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Originally published In Press as doi:10.1074/jbc.M508526200 on March 1, 2006

J. Biol. Chem., Vol. 281, Issue 19, 13021-13029, May 12, 2006
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Functional Relevance of Urinary-type Plasminogen Activator Receptor-{alpha}3beta1 Integrin Association in Proteinase Regulatory Pathways*

Supurna Ghosh{ddagger}, Jeff J. Johnson{ddagger}, Ratna Sen{ddagger}, Subhendu Mukhopadhyay{ddagger}, Yueying Liu{ddagger}, Feng Zhang§, Ying Wei§, Harold A. Chapman§, and M. Sharon Stack{ddagger}1

From the {ddagger}Department of Cell and Molecular Biology and the Robert H. Lurie Comprehensive Cancer Center, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611 and the §Division of Pulmonary and Critical Care Medicine, University of California, San Francisco, California 94143

Received for publication, August 3, 2005 , and in revised form, February 28, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Squamous cell carcinoma of the oral cavity is characterized by persistent, disorganized expression of integrin {alpha}3beta1 and enhanced production of urinary-type plasminogen activator (uPA) and its receptor (uPAR) relative to normal oral mucosa. Because multivalent aggregation of {alpha}3beta1 integrin up-regulates uPA and induces a dramatic co-clustering of uPAR, we explored the hypothesis that lateral ligation of {alpha}3beta1 integrin by uPAR contributes to uPA regulation in oral mucosal cells. To investigate mechanisms by which uPAR/{alpha}3beta1 binding enhances uPA expression, integrin-dependent signal activation was assessed. Both Src and ERK1/2 were phosphorylated in response to integrin aggregation, and blocking Src kinase activity completely abrogated ERK1/2 activation and uPA induction, whereas inhibition of epidermal growth factor receptor tyrosine kinase activity did not alter uPA expression. Proteinase up-regulation occurred at the transcriptional level and mutation of the AP1 (–1967) site in the uPA promoter blocked the uPAR/integrin-mediated transcriptional activation. Because uPAR is redistributed to clustered {alpha}3beta1 integrins, the requirement for uPAR/{alpha}3beta1 interaction in uPA regulation was assessed. Clustering of {alpha}3beta1 in the presence of a peptide ({alpha}325) that disrupts uPAR/{alpha}3beta1 integrin binding prevented uPA induction. Depletion of cell surface uPAR using small interfering RNA also blocked uPA induction following integrin {alpha}3beta1 clustering. These results were confirmed using a genetic strategy in which {alpha}3 null epithelial cells reconstituted with wild type {alpha}3 integrin, but not a mutant {alpha}3 unable to bind uPAR, induced uPA expression upon integrin clustering, confirming the critical role of uPAR in integrin-regulated proteinase expression. Disruption of uPAR/{alpha}3beta1 binding using peptide {alpha}325 or small interfering RNA blocked filopodia formation and matrix invasion, indicating that this interaction stimulates invasive behavior. Together these data support a model wherein matrix-induced clustering of{alpha}3beta1 integrin promotes uPAR/{alpha}3beta1 interaction, thereby potentiating cellular signal transduction pathways culminating in activation of uPA expression and enhanced uPA-dependent invasive behavior.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Oral squamous cell carcinoma (OSCC)2 is the most common malignancy of the oral cavity, with a distinctly low (50%) 5-year survival rate (www.NIH.gov). Advanced OSCC is associated with high mortality resulting from local, regional, and distant metastasis (2); however, the cellular and biochemical factors that underlie OSCC dissemination are poorly understood. Recent studies have used cDNA microarray analysis for genome-wide monitoring of genetic and epigenetic changes associated with OSCC primary tumors and lymph node metastases. These studies have identified enhanced expression of the proteinase urinary type plasminogen activator (uPA) and the cell-matrix adhesion molecule {alpha}3 integrin as key candidate biomarkers for prediction of poor disease outcome (3, 4).

The serine proteinase uPA converts plasminogen to plasmin, a broad spectrum proteinase active against numerous matrix substrates and additional proteinase zymogens. Acquisition of uPA activity is the net result of regulatory interplay between uPA, its cell surface receptor (uPAR), and its physiological inhibitor, plasminogen activator inhibitor-1 (5). These components have been extensively studied for their contribution to cell migration and invasion in various physiological and pathological conditions, particularly in cancer (6). The uPAR is a cell surface glycosylphosphatidylinositol-anchored molecule that binds uPA and "focalizes" proteolytic activity to the cell-matrix interface (7). Increased pericellular proteolysis of basement membrane proteins can then remove growth constraints imposed by the mechanical properties of a three-dimensional matrix and potentiate invasion and metastasis (8–10). However, recent studies have identified other interesting biological functions of uPAR, including regulation of cell adhesion, proliferation, and differentiation via interaction with other cell surface molecules including integrins and growth factor receptors (11–13). Recently published x-ray crystallography data suggest that uPAR does not undergo significant conformational changes upon uPA binding to its central cavity. Thus, uPAR, bound to uPA in its central core, retains a large outer receptor surface accessible to additional binding partners such as integrins, which in turn may mediate other biological functions of uPAR (14).

Integrins are transmembrane heterodimeric proteins comprised of {alpha} and beta subunits that mediate adhesion to matrix proteins through the extracellular domain and modulate signaling and cytoskeletal organization through the cytoplasmic tail. Numerous studies have demonstrated that the glycolipid-anchored uPAR can form lateral associations with transmembrane integrins (15–21), and uPAR/integrin binding has been shown to initiate or potentiate integrin signaling through focal adhesion kinase and/or Src kinases, downstream to activate MEK/ERK pathways (13, 18, 19, 22). These data support a mechanism whereby the glycolipid-anchored uPAR can couple to cytoplasmic signaling pathways via binding to transmembrane integrins and thereby regulate gene expression and cell behavior (13).

