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Tumor Biology |
v
5 Vitronectin Receptor, Promoting Urokinase-dependent Cell Migration in Breast Cancer1
National Cancer Institute [M. V. C., M. C., P. F., A. Z., G. B., G. D.], Nuclear Medicine Center (C. N. R.), University Federico II [S. D. V., M. S.], and International Institute of Genetics and Biophysics [L. F., M. P. S.], 80131 Naples, Italy
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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v
3, and v5 Vn receptors in 10 human breast carcinomas. Here, we show the ability of uPAR to physically associate with v5 in the breast carcinomas examined. The functional effects of this interaction were studied using HT1080 human fibrosarcoma and MCF-7 human breast carcinoma cell lines, both exhibiting a urokinase-dependent physical association between uPAR and v5. Both cell lines respond to urokinase or to its noncatalytic amino-terminal fragment by exhibiting remarkable cytoskeletal rearrangements that are mediated by v5 and require protein kinase C activity. On the contrary, binding of Vn to v5 results in the protein kinase C-independent formation of F-actin containing microspike-type structures. Furthermore, v5 is required for urokinase-directed, receptor-dependent MCF-7 and HT1080 cell migration. These data show that uPAR association with v5 leads to a functional interaction of these receptors and suggest that uPAR directs cytoskeletal rearrangements and cell migration by altering v5 signaling specificity. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A large body of evidence assigns to urokinase [urokinase-type plasminogen activator (uPA)] a key role in tumor progression and invasion, by virtue of its ability to activate plasminogen, which degrades many ECM components, such as fibronectin, laminin, and proteoglycans, and activates latent collagenases (2) . The inoculation of metastatic Lewis lung carcinoma cells into plasminogen-deficient mice results in the formation of smaller and less hemorrhagic tumors than in control wild type mice (3) . Furthermore, the absence of uPA negatively affects the progression of chemically induced melanocytic neoplasms in mice (4) .
In addition to its proteolytic role, uPA can regulate cell mobilization, adhesion, proliferation, and transcription of specific genes through a catalytic-independent mechanism (5, 6, 7)
. A key player in this process is the specific cell surface uPAR, which binds with high affinity the ATF of uPA (8)
. uPAR is a highly glycosylated 55,000–60,000 Mr protein that includes the N-terminal uPA binding domain, designated D1, a connecting domain (D2), and the COOH-terminal domain bearing a glycosylphosphatidylinositol anchor (D3; Ref. 2
). D2 and D3 domains have the property to recognize the matrix-like form of Vn (9)
. The multiple molecular events following uPAR ligation with uPA include diacylglycerol formation in endothelial cells, activation of PKC, and phosphorylation of cytokeratins 8 and 18 in human epithelial cells (10
, 11)
. A transient modification of the Src-family kinase p56/59hck activation state has been also reported in mielomonocytic cells (12
, 13)
. All of these cell responses raise a question concerning the modality of uPAR signaling transmission, as this is restricted to the outer leaflet of the membranous bilayer and therefore requires a transmembrane "adaptor." Coimmunoprecipitation studies show that uPAR is associated in large molecular complexes with integrins, caveolin, and Src kinases (14, 15, 16)
. The reversible association with other receptors is supported by the uPAR lateral mobility in the plasma membranous bilayer and its focal redistribution upon interaction with uPA (17)
. The relevance of uPAR lateral mobility to signaling is further sustained by the finding that a nonsignaling uPA variant is also unable to mobilize the receptor (18)
. Integrin receptors are composed of and subunits that heterodimerize to produce more than 20 different receptors, capable of mediating a variety of cell responses, such as spreading and migration, control of gene expression, growth, and differentiation (19, 20, 21)
. Changes in integrin structure and/or expression are frequently associated with malignant transformation and tumor progression (22)
. Overexpression of v integrins occurring in human mammary carcinomas is associated to a widespread deregulated expression of other integrins, such as 2 and 64 (23)
.
uPAR and integrins share the ability to activate members of the Src family, as well as pp125FAK, further supporting the possibility that uPAR impinges on cell function via integrins (24)
. Following engagement with uPA, uPAR colocalizes or physically associates with 1, 2, 3, or 5 integrins both in vivo and in vitro (15
, 25
, 26)
. It is presently known that uPAR association suppresses the normal adhesive function of different integrins, suggesting that they may acquire new functional properties (16)
.
