Fibronectin assembly into a detergent-insoluble matrix was determined as described previously (38). Cultures were incubated with 125I-fibronectin (1 µg/ml; 1 x 106 cpm/ml) in DMEM at 37 °C in the presence of either P- or S-25. Incubation times and peptide doses and/or inhibitors are as designated in the figure legends. After incubation, cells were rinsed three times in PBS, and cell layers were scraped directly into 1 N NaOH to determine the total cell layer-associated fibronectin. In some experiments, the detergent-insoluble extracellular matrix was isolated by extraction of cell layers in 1% deoxycholate (DOC). DOC extractions were done in a 20 mM Tris (pH 8.8) buffer containing 2 mM phenylmethylsulfonyl fluoride, 2 mM EDTA, 2 mM N-ethylmaleimide, and 2 mM iodoacetic acid. Cell layers were scraped into 1% DOC and centrifuged at 18,000 rpm for 30 min. To localize fibronectin, cell layers were incubated overnight with Alexa Fluor 488-derivatized plasma fibronectin (2 µg/ml) in serum-free medium containing 50 µM P- or S-25. Cell layers were rinsed, fixed with 3% paraformaldehyde, permeabilized with 0.1% Triton, and incubated with IST-9 antibody against cellular fibronectin, followed by goat anti-mouse Alexa Fluor 594-labeled secondary antibody. Fluorophores were visualized using an Olympus BMX-60 microscope equipped with a cooled LCD sensi-camera. Images were acquired using Slidebook Software (Intelligent Imaging Innovation, Inc., Denver, CO) and processed using Adobe Photoshop.

Fibronectin Fragment Binding Assays—Cell layers were preincubated with peptides for 1 h prior to the addition of 125I-labeled 70-kDa fragment (100 ng/ml) or 125I-labeled 120-kDa (12.5-100 ng/ml) fragment in serum-free medium. Following incubation with fragments, cell layers were washed three times with PBS, solubilized in 1 N NaOH, and cell-associated radioactivity was determined by {gamma} scintillation. Nonspecific binding of the 70- and 120-kDa fragments was determined in the presence of excess (50 or 100 µg/ml) unlabeled protein and was subtracted from total binding. Nonspecific binding typically ranged between 20 and 30% of the total. In some experiments, cells were plated directly onto substrates coated with either vitronectin or fibronectin. To minimize endogenous fibronectin levels during experimental procedures, cell layers were washed three times with serum-free DMEM and pretreated for 3.5 h with cycloheximide (20 µg/ml) in DMEM containing ITS+2 (Sigma) as described previously (39). To remove cell surface uPAR, cycloheximide-treated cells were suspended in PBS containing 0.9 mM Ca2+, 0.5 mM Mg2+, 0.2% bovine serum albumin, 10 mM glucose, 20 µg/ml cycloheximide, and 1.0 unit/ml phosphatidylinositol-specific phospholipase C for 1 h at 37 °C. Cells were replated onto coated substrates at 105 cells/well and allowed to adhere for 2 h. To coat substrates, fibronectin or vitronectin was diluted to 10 µg/ml in PBS and coated onto tissue plates (Corning/Costar) for 3 h at 37 °C.

Determination of the Transfer Rate Constant—The transfer rate constant for fibronectin matrix assembly was determined as described previously (34). The transfer rate constant for matrix assembly defines the probability of surface-bound detergent-soluble fibronectin (pool I) being incorporated into the matrix (pool II) over a 1-min period, based on the equation,

(Eq. 1)
where [FN]I and [FN]II represent the concentration of FN in pools I and II, respectively, at time t. This equation can be solved by a straight line with a slope of Kt.