While investigating the regulation of uPA activity in premalignant oral keratinocytes, we observed that multivalent aggregation of integrin {alpha}3beta1 up-regulates uPA expression via a MEK/ERK1/2-dependent pathway, suggesting a mechanism whereby cell-matrix adhesion may regulate subsequent invasive behavior (23). Integrin clustering was accompanied by a redistribution of endogenous uPAR to sites of clustered {alpha}3beta1 integrins and formation of a uPAR/{alpha}3beta1complex as demonstrated using confocal immunofluorescence microscopy and co-immunoprecipitation analysis. This finding was supported by studies showing binding of exogenous uPAR to {alpha}3beta1 in 293 cells and identification of a peptide, designated {alpha}325, that blocks uPAR/{alpha}3beta1 integrin binding (24). More recently, the uPAR-binding site was mapped to loop 4 in the {alpha}3 subunit beta-propeller region (residues 242–246), and a point mutation (H245A) in the {alpha}3 subunit was identified that abrogates uPAR binding (25). The objective of this study was to evaluate the functional contribution of uPAR/{alpha}3beta1 interaction to regulation of uPA expression and activity. Our data support a model wherein matrix-induced clustering of {alpha}3beta1 integrin promotes uPAR/{alpha}3beta1 interaction, thereby potentiating cellular signal transduction pathways culminating in enhanced uPA expression and uPA-dependent invasive behavior in premalignant oral keratinocytes.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Plasminogen and plasmin were purified by affinity chromatography from outdated human plasma as described previously (23). Anti-human integrin {alpha}3 and beta1 monoclonal antibodies (P1B5 and P5D2) were obtained from Chemicon (Temecula, CA). Affinity-purified polyclonal antibody specific for phosphorylated p42/p44 mitogen-activated protein kinase (anti-ACTIVE® MAPK p42/p44 or ERK1/2) was purchased from Promega (Madison, WI). Anti-ERK1/2 (anti-p42/p44) antibodies, which recognize both phosphorylated and nonphosphorylated p42/p44, were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Phospho-Src (Tyr416) antibody was obtained from Cell Signaling Technology (Beverly, MA). Pharmacological inhibitors of MEK1/2 (PD98059), Src kinase (PP2), PI3K (LY294002), p38 MAPK (SB203580), and EGFR kinase (AG1478) were purchased from Calbiochem. Anti-human uPAR antibodies (3936, 3937, and 399R) and anticatalytic uPA antibodies (394) were obtained from American Diagnostica (Greenwich, CT). Hydrobond-P polyvinylidene difluoride membrane and SuperSignal enhanced chemiluminescence reagents were obtained from Amersham Biosciences and Pierce, respectively.

Cell Culture—HPV16 immortalized premalignant oral keratinocytes (pp126 cells) were a gift from Dr. D. Oda (University of Washington, Seattle, WA) (26). Telomerase reverse transcriptase-immortalized normal oral keratinocytes (OKF6/T cells) were kindly provided by Dr. J. Rheinwald (Brigham & Women's Hospital, Harvard Institutes of Medicine, Boston, MA) (27), and SCC25 cells derived from squamous cell carcinoma of the oral cavity were obtained from American Type Culture Collection. pp126 cells were maintained in keratinocyte serum-free medium (Invitrogen) supplemented with 100 units/ml penicillin, 100 µg/ml streptomycin, 5 ng/ml epidermal growth factor, 50 µg/ml bovine pituitary extract supplied with the medium, and 0.09 mM CaCl2. OKF6/T cells were maintained in keratinocyte serum-free medium supplemented with 100 units/ml penicillin, 100 µg/ml streptomycin, 25 µg/ml bovine pituitary extract (supplied with the medium), 0.2 ng/ml epidermal growth factor, and 0.31 mM CaCl2. SCC25 were routinely maintained in Dulbecco's modified Eagle's medium and Ham's F-12 medium (1:1) containing 10% fetal calf serum and supplemented with 100 units/ml penicillin and 100 µg/ml streptomycin.

A kidney epithelial cell line derived from {alpha}3 integrin-null mice, as well as these cells reconstituted with wild type human {alpha}3 integrin or mutant (H245A) human {alpha}3 integrin were developed as previously described (25, 28).{alpha}3 integrin-deficient cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Hyclone), and the {alpha}3-reconstituted cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 50µg/ml of zeocin (Invitrogen).

Prior to treatment with antibody-coated beads, the cell monolayers were released from culture flasks by the addition of trypsin/EDTA, seeded at a constant density of 0.7 x 105 cells/well into 12-well tissue culture plates, and allowed to attach overnight in the medium described above. The cells were then washed twice with PBS, incubated for 15 h in medium lacking bovine pituitary extract and EGF, and supplemented with fresh bovine pituitary extract/EGF-free medium prior to treatment with antibody-conjugated latex beads (20 µl of bead slurry). Following incubation for the indicated time periods, the conditioned media were collected for uPA activity determination. Alternatively, the cell lysates were prepared using 50 mM Tris, 150 mM NaCl, 1% Nonidet P-40, 0.1% SDS (mRIPA). The total protein concentration of the lysates was analyzed. In some experiments, inhibitors of signaling pathways including PD98039, PP2, AG1478, LY294002, SB203580, or Me2SO (vehicle control) were added to culture wells 120 min prior to the introduction of integrin clustering beads. The cells were found to be >95% viable by exclusion of trypan blue at the highest concentrations of all the inhibitors used.

Preparation of Antibody-coated Latex Beads—Integrin antibodies and their IgG controls were covalently linked to 3.0-µm latex beads using a kit from Polysciences, Inc. (Warrington, PA) following the manufacturer's instructions. In brief, 250 µl of 2.5% bead slurry (Polysciences, Inc.) was used for each batch bead preparation. The beads were washed in PBS and resuspended in 500 µl of 8% gluteraldehyde. The bead mixture was rotated for 4–6 h at room temperature, centrifuged, washed thoroughly, and finally resuspended in 700 µl of PBS. 100 µg of antibody was added to the beads in PBS and incubated overnight on a rotator, following which beads were washed of excess, unbound antibodies, blocked with 0.2 M ethanolamine and bovine serum albumin and finally resuspended in 250 µl of storage buffer.