A shared ligand between integrins and uPAR is Vn that binds and stabilizes plasminogen activator inhibitor PAI-1 (27)
. Both, uPAR and PAI-1 bind to the somatomedin-like domain of Vn, and PAI-1 prevents Vn binding to the VnR, therefore stimulating cell detachment (28
, 29)
. The physical linkage between uPAR and VnR is supported by the colocalization of uPAR and v5 at focal contacts of human keratinocytes (30)
. Furthermore, Vn-dependent migration of human pancreatic carcinoma cells is inhibited by anti-uPAR or by anti-v5 antibody, suggesting a functional coupling between these two receptors (31)
.
The two known VnRs exhibit different functional properties, as they regulate two distinct pathways in angiogenesis (32
, 33) . Unlike v3, v5 can direct cell migration, as well as redistribution of talin, vinculin, and -actinin only in the presence of PKC activators (31
, 34)
.
We have previously shown the ability of membrane-associated uPARs to form high-affinity ternary complexes with ATF and Vn both in tumor cell lines and in breast carcinomas (35 , 36) .
The aim of this study was to investigate whether v3 and v5 VnRs play a role in the uPA-dependent tumor cell spreading and migration. First, we show that v5 copurifies with uPAR in breast carcinoma samples expressing high levels of these receptors. Second, we studied the functional consequences of uPAR-integrins interaction in HT1080 human fibrosarcoma and MCF-7 human breast adenocarcinoma cell lines, both of which express uPAR (36)
. Here, we report that treatment of both cell lines with uPA promotes the physical association of uPAR with v5 and that v5 is required for uPAR-directed cell migration. We also show that uPA promotes cytoskeletal rearrangements that are mediated by v5 in a PKC-dependent manner. Finally, the finding that PKC inhibitors selectively prevent uPA-dependent and not Vn-dependent effects on cytoskeleton suggest that uPAR association modifies normal v5 signaling specificity.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Antibodies.
Anti-uPAR 399 rabbit polyclonal antibody was from American Diagnostica (Greenwich, CT). Anti-uPAR R4 mAb was a gift of Dr. Gunilla Hoyer-Hansen (Copenhagen, Denmark; Ref. 37
). Anti-v5 mAb (clone P1F6) and anti-v chain mAb (clones VNR147 and VNR139) were from Life Technologies (Milan, Italy; Refs. 38
and 39
). Anti-v3 mAb (clone 23C6) was from Pharmigen (San Diego, CA; Ref. 40
). Anti-5 chain polyclonal antibody was from Chemicon Int. Inc. (Temecula, CA; Ref. 32
). Anti-3 chain mAb (clone 26; Ref. 41
), the positive control human fibroblast, and A431 extracts were from Transduction Laboratories (Lexington, KY). Anti-3 chain mAb (clone 61) was from DAKO (Copenhagen, Denmark; Ref. 25
).
Cell Lines and Culture Conditions.
HT1080 human fibrosarcoma and MCF-7 breast adenocarcinoma cell lines were grown in DMEM supplemented with 10% fetal bovine serum, 100 IU/ml penicillin, and 50 µg/ml streptomycin. The MCF-7/uPAR4 cell line is derived from the MCF-7 cell line, stably transfected with pcDNA3-uPAR, carrying the entire human uPAR cDNA excised from puPAR-1 (42)
. Briefly, 1 x 107 subconfluent MCF-7 cells were cotransfected with 30 µg of pcDNA3-uPAR and pRSVneo (10:1) by electroporation at 250 V and 960 microfarads. The neomycine-resistant clones were analyzed by radioreceptor binding assay with 125I-ATF, which revealed that MCF-7/uPAR4 clone produces about 5 times more uPAR than the parental cells (8)
.