Integrin Activation Assay—A1-F cells were grown to confluence in 48-well plates and then treated with P-25, S-25, or 2 mM MnCl2 in DMEM for 4 h. Cells were fixed with 3% paraformaldehyde, blocked with 2% bovine serum albumin, and incubated with either 100 ng/ml of 9EG7 or 100 ng/ml of HUTS-4 antibody, which recognizes the activated conformation of the {beta}1 integrin (40, 41). To assay for total {beta}1 integrin, cells were incubated with P5D2, an antibody that recognizes all forms of {beta}1 integrin. Cells were washed with PBS and incubated for 1 h with either goat anti-mouse HRP for P5D2 and HUTS-4 or goat anti-rat HRP for 9EG7. Freshly prepared substrate (0.1 M citrate buffer, 0.5 mg/ml o-phenylenediamine, 1 µl/ml 30% hydrogen peroxide (pH 5)) was added to each well, and the color was allowed to develop. The reaction was stopped with the addition of 2 N sulfuric acid, and the OD was measured at A490. Measurements were corrected for light scattering by subtracting the OD obtained at A630.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
To evaluate whether uPAR might regulate FN matrix assembly in skin fibroblasts, cells were incubated with increasing doses of a peptide, P-25, known to bind to uPAR and modulate activity of {beta}1 integrins (24, 28, 31). The addition of 50 µM P-25 to monolayers of skin fibroblasts resulted in an ~15-fold increase in the accumulation of exogenous fibronectin into fibroblast monolayers as compared with control experiments done in the presence of a scrambled peptide, S-25 (Fig. 1A). Additional control experiments indicated that the scrambled peptide had no effect on basal levels of fibronectin matrix assembly (data not shown). Assembly of fibronectin into a matrix depends on the binding of the amino-terminal region of FN to matrix assembly sites on the cell surface (4, 42). To determine whether the effects of P-25 on matrix assembly resulted from effects on matrix assembly-site expression, cell layers were incubated with the 70-kDa amino-terminal fragment of fibronectin in the presence of the P-25 peptide. Fig. 1B shows an increase in the binding of the 70-kDa fragment to cells in the presence of P-25 with levels of 70-kDa binding reaching 7-8-fold higher in the presence of P-25. The scrambled peptide, S-25, did not affect the binding of the 70-kDa fragment to cells. The binding of fibronectin to matrix assembly sites is then followed by intermolecular homophilic binding events that render the fibronectin detergent-insoluble (8-11, 38, 43). To determine the effect of P-25 on the assembly of fibronectin into the detergent-insoluble extracellular matrix, 125I-fibronectin was incubated with cell layers for 6 h after which matrix was isolated by extraction of cells with deoxycholate. Fig. 1C shows a dose-dependent increase in the assembly of 125I-fibronectin into the detergent-insoluble matrix in the presence of P-25. Between the doses of 10 and 100 µm P-25, there was a 5-40-fold increase in the level of fibronectin incorporated into the detergent-insoluble matrix.



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  		FIG. 1.
Effect of P-25 peptide on fibronectin matrix assembly. A, confluent fibroblast monolayers were incubated in DMEM containing 125I-fibronectin in the presence of 50 µM P- or S-25. At the indicated time points, the medium was removed from the cells, the cells were rinsed in PBS, and the cell layer was solubilized in 1 N NaOH. 125I-FN associated with the cell layer was determined by {gamma} scintillation. B, cell layers were incubated in DMEM containing increasing concentrations of P- or S-25 for 1 h. The 125I-labeled 70-kDa fibronectin fragment was then added to the medium and incubated with the cells for an additional hour. Cell layers were rinsed in PBS and solubilized in 1 N NaOH. The 125I-labeled 70-kDa fragment associated with the cell layer was determined by {gamma} scintillation. C, cell layers were incubated with increasing concentrations of P- or S-25 in the presence of 125I-FN for 6 h. Cell layers were extracted with 1% deoxycholate, and soluble and insoluble material was separated by centrifugation. The amount of 125I-FN incorporated into the detergent-insoluble matrix was determined by {gamma} scintillation.