uPA Activity Assay—Net plasminogen activator activity in conditioned media was quantified using a coupled assay to monitor plasminogen activation and the resulting plasmin hydrolysis of a colorimetric substrate (D-Val-Leu-Lys-p-nitroanilide; Sigma) as described previously (23). Because the assay measures both uPA and tissue-type plasminogen activator activity, control reactions contained 10 µg/ml of the anti-catalytic uPA antibody (American Diagnostica) to assess the tissue-type plasminogen activator levels in cells. None of the oral cell lines tested expressed any tissue-type plasminogen activator, and all of the plasminogen converting activity in the conditioned medium was thus attributed to uPA.

uPA Promoter Assays—p-uPA-CAT-2350 containing the sequence –2350 to +30 from the start site of the uPA gene was kindly provided by Dr. Douglas Boyd (M. D. Anderson Cancer Center, Houston, TX). The construct containing the uPA promoter was digested with SmaI and cloned into the SmaI site of the promoterless luciferase reporter vector pGL3 (Promega) to generate the 2350 uPA-Luc construct. The clones were verified by sequencing. To generate the –1967 AP1-Luc, a QuikChange site-directed mutagenesis kit (Stratagene) was used. The oligonucleotides used to generate this mutation were: sense, CCT CTT TGT CCA GGA AAT GAA aga ATC TGT CCT CAG CAA TCA GCA TGA, and antisense, GTC ATG CTG ATT GCT GAG GAC AGA tct CTT CAT TTC CTC CTG GAC AA GAG G. The mutated bases are shown as lowercase letters. The clones were verified by restriction digestion and confirmed by sequencing.


Figure 1
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  		FIGURE 1.
Integrin aggregation promotes uPA expression. Premalignant oral keratinocytes pp126 or OKR6/T, asindicated (0.7 x 105 cells/wellof a 12-well dish), were cultured for 15–18 h and shifted to growth factor-free medium for an additional 15 h. The cells were then treated with 20 µl of antibody-coated (IgG or {alpha}3 or beta1) latex beads or left untreated (designated 0). After 22 h, conditioned medium was collected, and uPA activity was determined by a colorimetric assay.

 
Subconfluent OKF6/T cells were grown in their normal growth medium and transfected with 2 µg of the reporter constructs using TransIT keratinocyte transfection reagent (Mirus, WI). The cells were then washed with growth factor-free medium after 12 h and treated with the integrin clustering beads coated with IgG, {alpha}3 integrin, or beta1 integrin antibodies. After 18 h, the cells were washed with cold PBS and lysed, and the luciferase activities were determined by luminometry. The results were normalized to protein concentration and expressed relative to control cells.

Detection of p44/42 ERK and Src Activation—To evaluate the phosphorylation state of ERK and Src, the cells (0.7 x 105) were cultured overnight in growth factor-free medium followed by treatment with IgG-, {alpha}3-, or beta1-coated beads as described above. At varying time points, the cells were lysed with mRIPA buffer with 1 mM sodium orthovanadate and extracted on ice for 20 min. The lysates were centrifuged, protein concentration was determined using the Bio-Rad protein assay kit, and equal amounts of cellular protein (20 µg) were electrophoresed on SDS-polyacrylamide gels and electroblotted to Immobilon. The blots were then probed with anti-ERK1/2 antibody (1:1000) to detect total ERK1/2 expression or with anti-ACTIVE-MAPK p42/p44 (1:2000) to detect the phosphorylated, active forms of ERK or anti-phospho Src (1:1000) to estimate the level of active Src.

Development of uPAR-deficient OKF6/T Cells—An siRNA knock-down approach was used to generate cells with reduced levels of surface uPAR. The paired oligonucleotides indicated below were annealed and ligated to BbsI-cut vector (psiRNAhH1neo from Invivogen) and transformed into HB101 competent cells. target seq2 oligonucleotide 4A (5'-tcccaagccgttacctcgaatgcatttcaagagaatgcattcgaggtaacggctttt-3') and target seq2 oligonucleotide 4B (5'-caaaaaaagccgttacctcgaatgcattctcttgaaatgcattcgaggtaacggctt-3') DNA was isolated (Qiaprep spin miniprep kit; Qiagen), and the identities of the clones were confirmed by restriction digestion and sequencing with primer OL381 (sequencing primer oligonucleotide OL381, 5'-ccctaactgacacacattcc-3'). Selected clones were then grown in 500-ml cultures, and DNA isolations were done using a nuclease-free DNA isolation kit (Qiagen). OKF6/T cells were transfected by electroporation using the human keratinocyte nucleofector kit and device (Amaxa) following the recommended protocol. Briefly, the cells were cultured to ~65% confluence, trypsinized, and resuspended at a density of 500,000 cells/100 µl nucleofector solution. DNA (1.5 µg in less than 5 µl) was added to each aliquot of cells and gently mixed, and each aliquot was electroporated. After 24 h under nonselective conditions, the medium was replaced with medium containing 35 µg/ml G418. During outgrowth, the cells were subcultured before reaching 70% confluence, and the selection medium was replaced every 2–3 days. Colonies were picked at the 100 cell stage and grown until they were numerous enough to test for knockdown of uPAR by flow cytometry.

Flow Cytometry—Surface expression of uPAR was determined using flow cytometry by incubating 1.8 x 105 cells/100 µl of medium containing antibodies against human uPAR (3937 from American Diagnostica, 1:100) for 45 min at room temperature. The cells were then washed twice with PBS and incubated with the corresponding fluorescein isothiocyanate-conjugated secondary antibody (anti-mouse IgG fluorescein isothiocyanate GM488 from Molecular Probes (Eugene, OR)) at a 1:1000 dilution for 30 min in the dark at room temperature. The cells were washed twice with PBS and resuspended in medium for fluorescence analysis on an Epics XL-MCL flow cytometer (Beckman Coulter, Hialeah, FL). Control experiments contained only the appropriate secondary antibody.

Cell Surface Biotinylation—Cells transfected with uPAR siRNA were grown in a 6-well plate, washed with ice-cold PBS, and incubated at 4 °C with gentle shaking for 30 min with 0.5 mg/ml cell-impermeable Sulfo-NHS-Biotin in ice-cold PBS, followed by washing with 100 mM glycine to quench free biotin. The cells were then detached by scraping, lysed in modified RIPA buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, and 0.1% SDS) with proteinase inhibitors, and clarified by centrifugation. To isolate biotinylated cell surface proteins, an equal amount of protein from each of the samples was incubated with streptavidin beads at 4 °C for 14 h, followed by centrifugation. After boiling in Laemmli sample dilution buffer to dissociate streptavidin bead-biotin complexes, the samples were analyzed by SDS-PAGE (9% gels) and immunoblotted for uPAR (1:1000; American Diagnostica, clone 399R).