Tissue Preparation.
Ten tumor biopsy specimens were obtained from patients undergoing surgery for a breast lump. Tumors included a total of seven ductal and three lobular carcinomas, whereas benign breast lesions consisted of five fibrocystic diseases. The specimens were immediately frozen in liquid nitrogen and stored at -80°C until used for immunocytochemistry and crude lysate preparation.
Immunostaining.
For immunohistochemical analysis of breast tumor specimens, 5-µm-thick frozen serial sections corresponding to the largest cross-sectional area of the tumor were cut, placed on clean glass slides, air dried, and subjected to immunostaining with the streptavidin-biotin-peroxidase method as described previously (35
, 36)
. For immunocytochemical staining, HT1080 and MCF-7 cells were grown on glass slides in the presence of serum. Before VnR immunostaining, samples were fixed with 3.5 formaldehyde, 0.1% Triton X-100 in PBS for 10 min at 4°C. In all cases, slides were incubated overnight at 4°C with 10 µg/ml R4 anti-uPAR, anti-v5, anti-v3 or anti-v chain clone VNR 147 mAbs. Photographs were taken on Kodak 100 ASA film at x400 magnification.
The intensity of immunostaining with anti-uPAR and anti-v5 mAbs was graded from 1 to 3+ when faint, moderate, or intense staining was observed in epithelial tumor cells.
Western Blot Analysis of uPAR Containing Immunocomplexes.
Two hundred mg of breast carcinoma samples were lysed using 600 µl of lysis buffer (10 mM Tris-HCl, pH 8.1, 140 mM NaCl, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 0.5% NP-40, 1% Triton X-100) and extracted for 30 min at 4°C. Lysates were cleared by centrifugation at 13,000 rpm for 10 min at 4°C, and the protein content was determined by the Bradford method. Eight hundred µg of each sample were preabsorbed with 20 µl of a 1:1 suspension of protein G-Sepharose for 2 h at 4°C and then immunoprecipitated overnight at 4°C using 10 µg/ml R4 or 399 anti-uPAR antibodies. The immunoprecipitates were recovered by absorption to protein G-Sepharose as described (35)
.
For analyzing uPAR-containing complexes from cell lines, acid-treated HT1080 cells were incubated for 1 h at 22°C with 10 nM recombinant uPA, with or without 500 nM urea-denatured Vn. Cells (5 x 106 cells/sample) were lysed in 500 µl of 10 mM Tris-HCl, pH 8.1, 140 mM NaCl, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 0.5% NP-40, 1% Triton X-100 for 30 min at 4°C. Lysates were then cleared by centrifugation, and 800 µg of each sample were immunoprecipitated with anti-uPAR 399 polyclonal antibody. Alternatively, 50 µg of acid-pretreated cell membrane fractions were incubated with 10 nM ATF in the presence or in the absence of 500 nM urea-denatured Vn and cross-linked using disuccinimidyl suberate according to a previously published procedure (36 , 43) . The samples were then immunoprecipitated with the indicated anti-uPAR antibodies.
In all cases, the immunoprecipitated proteins were separated by 6% SDS-PAGE under unreducing conditions and transferred to nitrocellulose membranes. Blots were blocked overnight with 5% nonfat dry milk, 3% BSA and then incubated with a 2 µg/ml concentration of the indicated antibody for 2 h at 4°C. After washing with 0.1% Tween-20 in PBS, the filters were incubated with 1:4000 biotinylated antimouse immunoglobulins for 1 h at 22°C. They were extensively washed and finally analyzed using the ECL system, according to the manufacturer’s recommendations.
Analysis of Cytoskeleton.