 
Incorporation of exogenous fibronectin into matrix fibronectin can be separated into two steps. In the first step, fibronectin is associated with the cells in a soluble, reversible manner (pool I). Cell-associated fibronectin is then transferred to a detergent-insoluble, irreversibly bound form (pool II) in the extracellular matrix (38, 44). The rate of transfer of pool I-bound fibronectin into the detergent-insoluble matrix can be described by a transfer rate constant, Kt, which measures the probability of fibronectin moving from pool I to pool II over a 1-min period (34). Kt determinations for both P-25- and S-25-treated cells were determined over a 6-h period (Fig. 2). The Kt for S-25-treated cells was 0.000304 min-1. The Kt for P-25-treated cells was 0.01141 min-1, making the transfer rate constant for P-25-treated cells 37 times greater than that of S-25-treated cells. This result indicates that once fibronectin associates with cell layers, P-25-treated cells are 37 times more efficient than S-25-treated cells at transferring fibronectin into a detergent-insoluble matrix.



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  		FIG. 2.
P-25 enhances the matrix assembly rate constant. Fibroblast cell monolayers were incubated for the indicated times in DMEM containing 125I-FN and 50 µM S- or P-25. Radiolabeled medium was removed, and cell layers were rinsed and extracted in 1% deoxycholate. Deoxycholate-soluble 125I-FN (Pool I) and deoxycholate-insoluble 125I-FN (Pool II) were separated by centrifugation. a and b show the transfer rate constant (Kt) that was derived from the slope of the line of the ratio of fibronectin binding in Pool II/Pool I plotted as a function of time. Lines shown in a and b were obtained from data shown in A and B, respectively.

 
Experiments were done to assess the localization of exogenous fibronectin within the cell layer following P-25 treatment particularly with reference to endogenous cell synthesized fibronectin. As shown in Fig. 3, the assembly of exogenous Alexa Fluor-derivatized fibronectin into the matrix has been enhanced in the presence of P-25 (compare A with B), and it co-localizes with endogenous cell-derived fibronectin as shown in C and D. Endogenous fibronectin was visualized using the IST-9 monoclonal antibody, which recognizes the alternatively spliced type III repeat, EDA, which is not found in plasma fibronectin (45). The merged images shown in yellow (E and F) indicate complete co-localization with the endogenous matrix.



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  		FIG. 3.
P-25-dependent fibronectin matrix assembly co-localizes with endogenous fibronectin matrix. A1-F cells were cultured in complete medium for 24 h. Cells were then incubated for an additional 24 h in DMEM containing 2 µg/ml fibronectin (A, B) derivatized with Alexa Fluor 488 in the presence of 50 µM P-25 (A, C, and E) or S-25 (B, D, and F). Cells were fixed and permeabilized, and endogenous fibronectin was visualized by indirect immunofluorescence using the IST-9 antibody specific for cell-derived fibronectin (C, D). Panels E and F show merged images.

 
Previous studies have shown that the adhesion of cells to vitronectin down-regulates the expression of matrix assembly sites on the surface of adherent cells (46-48). To determine whether P-25 could affect the expression of matrix assembly sites under these conditions, cells newly adherent to either fibronectin or vitronectin were incubated with the 70-kDa fragment of fibronectin in the presence or absence of the P-25 peptide. Fig. 4 shows that similar to previously published observations (47, 48), cells adherent to vitronectin exhibit a 50% decrease in the expression of 70-kDa binding sites as compared with cells adherent to fibronectin. The addition of P-25 to cells adherent to either vitronectin or fibronectin resulted in a severalfold increase in expression of 70-kDa binding sites. To verify that the effects of P-25 on matrix assembly-site expression were dependent on uPAR, adherent cells were pretreated with phospholipase C to remove uPAR prior to the addition of P-25. Treatment of cells with phospholipase C resulted in a 40% decrease in the amount of P-25-induced enhancement of 70-kDa binding sites on cells adherent to fibronectin and a 60% decrease in 70-kDa binding sites on cells adherent to vitronectin. Interestingly, phospholipase C had no effect on 70-kDa binding in the absence of P-25. Western blotting of phospholipase-treated cells indicated that ~80% of uPAR was removed from the cell surface (data not shown). These results suggest that ligation of uPAR with P-25 rescues the inhibitory effect of vitronectin on the expression of matrix assembly sites. Experiments were also done to determine whether the P-25-enhanced incorporation of intact fibronectin into a detergent-insoluble matrix was dependent on uPAR. In these experiments, cells were preincubated with the R2 monoclonal antibody directed against uPAR prior to treatment with P-25. This antibody has been used in other studies to modulate the effects of uPAR on integrin function (28). As shown in Fig. 5, the R2 antibody was able to block ~65% of the P-25-dependent increase in matrix assembly. The R2 antibody had no significant effect on matrix assembly in the absence of P-25. The observation that both phospholipase C and anti-uPAR antibody block only the P-25-dependent increase in matrix assembly is consistent with a model in which basal levels of matrix assembly are not under uPAR regulation.