Analysis of Filopodial Proteins—The relative localization of proteins in cellular versus filopodial fractions was evaluated following biochemical isolation of filopodia as described (29). Briefly, six-well plates containing 1.0-µm pore size membrane inserts were coated on both top and bottom sides with rat tail collagen (BD Biosciences) to a concentration of 10 µg/ml. The collagen was diluted with 0.1 M Na2CO3, pH 9.6, and kept overnight at 4 °C for coating. Before the experiment, the inserts were washed twice with PBS. Nearly confluent dishes of OKF6/T cells were first serum-starved overnight. The cells were then trypsinized and neutralized with soybean trypsin inhibitor, centrifuged, and dissolved in serum-free medium. The cells (1 x 106) were then plated on top of each insert in growth factor-free medium. Each bottom chamber contained 2.5 ml of serum-free medium containing EGF (.25 µg/ml) as a chemoattractant. Following a 12-h incubation (empirically determined based on the lack of visible nuclei in the "bottom" fraction), total cell lysate was collected from the top and bottom of paired inserts using lysis buffer (100 mM Tris, 4.5 mm EDTA, 150 mM NaCl, 1% SDS, and protease inhibitor mixture). The top and bottom lysates were collected and pooled from the replicate chambers, protein concentrations were determined, and samples (40 ug) were then analyzed by Western blotting (29, 30). The effect of peptide {alpha}325 or scrambled control peptide on filopodial protrusion was evaluated by performing the above assay in the presence of peptide (20 µM) followed by analysis of total filopodial protein as described above.


Figure 2
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  		FIGURE 2.
Induction of uPA activity by integrin clustering requires activation of ERK and Src kinases. To identify the signaling pathways involved in integrin mediated induction of uPA activity, the cells were cultured as described in Fig. 1 following preincubation with MEK inhibitor PD98059 (A, 10 µM) or Src kinase inhibitor PP2 (B, 10 µM) or dimethyl sulfoxide (DMSO) control (1:1000) for 2 h. The cells were then treated with antibody- or IgG-coated beads, as indicated, in the continued presence of the inhibitors. Conditioned medium was collected after 22 h and assayed for uPA activity (A and B). Alternatively, the cells were lysed after 1.5 h (C and D), the total protein levels were determined, and 20 µg of protein was analyzed in Western blot for phoshpho-ERK1/2 and phospho-Src levels as described under "Experimental Procedures." The blots were stripped and reprobed for total ERK or Src as loading controls. Similar responses were observed with both pp126 and OKF6/T cells, and representative data from OKF6/T cells are shown. E and F, semi-quantitative densitometric scanning of data in D normalized to total ERK levels.

 
Analysis of Invasion—Invasive activity was quantified using a Boyden chamber (8-µm pore size) coated with Matrigel (10 µg for 1 h at room temperature) as described earlier (23, 31). The cells (2 x 105) were added to the upper chamber in 500 µl of serum-free medium. Following 24 h of incubation at 37 °C, the noninvading cells were removed from the upper chamber with a cotton swab, the filters were fixed and stained with Diff-Quik stain, and the invading cells adherent to the underside of the filter were enumerated using an ocular micrometer and counting a minimum of 10 high powered fields. The data are expressed as relative migration (number of cells/field). Control experiments contained anti-catalytic uPA function blocking antibody (antibody 394; American Diagnostica), uPAR blocking antibody (antibody 3936; American Diagnostica), murine IgG (all antibodies, 20 µg/ml), or exogenous uPA (0.05 µg/ml).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Adhesion-regulated Proteinase Expression—Integrin clustering occurs at sites of cellular contact with three-dimensional matrices, such as may be encountered by cells penetrating an extracellular matrix barrier (32, 33). Integrins signal cellular responses by regulating the formation of signal transduction complexes on a cytoskeletal framework, and this integration of signaling and cytoskeletal events is dictated by the physical nature of the integrin-ligand interaction. Low valency integrin occupancy can be induced by matrix protein fragments or soluble integrin subunit-specific antibodies and results in redistribution of the integrin to focal adhesions without activation of tyrosine kinase signaling or accumulation of cytoskeletal components (32, 33). Integrin occupancy by a multivalent ligand such as presented by intact three-dimensional extracellular matrix leads to a more robust cytoplasmic response characterized by the accumulation of a large variety of cytoskeletal (e.g. talin, {alpha}-actinin, paxillin) and signaling (e.g. Src, ERK, c-Jun N-terminal kinase) molecules at the cytoplasmic face of the integrin. This response can be mimicked by integrin subunit-specific antibodies immobilized on beads (32, 33). We have previously demonstrated preferential adhesion of premalignant oral keratinocytes to laminin-5 and collagen I substrata via beta1 integrins, predominantly {alpha}3beta1 (23). Furthermore, culturing OKF6/T or pp126 cells in three-dimensional collagen gels or in the presence of beads coated with type I collagen or laminin-5 results in a significant increase in uPA activity (not shown and Ref. 23). Thus, to model cellular interaction with a three-dimensional matrix that promotes multivalent {alpha}3beta1 integrin aggregation, integrin subunit-specific antibody-coated beads were used to cluster {alpha}3beta1 integrins on the cell surface, followed by evaluation of uPA activity in conditioned medium as previously described (23, 31, 34). A significant increase in uPA activity was observed in the conditioned medium of premalignant pp126 and OKF6/T oral keratinocytes in response to {alpha}3 or beta1 integrin clustering (Fig. 1), relative to IgG beads (gray bars) or untreated controls (open bars). In control experiments, neither soluble antibodies nor bead-immobilized antibodies directed against the {alpha}2, {alpha}5, {alpha}6, or beta4 integrin subunits altered uPA expression levels (Ref. 23 and not shown).