HT1080 and MCF-7 cells were harvested by a mild trypsinization and incubated with 10% fetal bovine serum/DMEM for 1 h at 37°C, 5% CO2. Then, cells were acid treated, washed with PBS, and incubated in serum-free medium with 10 nM recombinant uPA or ATF in the presence or in the absence of 500 nM urea-denatured Vn for 1 h at 22°C. When specified, the cells were pretreated with a 5 µg/ml concentration of the indicated antibodies or with 200 nM calphostin C, 20 µM bisindolylmaleimide (GF109203X), or 10 µg/ml cycloheximide for 1 h at 22°C. The cells were fixed with 3.5% formaldehyde for 10 min on ice, permeabilized with 0.1% Triton X-100, and incubated for 40 min with 0.1 µg/ml rodhamine-conjugated phalloidin. Finally, cell cytoskeleton was examined by a fluorescence microscope (Axioplan Zeiss), and photographs were taken on Kodak 400 ASA film at x1000 magnification.
Cell Migration Assay.
Cell migration assays were carried out in Boyden chambers under serum-free conditions as described previously, with minor modifications (12)
. The 8 µm pore size polycarbonate filters were coated with either 5 µg/ml Vn or 50 µg/ml collagen. Subconfluent HT1080 or MCF-7/uPAR4 cells were harvested, acid treated, and incubated with or without a 10 µg/ml concentration of the indicated anti-VnR antibodies for 1 h at 22°C in serum-free medium. In all cases, 2 x 105 viable cells/sample were allowed to migrate toward 10 nM recombinant uPA or ATF for 3 h at 37°C. Then, cells were fixed in ethanol and stained with hematoxylin, and 10 random fields/filter were counted at x200 magnification.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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v5, and v3 VnRs in 10 human breast carcinomas and in 5 benign breast lesions by immunohistochemical staining with R4 anti-uPAR, P1F6 anti-v5, and 23C6 anti-v3 mAbs. Table 1
reports the clinical data and pathological findings of 10 human breast carcinomas (7 ductal and 3 lobular carcinomas). The intensity of both uPAR and v5 staining of epithelial tumor cells was graded as faint (grading 1), moderate (grading 2), or intense (grading 3; Table 1
). In agreement with our previous findings (35)
, each individual tumor showed a heterogeneous pattern of staining with anti-uPAR mAb (Table 1)
. A diffuse staining of the epithelial tumor cells in sections from ductal and lobular carcinomas was obtained using anti-v5 mAb. In positive tumor cells, a prominent staining of plasma cell membranes was often observed. Three representative cases are shown in Fig. 1A
. Both anti-uPAR and anti-v5 mAbs were slightly reactive to the five benign breast lesions examined, weakly staining ductal cells (not shown). On the contrary, anti-v3 mAb was poorly reactive toward the epithelial tumor cells, showing a strong reaction to the endothelial cells (Fig. 1A)
as well as to normal ductal cells (not shown).
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v chain of VnRs can be copurified with uPAR, crude lysates were prepared from breast tumor samples and subjected to immunoprecipitation with anti-uPAR antibodies. Then, the immunoprecipitated proteins were analyzed by immunoblotting for their v content. Anti-v VNR139 mAb reacted with a Mr 120,000 protein, indicating a physical association between uPAR and v (Fig. 1B)
. Similar results were obtained with 399 anti-uPAR polyclonal antibodies (not shown). Furthermore, we noticed that the relative intensity of the v band copurified with uPAR from T1, T2, and T3 tumors is consistent with their relative v5 grading. The absence of a specific reaction in samples incubated in the absence of the primary antibody was also observed.
uPA- and Vn-dependent Physical Association of uPAR and v5 in HT1080 and MCF-7 Tumor Cell Lines.
The results of the previous experiments suggest the occurrence of uPAR-v5 physical association in the epithelial tumor cells of breast carcinomas. To test whether this association may lead to a functional interaction, MCF-7 and HT1080 cell lines were preliminarily analyzed, by immunohistochemical staining, for the expression of uPAR, v3, v, and v5 using anti-uPAR R4, anti-v5, anti-v3, or anti-v chain clone VNR147 mAbs (Fig. 2
, top panel). Both cell lines express, although to a different extents, v5, v chain and uPAR. Anti-v3 mAb did not stain HT1080 cells, whereas it appreciably reacted with MCF-7 cells. Accordingly, when various anti-3 mAbs, such as clones 26 and 61, were incubated with HT1080 cells, again, a scarce specific staining was observed (not shown).