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  		FIG. 4.
uPAR is required for the P-25-mediated increase in matrix assembly-site expression. Cycloheximide-treated fibroblasts were incubated with phosphatidylinositol-specific phospholipase C (PI-PLC) for 3 h to remove cell surface uPAR and seeded at confluence on wells coated with either fibronectin or vitronectin. Control and enzyme-treated cell layers were preincubated with 100 µM P- or S-25 for 2 h. A 125I-labeled 70-kDa fragment was then added for an additional hour in the continued presence of phosphatidylinositol-specific phospholipase C and cycloheximide. Cell layers were rinsed and then solubilized in 1 N NaOH, and the 125I-labeled 70-kDa fragment associated with the cell layer was determined by {gamma} scintillation. Error bars indicate the S.E. of four wells.

 



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  		FIG. 5.
uPAR is required for P-25-mediated increase in fibronectin matrix. Confluent monolayers of A1-F cells were pretreated for 30 min with R2 (10 µg/ml) monoclonal antibody against uPAR or with control anti-mouse IgG. Cells were then incubated in 125I-fibronectin-containing medium in the presence of 50 µM P- or S-25 for 6 h. Cells were extracted in 1% DOC, and detergent-insoluble matrix fibronectin was isolated by centrifugation and measured by {gamma} scintillation. Error bars indicate the S.E. of four wells.

 
The assembly of exogenous fibronectin into matrix as well as the expression of 70-kDa binding sites on the surface of most cell types is regulated by the {alpha}5{beta}1 integrin receptor for fibronectin (2, 6). To determine whether the P-25 enhancement of the matrix assembly was dependent on the {beta}1 integrin, a function blocking monoclonal antibody, P5D2, against the {beta}1 integrin was tested for its ability to block P-25-induced matrix assembly-site expression. As shown in Fig. 6A, P5D2 was able to inhibit the P-25-induced increase in the binding of the 70-kDa fragment. Similar results were seen when P5D2 was used to block the P-25-induced increase in incorporation of exogenous fibronectin into the detergent-insoluble matrix. As shown in Fig. 6B, the addition of P-25 to confluent monolayers resulted in a 35-fold increase in the assembly of FN into a detergent-insoluble matrix. In the presence of the blocking antibody against the {beta}1 integrin, the P-25-induced increase was attenuated by ~65%. Similar results were seen using mAb13, another function-blocking antibody to {beta}1 integrin (data not shown). Assembly of fibronectin into the matrix also requires the interaction of the amino-terminal matrix-assembly domain of fibronectin with matrix assembly sites on the cell surface. Addition of this fragment to cells results in the inhibition of matrix assembly (4, 42). To determine whether the P-25-dependent increase in matrix assembly was dependent on the amino-terminal region of fibronectin, cells were incubated with 125I-fibronectin in the presence of a 50 µg/ml unlabeled 70-kDa fragment. As shown in Fig. 6C, the P-25-dependent increase in fibronectin incorporation into the DOC-insoluble matrix was nearly completely inhibited in the presence of the 70-kDa fragment. These results indicate that the effects of P-25 on both the expression of matrix assembly sites as well as the assembly of fibronectin into a detergent-insoluble matrix are dependent on the {beta}1 integrin as well as the matrix-assembly domain of fibronectin.