To evaluate potential mechanisms by which integrin clustering may up-regulate uPA expression, the cells were treated with pharmacologic inhibitors of second messenger pathways known to be activated by integrin signaling. Following overnight culture in growth factor-free medium, the cells were preincubated for 2 h with specific inhibitors of MEK (PD98059, 50 µM), Src family kinases (PP2, 10 µM), p38 MAPK (SB202190, 10 µM), PI3K (LY294002, 10 µM), EGF receptor tyrosine kinase (AG1478, 10 µM), or Me2SO control, followed by treatment with integrin antibody beads for 22 h to allow for accumulation of measurable uPA. Conditioned media were then collected, centrifuged, and assayed for uPA activity. Similar responses were observed with both pp126 and OKF6/T cells, and representative data from OKF6/T cells are shown (Figs. 2 and 3). Treatment of cells with both the MEK inhibitor PD98059 and the Src inhibitor PP2 abrogated the production of uPA in response to {alpha}3beta1 integrin clustering (Fig. 2, A and B). Clustering of integrin {alpha}3beta1 induced phosphorylation of both Src (2.5–5.3-fold increase) and p44/42 ERK (3.0–4.6-fold increase) (Fig. 2, C–F), and inhibition of Src kinase activity with PP2 abrogated the phosphorylation of ERK1/2, implying that the activation of MEK is downstream of Src kinase activity. In contrast, pharmacologic inhibitors of PI3K (LY294002) or p38 MAPK (SB202190) were ineffective in blocking {alpha}3beta1 integrin-induced uPA expression (Fig. 3A).


Figure 3
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  		FIGURE 3.
{alpha}3beta1 integrin-mediated induction of uPA activity does not require p38 MAPK, PI3K, or EGFR kinase activities. A, following overnight culture in growth factor-free medium, the cells were pretreated with dimethyl sulfoxide (DMSO) (1:1000), p38 MAPK inhibitor SB202190 (10 µM), or PI3K inhibitor LY294002 (10 µM). After 2 h, the cells were treated with designated antibody coated beads in the continued presence of the inhibitors. After 22 h, conditioned media were collected and assayed for uPA activity as described. B and C, cells were cultured for 15 h without growth factors, pretreated with EGFR kinase inhibitor AG1478 where indicated (AG) for 2 h, and then treated with either 10 nM EGF (B) or 20 µl IgG or beta1 integrin antibody-coated beads (C). After 22 h, conditioned media were assayed for uPA activity. Similar responses were observed with both pp126 and OKF6/T cells, and representative data from OKF6/T cells are shown.

 
Because beta1 integrin clustering can induce ERK1/2 phosphorylation via transactivation of the EGFR (35, 36) and EGF treatment has been reported to induce uPA expression in many cell types including oral squamous cells (37–39), the role of EGFR transactivation in integrin-mediated uPA regulation was evaluated. In control experiments, EGF treatment increased uPA activity in pp126 cells, and this induction was blocked by the EGFR tyrosine kinase inhibitor AG1478 (Fig. 3B). In addition, clustering of {alpha}3beta1 integrin resulted in transactivation (phosphorylation) of the EGFR (not shown). However inhibition of EGFR kinase activity by AG1478 did not alter the up-regulation of uPA expression observed following integrin clustering (Fig. 3C), suggesting independent pathways of uPA regulation by EGF and {alpha}3beta1 integrin.


Figure 4
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  		FIGURE 4.
Integrin {alpha}3beta1 induces transcriptional activation of the uPA promoter via the –1967 AP1 site. OKF6/T cells were transiently transfected with a uPA promoter/luciferase construct (–2350 to +30), either wild type (top bars) or mutated at the AP1 site (–1967, bottom bars). After 14 h of transfection, the cells were washed and incubated in growth factor-free medium for an additional 8 h, followed by treatment with IgG- or integrin antibody-coated beads as indicated. After 18 h, the cell lysates were evaluated for luciferase activity as described under "Experimental Procedures."

 
ERK1 phosphorylation induces uPA promoter activation in oral SCC cells (40), and we previously reported that integrin-stimulated uPA expression is blocked by the general transcription inhibitor actinomysin D (23). To determine whether integrin-induced uPA expression was due to transcriptional activation of the uPA gene, luciferase reporter gene assays were performed using the sequence –2350 to +30 of the uPA promoter. OKF6/T cells were transiently transfected with the uPA promoter-luciferase construct, serum-starved overnight, and then treated with the {alpha}3 or beta1 antibody-coated or IgG control beads for an additional 18–20 h prior to lysis and measurement of luciferase activity. Regulation of uPA expression occurred at the transcriptional level, because clustering of {alpha}3beta1 integrin led to an increase in luciferase activity in cells transfected with the uPA promoter/luciferase reporter construct (Fig. 4). Mutation of the AP-1-binding site (–1967), previously shown to be required for maximal ERK1-mediated promoter activation in squamous cells (40, 41), blocked integrin-regulated transcriptional activation (Fig. 4). Basal promoter activity from untreated or IgG bead-treated cells was unaffected by mutation of the AP-1 site (not shown).

uPAR Is an Essential Contributor to Integrin {alpha}3beta1-induced uPA Expression—Our results demonstrate that multivalent aggregation of {alpha}3beta1 integrin leads to up-regulation of uPA expression via a Src/MEK/ERK1-dependent pathway, suggesting a mechanism whereby matrix status may influence pericellular proteolytic potential. Because integrin clustering also induces a dramatic redistribution of uPAR to sites of clustered integrins and formation of co-immunoprecipitable complexes between {alpha}3beta1 and uPAR (23), the requirement for direct uPAR/{alpha}3beta1 integrin interaction in regulation of proteinase expression was evaluated using three distinct approaches. First, a blocking strategy was employed using a peptide derived from the W4 repeat region of {alpha}3 integrin ({alpha}325) that was demonstrated to block physical association between uPAR and {alpha}3 integrin and subsequent signaling events (24). It was previously reported that peptide {alpha}325, but not homologous sequences from {alpha}5 or {alpha}v integrins, abrogated uPAR binding to recombinant {alpha}3beta1 (24), indicating that {alpha}325 specifically interferes with uPAR binding to {alpha}3beta1 integrin rather than other integrins. The cells were preincubated with {alpha}325 peptide or a scrambled sequence control (sc325) for 2 h prior to the addition of integrin antibody-coated beads. Clustering of {alpha}3beta1 in the presence of {alpha}325 abolished integrin-induced uPA expression (Fig. 5), suggesting that uPAR/{alpha}3beta1 binding is required for initiation or potentiation of signaling events that lead to induction of uPA expression. The scrambled peptide had no effect on {alpha}3beta1 integrin-induced uPA expression (Fig. 5). In additional controls, clustering of uPAR alone, using bead-immobilized anti-uPAR antibodies, was not sufficient to induce uPA expression (not shown).

In the second approach, a genetic strategy was employed to test the functional contribution of uPAR/{alpha}3beta1 integrin interaction to uPA regulation. Epithelial cells from mice genetically deficient in {alpha}3 integrin expression (28) were reconstituted with wild type or mutant human (H245A) {alpha}3 integrin. His245 in the W4 repeat region of {alpha}3 integrin has been shown to be essential for uPAR/{alpha}3beta1 complex formation and signaling (25). Although the {alpha}3-deficient R10 cells failed to respond to clustering (negative control; Fig. 6A), reconstitution of cells with wild type human integrin {alpha}3 subunit restored integrin-regulated uPA expression (Fig. 6A). Furthermore, this induction was inhibited by the uPAR-blocking peptide {alpha}325, but not the scrambled control peptide sc325 (Fig. 6A). This result was confirmed by reconstitution of knockout cells with an {alpha}3 H245A mutant that retains the matrix-binding site but is incapable of forming complexes with uPAR (25). Although {alpha}3 antibody beads bound indistinguishably to cells expressing either wild type or H245A {alpha}3 and precipitated similar levels of the {alpha}3 integrin in control experiments (Fig. 6, C and D), uPA expression was not induced in the H245A mutant reconstituted cells that lack uPAR/{alpha}3beta1 lateral association (Fig. 6B), confirming the critical role of uPAR in integrin-regulated proteinase expression in this system.


Figure 5
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  		FIGURE 5.
Disruption of {alpha}3beta1/uPAR interaction abolishes integrin-mediated induction of uPA activity. OKF6/T cells were preincubated in the presence of {alpha}325 peptide (20 and 40 µM as indicated) to disrupt uPAR/{alpha}3 integrin binding, equal concentration of scrambled peptide, or 1:1000 dilution of vehicle control (DMSO) for 2 h. Following preincubation, the cells were treated with bead-immobilized IgG or {alpha}3 integrin antibodies, as indicated, in the continued presence of the peptides. After 22 h, conditioned media were assayed for uPA activity using a coupled colorimetric assay.

 
Lastly, depletion of surface uPAR using siRNA was also used to examine the functional relevance of uPAR/{alpha}3beta1 integrin interaction in proteinase induction. A relative decrease in surface uPAR of 30–60% was achieved using this strategy as quantified by FACS analysis (Fig. 7, A and C) and surface biotinylation, shown for two representative clones in Fig. 7A. Down-regulation of uPAR did not affect the surface levels of {alpha}3 or beta1 integrins (Fig. 7A) or alter basal uPA activity (data not shown). However, significant changes were observed in responsiveness to integrin clustering as a result of uPAR knockdown. Whereas {alpha}3beta1 integrin clustering results in activation of ERK in untransfected (Fig. 2, C and D) or vector-transfected cells (Fig. 7B), robust ERK activation was not observed in uPAR knockdown cells, shown for a representative clone (Fig. 7B, fourth lane). Consistent with the lack of ERK1/2 phosphorylation, down-regulation of surface uPAR also resulted in the loss of uPA induction following {alpha}3beta1 integrin clustering (shown for three representative clones; Fig. 7C), further supporting the hypothesis that uPAR/{alpha}3beta1 integrin interaction potentiates integrin signaling that controls proteinase regulation pathways in response to microenvironmental cues.


Figure 6
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  		FIGURE 6.
Analysis of murine {alpha}3–/– cells reconstituted with wild type and mutant human {alpha}3 integrin. A, epithelial cells from {alpha}3–/– mice and the same cells stably transfected with human {alpha}3 integrin (25) were treated with beads coated with IgG or anti-{alpha}3 antibodies as described above, and 22 h conditioned media were assayed for uPA activity. The results are normalized relative to IgG-bead treated cells (designated 1). Pretreatment with peptide {alpha}325 (40 µM) but not the scrambled control abrogates uPA induction in response to integrin clustering. B, {alpha}3–/– murine epithelial cells stably transfected with wild type (Hu-{alpha}3) or mutant (Hu-{alpha}3-H245A) {alpha}3 integrin were treated with beads coated with IgG or anti-{alpha}3 antibodies, and conditioned media were analyzed as described above. Changes in uPA activity are expressed as fold difference from the IgG-bead control. C, {alpha}3–/– murine epithelial cells stably transfected with wild type (Hu-{alpha}3) or mutant (Hu-{alpha}3-H245A) {alpha}3 integrin were incubated with {alpha}3 integrin antibody beads or isotype-matched control IgG beads and examined visually using phase contrast microscopy. Integrin {alpha}3 subunit antibody-coated beads bound avidly (panels b and d) to cells reconstituted with both wild type (panel b) and H245A mutant {alpha}3 integrin (panel d), relative to loosely adherent IgG control beads (panels a and c). Similar results were obtained using premalignant oral keratinocytes (23). D, murine epithelial cells treated with IgG or anti {alpha}3 integrin beads were lysed with mRIPA buffer, and the beads were precipitated by centrifugation, washed with PBS, and analyzed by Western blotting to demonstrate that the relative efficiency of {alpha}3 integrin antibody bead binding to either wild type or H245A mutant {alpha}3 integrin is identical.