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  		FIG. 6.
P-25-mediated matrix assembly requires the amino terminus of fibronectin and the {beta}1 integrin. A, cell monolayers were preincubated for 30 min with 20 µg/ml of clone P5D2, a function-blocking monoclonal antibody against the {beta}1 integrin. Cells were then treated with 50 µM P- or S-25 for 1 h and 125I-labeled 70-kDa fragment for an additional hour. Medium was removed, and cells were washed and solubilized in NaOH. Bound 70-kDa fragment was determined by {gamma} scintillation. B, cell monolayers were preincubated with P5D2 for 30 min and then treated with 50 µM P- or S-25 in the presence of 125I-fibronectin (FN) for 6 h. Cell layers were extracted in 1% DOC, and 125I-FN that was incorporated into the detergent-insoluble matrix was recovered by centrifugation and measured by {gamma} scintillation. C, confluent monolayers of A1-F cells were incubated in 125I-FN-containing medium in the presence of an excess of 70-kDa fragment (50 µg/ml) and 50 µM P- or S-25 for 6 h. Incorporation of 125I-FN into the detergent-insoluble matrix was obtained as described in the legend to B.

 
{alpha}5{beta}1 integrins are thought to exist in distinct activation states that are associated with different functional activities (49). As uPAR has been reported to modulate the adhesive function of the {alpha}5{beta}1 integrin (24), we investigated whether the effects of P-25 on matrix assembly might be because of changes in the activation state of the {alpha}5{beta}1 integrin. Increases in integrin activation have been shown to result in an increase in ligand binding affinity (50). To evaluate changes in integrin activation, a binding assay using the 120-kDa cell-binding fragment of fibronectin was performed to determine whether P-25 had any effect on the binding of this {alpha}5{beta}1 ligand to cell surfaces. As shown in Fig. 7, incubation of fibroblast monolayers with increasing doses of the 120-kDa fragment resulted in a dose-dependent increase in specific binding of this fragment to cells. In the presence of the P-25 peptide, the binding of this fragment was increased up to 10-fold. Blocking antibodies to the {beta}1 integrin blocked this increase by ~60%. These results indicate that P-25 increases the binding of the 120-kDa fragment to cells and suggests that P-25 causes an increase in affinity of the {alpha}5{beta}1 for fibronectin. Changes in integrin affinity are associated with conformational changes in the integrin resulting in the exposure of neoepitopes, which are recognized by specific antibodies (51). To evaluate the effect of P-25 on integrin conformation, an enzyme-linked immunosorbent assay was performed on P-25-treated fibroblast monolayers using monoclonal antibodies 9EG7 and HUTS-4, which recognize only the active conformation of the {beta}1 integrin. As shown in Fig. 8A, the addition of P-25 to cells resulted in a dose-dependent increase in the level of 9EG7 binding as compared with control (S-25)-treated cell layers. Increased binding of 9EG7 ranged from 2- to 6-fold over a 10-100 µM dose of P-25. Mn2+, a known activator of the {beta}1 integrin, was used as a positive control. Similar results were seen using another antibody, HUTS-4, which recognizes the active conformer of {beta}1 (Fig. 8B). To determine whether P-25 might increase the total number of {beta}1 integrins on the cell surface, cells were incubated with P-25, and levels of {beta}1 were assessed using P5D2, an antibody that recognizes all forms of {beta}1. As shown in Fig. 8C, there was no difference between the total amount of {beta}1 present on P-25- or S-25-treated cells. These data demonstrate that the addition of the P-25 peptide to fibroblast monolayers results in an increase in the activation state of the {beta}1 integrin.



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  		FIG. 7.
P25 enhances binding of the 120-kDa fibronectin fragment. Cells were preincubated with DMEM containing 50 µM P- or S-25 for 1 h, and the 125I-labeled 120-kDa fragment was then added for an additional hour in the continued presence of the peptides. Medium was removed, and the cells were washed with PBS and solubilized in NaOH. Bound 120-kDa fragment was determined by {gamma} scintillation. In one set of wells, cell monolayers were preincubated for 30 min with 20 µg/ml of clone P5D2 (P-25+P5D2), a function-blocking monoclonal antibody against the {beta}1 integrin, prior to the addition of P-25 and 125I-labeled 120-kDa fragment.