 


Figure 7
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  		FIGURE 7.
Down-regulation of uPAR expression blocks integrin-mediated uPA induction. A, vector control and uPAR siRNA expressing clones (KD7 and KD51) were cultured on six-well dishes and surface-labeled with cell-impermeable Sulfo-NHS-Biotin. Labeled proteins were then precipitated with streptavidin ("Experimental Procedures"), and surface uPAR and beta1 integrin expression levels were analyzed by Western blotting. Relative surface expression levels of {alpha}3 integrin and uPAR were also measured by flow cytometric analysis using primary antibodies P1B5 and 3937, respectively, and GM488 secondary antibody and mean fluorescence index values are shown. Note that although relative uPAR levels are down-regulated, the relative levels of {alpha}3 integrin expression are not altered by uPAR siRNA. B, a representative vector control and a uPAR knockdown clone were treated with beads coated with control IgG or anti-{alpha}3 integrin antibody for 1.5 h prior to lysis and immunoblotting with antibodies directed against phospho-ERK (P-ERK) or total ERK1/2 as indicated. C, vector control (V22 and V29) or uPAR knockdown clones (KD7, KD11, and KD51) were treated with bead-immobilized IgG, anti-{alpha}3, or anti-beta1, as indicated, and 22 h conditioned media were assayed for uPA activity. All of the knockdown clones had a 30–60% decrease in surface uPAR, as quantified by FACS (indicated below the clone numbers). The results are normalized relative to V22/IgG control (designated 1).

 


Figure 8
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  		FIGURE 8.
Functional relevance of uPAR/{alpha}3beta1 interaction. A, OKF6/T cells were seeded onto a tissue culture insert containing a porous membrane (1-µm pores) coated on the underside with type I collagen. After 24 h, filopodial protrusions were purified by scraping the underside of the membrane, solubilized, and analyzed for protein concentration. An equal amount (40 ug) of filopodial (bottom) or total cellular (top) protein was evaluated by Western blotting for {alpha}3 integrin, uPAR, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). B, OKF6/T cells were seeded as in A in the presence of peptide {alpha}325 (20µM, white bar) or scrambled peptide (20µM, black bar) as indicated. After 24 h, total cell lysate was collected from the top and bottom of paired inserts, protein concentration determined, and the relative amount of filopodial protein (bottom) is shown. C, following overnight growth in growth factor-free medium, OKF6/T cells were plated in the top chamber of Boyden chamber (8-µm pore size, coated with Matrigel) with peptide {alpha}325 (white bar) or control scrambled peptide (black bar). After 36 h, invading cells at the bottom chamber were stained and counted. The data are expressed as percentages of invasion relative to untreated control (designated 100%). D, representative clones of vector control and uPAR knockdown (KD) SCC25 cells were added to the top of Matrigel-coated Boyden chambers in the presence or absence of anti-catalytic uPA antibody (designated uPA Ab), function blocking uPAR antibody (designated uPAR Ab), or exogenous uPA (0.05µg/ml), as indicated. Invasion was evaluated as in C. The results are shown as percentages of invasion relative to vector control (Con, designated 100%).

 
Functional Relevance of uPAR/{alpha}3beta1 Interaction—We have previously reported that uPA contributes to the invasive behavior of premalignant and malignant oral keratinocytes (23, 31). Invasion of a three-dimensional matrix barrier is initiated by the protrusion of filopodia that serve as traction sites for migration (29). Activated integrins preferentially localize to the leading edge of the cell, initiate formation of focal adhesions, and transduce signals that regulate migration. Subsequent alterations in pericellular proteolytic potential can affect invasion of three-dimensional matrices by removal of matrix constraints. Biochemical analysis of filopodial protrusions indicated that although uPAR is distributed relatively equally in the cellular versus filopodial fractions (Fig. 8A, second panel), {alpha}3 integrin was preferentially localized in the filopodial pool (Fig. 8A, top panel). Because the uPAR is also found in the filopodial fraction, the opportunity for uPAR/{alpha}3beta1 interaction is likely substantially enhanced in this compartment. However, unlike {alpha}3 integrin, the entire population of cellular uPAR is not localized in filopodia, suggesting the existence of distinct cellular pools of uPAR. In control experiments, caveolin-1 was found in both the cellular and filopodial fractions, consistent with its localization in membrane lipid rafts (Fig. 8A, third panel), whereas proliferating cell nuclear antigen was not detected in the filopodia fraction (not shown). Disruption of uPAR/{alpha}3beta1 binding using peptide {alpha}325 blocked filopodial protrusion, resulting in a 50% decrease in protein content in the filopodial fraction (Fig. 8B). As a consequence, a significant decrease in Matrigel invasion was observed in the presence of peptide {alpha}325 (Fig. 8C), whereas the scrambled peptide sc325 has no effect on invasion (Fig. 8C). This is likely due to the loss of uPA induction, because function-blocking antibodies directed against both uPA and uPAR significantly inhibited invasion (Fig. 8D). A similar striking effect on invasive activity of malignant SCC25 cells is observed when comparing wild type to uPAR knockdown clones. In the representative experiment shown, a 6-fold decrease in invasive activity was observed relative to wild type cells (Fig. 8D) using a SCC25 uPAR knockdown clone with a 56% reduction in surface uPAR (as determined by FACS), and this is partially restored by the addition of exogenous uPA, providing additional support for the hypothesis that uPAR/{alpha}3beta1 interaction is an important regulator of cellular invasion.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
OSCC is the most common malignancy of the oral cavity, causing more deaths than any other oral disease. Approximately 30,000 new cases are diagnosed in the United States each year, resulting in patient deaths at a rate of 1 person/h, and the 5-year survival rate has not improved appreciably in over 20 years, remaining at a low 50% (www.NIH.gov). These statistics reflect a limited understanding of the molecular events that govern disease progression. Thus, a more detailed analysis of the cellular and biochemical processes that contribute to OSCC metastasis is a necessary prerequisite to the development of novel early detection and treatment strategies that favorably impact survival of OSCC patients. To this end, recent studies have utilized cDNA microarray analysis for genome-wide monitoring of genetic and epigenetic changes associated with OSCC primary tumors and lymph node metastases (3, 4). These studies have identified the proteinase uPA and the cell-matrix adhesion molecule {alpha}3 integrin as key candidate biomarkers for prediction of poor disease outcome. The current data support these findings and suggest a functional link between {alpha}3beta1 integrin-mediated matrix interaction and acquisition of uPA-dependent pericellular proteolytic activity.