 



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  		FIG. 8.
P-25 treatment increases the number of active {beta}1 integrin receptors. A, A1-F cells grown to confluence were incubated for 4 h in DMEM containing 50 µM P-25, 50 µM S-25, or 2 mM Mn2+. Cells were then fixed and incubated for 1 h with monoclonal antibodies that recognize either the active form of the {beta}1 integrin, 9EG7 (100 ng/ml) (A), and HUTS-4 (100 ng/ml) (B) or total {beta}1 integrin (C). Cell layers were washed and incubated with secondary antibodies linked to HRP. Bound antibody was detected by incubating cells with a substrate, and color development was measured at A490 in a plate reader.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The present study indicates that the urokinase receptor for plasminogen activator, uPAR, modulates the rate of fibronectin matrix assembly in human skin fibroblasts by regulating the activation state of the {alpha}5{beta}1 integrin receptor. Our data are consistent with a model by which an increase in integrin activation results in both an increase the number of cell surface fibronectin binding sites as well as an increase in the rate at which surface-bound fibronectin is transferred into a detergent-insoluble extracellular matrix. Treatment of human dermal fibroblasts with P-25 peptide, a uPAR ligand, resulted in a 7-8-fold increase in the expression of binding sites for the amino terminus of fibronectin. The P-25-induced increase in 70-kDa fragment binding was inhibited by blocking antibodies to the {beta}1 integrin, suggesting a role for {beta}1 in mediating the P-25-dependent binding of the amino terminus of fibronectin to the cell surface. Previous studies have shown that blocking antibodies to the {beta}1 integrin can inhibit binding of the 70-kDa amino-terminal fibronectin fragment to cells (6) and that the 70-kDa fragment can bind directly to the {alpha}5{beta}1 integrin (39). These data suggest that P-25-induced changes in integrin activation may be associated with an increase in affinity of the integrin for the amino terminus of fibronectin. This possibility is supported by earlier experiments demonstrating that the reorganization of actin-filament structure promotes the {alpha}5{beta}1-dependent adhesion to the amino terminus of fibronectin (39). Recent studies have identified distinct {alpha}5{beta}1 activation states that exhibit differential binding to the RGD and synergy binding domains in fibronectin (52). As uPAR has been shown to affect signaling pathways important in actin organization (53, 54), it is possible to speculate that the binding of the P-25 peptide to uPAR modulates the activation state of the integrin and its ligand binding specificity by regulating the association of integrin with the cytoskeleton or with cytoskeletal-associated signaling molecules.

In addition to increasing the expression of binding sites for the amino terminus of fibronectin, P-25 also increased the rate of transfer of cell surface-bound fibronectin into detergent-insoluble matrix. The mechanism by which surface-bound fibronectin is converted into a detergent-insoluble matrix is not well understood but is thought to involve homophilic binding events (9, 10, 55-57) as well as disulfide exchange (58, 59) resulting in the formation of high molecular weight disulfide-stabilized multimers. How these homophilic binding events are regulated is not known; however, recent studies (60-62) indicate that elasticity within the molecule, particularly within the type III repeats, coupled with changes in actin-based cellular contractility (7, 13) may contribute to the conversion of surface-bound fibronectin into detergent-insoluble multimeric fibronectin fibrils by regulating the unfolding of cryptic homophilic binding sites within the matrix (63, 64). Whether changes in integrin activation are associated with changes in the force distribution on the matrix is not known; however, changes in integrin activation are known to increase the adhesive strength of integrins (49). These changes in adhesive strength may contribute to the unfolding of modules thereby promoting homophilic events required for assembly of the matrix.

Numerous studies have indicated that uPAR can effect the function of several different integrin receptors including the {alpha}5{beta}1 receptor for fibronectin (reviewed in Refs. 15 and 16). The effect of uPAR on {alpha}5{beta}1 integrin function can be either positive or negative and likely depends on cellular context as well as uPAR levels (24). Therefore, the effects of P-25 on integrin-dependent cellular functions can be expected to be quite varied. For example, overexpression of uPAR in human kidney 293 cells decreased {alpha}5{beta}1-dependent cell adhesion to fibronectin (31). Adhesion was restored by addition of the P-25 peptide indicating that in these cells ligation of uPAR with P-25 could positively effect {alpha}5{beta}1 integrin function. Similar results were seen in MDA-MB-231 breast cancer cells wherein the addition of P-25 to cells increased the adhesion and spreading of these cells on fibronectin. In contrast, high levels of uPAR in human epidermoid carcinoma cells were associated with increased {alpha}5{beta}1-dependent signaling to extracellular signal-regulated kinase as well as increased deposition of the fibronectin matrix (28). Both {alpha}5{beta}1-dependent signaling to extracellular signal-regulated kinase as well as fibronectin matrix assembly were decreased in the presence of the P-25 peptide, indicating that in these cells ligation of uPAR with P-25 inhibited {alpha}5{beta}1 function. Our studies indicate that the P-25 peptide resulted in a large increase in the level of fibronectin matrix deposited by human skin fibroblasts. The reason for the discrepancy in the effects of P-25 on matrix assembly in these two studies is not known but may reflect differences in cellular levels of co-receptors or signaling molecules necessary for uPAR/integrin "cross-talk" (30). Nevertheless, both studies are consistent in that they indicate a role for uPAR in the regulation of fibronectin matrix assembly.

It is clear in the current study that P-25 causes an increase in the number of active integrins present on the surface of human skin fibroblasts. A role for {alpha}5{beta}1 integrin activation in the assembly of fibronectin into a detergent-insoluble matrix has been shown previously by Sechler et al. (14) who demonstrated that activation of integrins through the addition of Mn2+ increased the rate of matrix assembly by severalfold. Our study is the first to provide direct evidence that uPAR can regulate the rate of matrix assembly by increasing the activation state of {beta}1 integrins on the cell surface. The mechanism by which P-25 regulates the activation state of the integrin is not known. One model, which has been proposed, is that the direct binding of uPAR to the {alpha}5{beta}1 integrin stabilizes it in an active conformation. uPAR has been shown to bind directly to some integrins, but evidence for direct binding of uPAR to {alpha}5{beta}1 is lacking (30, 32, 33). Our data would be consistent with a model in which the effects of P-25 on integrin function may be indirect. One possibility is that the binding of P-25 to uPAR modulates the signaling pathways that have an impact on integrin activation and matrix assembly. As uPAR is not a transmembrane receptor, it has been proposed that uPAR may alter signaling pathways within the cell by complexing with co-receptors such as integrins (28), G-protein-linked receptors (65), or growth factor receptors such as epidermal growth factor (66, 67). Recent studies have suggested that complexes between uPAR and {alpha}3{beta}1 may regulate the function of the {alpha}5{beta}1 receptor through a G-protein-dependent signaling mechanism (30). uPAR-mediated signaling has been shown to alter the activity of small molecular weight GTPases such as Rac and Ras, which are known to regulate integrin function (68). uPAR has also been shown to participate in both Src and extracellular signal-regulated kinase-dependent signaling (23, 66), two pathways known to regulate matrix assembly (69, 70). Further studies are needed to understand the molecular basis underlying the regulation of integrin activation and matrix assembly by P-25 as well as other uPAR ligands and/or co-receptors. Delineating these pathways will help identify target molecules that can modulate pathologies resulting from disregulated matrix deposition such as fibrosis and tumor progression.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants CA-58626 and CA-69612 (to P. J. M.-L.). 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

{ddagger} Supported by NHLBI, National Institutes of Health Predoctoral Training Grant T32-HL-07194. Back

§ Supported by American Heart Association Postdoctoral Fellowship Award 0120273T. Back

To whom correspondence should be addressed: Center for Cell Biology and Cancer Research, Albany Medical College, 47 New Scotland Ave., Albany, NY 12208. Tel.: 518-262-5661; Fax: 518-262-5696; E-mail: mckeowp{at}mail.amc.edu.

1 The abbreviations used are: uPAR, urokinase-type plasminogen activator receptor; FN, fibronectin; PBS, phosphate-buffered saline; DOC, deoxycholate; HRP, horseradish peroxidase; DMEM, Dulbecco's modified Eagle's medium. Back



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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