The normal oral mucosa is supported by a laminin-5-rich basement membrane overlaying a connective tissue stroma with loose reticular collagen III and VI and thick fiber bundles of collagen I (42, 43). Hyperplastic premalignant lesions show enhanced laminin-5 staining intensity, whereas multifocal breaks in the basement membrane are observed in OSCC, indicative of proteolytic degradation (43, 44). Laminin-5 is also observed in stromal tissues adjacent to budding carcinoma cells, suggesting that OSCC invasion is guided by the laminin-5 matrix (44). This hypothesis is supported by tissue culture models, wherein invading OSCC cells deposit laminin-5 at the matrix interface (45). Two cellular integrins interact with laminin-5, {alpha}3beta1, and {alpha}6beta4. Although {alpha}6beta4 is involved in hemidesmosome formation (46), {alpha}3beta1 regulates adhesion, spreading, and motility (47). Indeed, the {alpha}3beta1/laminin-5 interaction was recently shown to be a major contributing factor to the arrest of circulating tumor cells in the lung vasculature during pulmonary metastasis (48). This finding is consistent with the microarray data described above, as well as with studies of human OSCC tumors that demonstrate persistent, albeit disorganized, staining for {alpha}3beta1 integrin (1, 49) and FACS analysis of OSCC cell lines that show enhanced {alpha}3beta1 integrin expression (31).

In addition to persistent expression of {alpha}3beta1 integrin, invasive and metastatic OSCC is also characterized by enhanced expression of uPA and its receptor uPAR relative to the normal oral mucosa. Highly invasive OSCC cells exhibit constitutive ERK activation, culminating in elevated basal uPA levels (31, 40); however, the cellular mechanisms that underlie dysregulated uPA expression in OSCC are not well characterized. Previous studies demonstrated that uPA/R-integrin binding may modify integrin function, providing a potential mechanism whereby the GPI-anchored uPAR may indirectly couple to cytoplasmic signaling pathways and thereby participate in regulation of gene expression, cell cycle progression, and/or motility (18, 21). Our data support these findings and demonstrate that {alpha}3beta1 integrin clustering initiates a Src/MEK/ERK-dependent signaling pathway, resulting in transcriptional activation of the uPA promoter. Complex formation between {alpha}3beta1 integrin and uPAR is necessary for integrin-induced uPA induction, because uPA expression is substantially attenuated by inhibition of {alpha}3beta1/uPAR binding with peptide {alpha}325 (residues 241–257 of {alpha}3 integrin), siRNA down-regulation of surface uPAR, and expression of a mutated {alpha}3 subunit (H245A) that disrupts lateral association with uPAR.

The current data support the hypothesis that uPAR/{alpha}3beta1 interaction potentiates integrin signaling for enhanced proteinase expression in response to integrin clustering. UPAR/integrin interaction has been previously demonstrated to alter the relative magnitude and duration of MAPK signaling, thereby altering tumor cell behavior (19). Additional studies have shown that the ability of uPAR to function as a cis-acting ligand for {alpha}3beta1 leads to sustained low level activation of Src, in contrast with matrix engagement alone that promotes transient integrin signaling (25). Together these data support a model wherein matrix-induced clustering of {alpha}3beta1 integrin promotes sustained uPAR/{alpha}3beta1 interaction, thereby potentiating cellular signal transduction pathways culminating in activation of the uPA promoter. Further, we speculate that uPAR/{alpha}3beta1 signaling is constitutively active in malignant OSCC (31), resulting in matrix-independent pericellular proteolysis and subsequent invasive behavior.


   FOOTNOTES
 
* This work was supported by Grants RO1CA85870 (to M. S. S.), PO1DE12328 (to M. S. S.), and RO1HL44712 (to H. A. C.) from the National Institutes of Health and by funds from the H Foundation for Basic Science Research (to M. S. S.). 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

1 To whom correspondence should be addressed: Dept. of Cell and Molecular Biology, Northwestern University Feinberg School of Medicine, 303 E. Superior St., Lurie Bldg. 3-111, Chicago, IL 60611. Tel.: 312-908-8216; Fax: 312-503-0386; E-mail: mss130{at}northwestern.edu.

2 The abbreviations used are: OSCC, oral squamous cell carcinoma; uPA, urinary-type plasminogen activator; uPAR, uPA receptor; ERK, extracellular signal-regulated kinase; EGF, epidermal growth factor; EGFR, EGF receptor; siRNA, small interfering RNA; MAPK, mitogen-activated protein kinase; MEK, MAPK/ERK kinase; PI3K, phosphatidylinositol 3-kinase; PBS, phosphate-buffered saline; FACS, fluorescence-activated cell sorter. Back



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C.-H. Tang, M. L. Hill, A. N. Brumwell, H. A. Chapman, and Y. Wei
Signaling through urokinase and urokinase receptor in lung cancer cells requires interactions with {beta}1 integrins
J. Cell Sci., November 15, 2008; 121(22): 3747 - 3756.
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 Clin. Cancer Res.Home page
A. P. Mazar
Urokinase Plasminogen Activator Receptor Choreographs Multiple Ligand Interactions: Implications for Tumor Progression and Therapy
Clin. Cancer Res., September 15, 2008; 14(18): 5649 - 5655.
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 BloodHome page
S. von Vietinghoff, G. Tunnemann, C. Eulenberg, M. Wellner, M. Cristina Cardoso, F. C. Luft, and R. Kettritz
NB1 mediates surface expression of the ANCA antigen proteinase 3 on human neutrophils
Blood, May 15, 2007; 109(10): 4487 - 4493.
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J. Symowicz, B. P. Adley, K. J. Gleason, J. J. Johnson, S. Ghosh, D. A. Fishman, L. G. Hudson, and M. S. Stack
Engagement of Collagen-Binding Integrins Promotes Matrix Metalloproteinase-9-Dependent E-Cadherin Ectodomain Shedding in Ovarian Carcinoma Cells
Cancer Res., March 1, 2007; 67(5): 2030 - 2039.
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Y. Wei, C.-H. Tang, Y. Kim, L. Robillard, F. Zhang, M. C. Kugler, and H. A. Chapman
Urokinase Receptors Are Required for {alpha}5beta1 Integrin-mediated Signaling in Tumor Cells
J. Biol. Chem., February 9, 2007; 282(6): 3929 - 3939.
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