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v5 and/or v3 VnRs can be copurified with uPAR in an in vitro assay. To this end, acid-treated membrane fractions from HT1080 and MCF-7 cells were incubated with ATF in the presence or in the absence of urea-denatured Vn, solubilized, and immunoprecipitated with 399 or R4 anti-uPAR antibodies. Then, these samples were analyzed by immunoblotting for their v, 5, and 3 content. Anti-v VNR139 mAb reacted with a Mr 120,000 protein, thereby suggesting the uPA-dependent physical association of v with uPAR, in the presence of Vn (Fig. 2A)
. In vitro, v does not copurify with uPAR, in the absence of ATF or uPA (not shown). The absence of a specific reaction in samples incubated with a nonspecific rabbit serum and the absence of cross-reactivity between each secondary antibody and anti-uPAR or anti-v antibodies were also observed.
Immunoprecipitation of lysates from MCF-7 cells with R4 anti-uPAR mAb, followed by probing with anti-5 polyclonal antibodies, showed a specific band at Mr 82,000, in agreement with the 5 chain Mr (Fig. 2B)
. Conversely, the molecular species immunoprecipitated with 399 anti-uPAR polyclonal antibody from MCF-7 lysates did not react with anti-3 mAb (Fig. 2C)
. As a positive control, the reactivity of anti-5 and anti-3 antibodies was tested employing commercial extracts from either A431 epidermoid carcinoma cell line or human fibroblasts, respectively. These results indicate that uPAR associates with v and 5 chains following exposure to ATF, in the presence of Vn. To test whether the interaction between uPAR and v5 may occur in vivo, coimmunoprecipitation experiments were carried out on intact cells. Acid-treated HT1080 cells were incubated with or without 10 nM recombinant uPA, in the presence or in the absence of 500 nM urea-denatured Vn, under serum-free conditions. Crude lysate extracts were subsequently immunoprecipitated with 399 anti-uPAR polyclonal antibody, and the resulting immunoprecipitates were tested for the v content. Fig. 2D
shows that in the absence of uPA, the low amount of v copurifying with uPAR is comparable to that obtained following the addition of Vn alone (Lanes 1 and 3). It is evident that cell exposure to uPA greatly enhances the extent of uPAR-associated v. The low amount of v copurified with uPAR in the absence of exogenous uPA may be due to traces of uPA possibly secreted from HT1080 cells during the 1-h incubation. The combined use of Vn and uPA neither reduced nor enhanced the extent of v copurified with uPAR (Fig. 2D
, Lane 4). This experiment suggests that in vivo Vn is dispensable for this association to occur. Our previous data indicated that Vn is required for uPAR-v association in isolated membranes; these apparently contradictory results may be reconciled considering the possibility that uPAR may exclusively associate with the v5 active conformer. Therefore, in vitro, Vn may be required to convert v5 to its active state, whereas in vivo, integrin affinity could be regulated by an inside-out mechanism that depends on cell metabolic activity. This experiment confirms the central role of uPA in triggering uPAR-v5 interaction and raises the possibility of a functional cooperation between these two receptors.
Role of VnRs in uPA- and/or Vn-dependent Cytoskeletal Rearrangements.
The physical interaction between v5 and uPAR prompted us to examine whether this uPA-dependent coupling may indeed affect cytoskeletal arrangement. HT1080 cells were harvested, acid treated, and incubated with uPA and/or Vn in the presence or in the absence of anti-v5, VNR147 anti-v, anti-5, or anti-v3 antibodies. Staining with rodhamine-phalloidin showed that exposure to uPA remarkably modified the F-actin distribution in at least 50–60% of the cell population with the appearance of peripheral, filamentous structures, often localized at one pole of the cell and possibly resembling lamellipodia-type structures (Fig. 3A)
. Unlike uPA, Vn treatment resulted in the formation of short, thin, and homogeneously distributed microspike-type structures in about half of the cell population. These effects are combined in cells treated with uPA and Vn, although it is difficult to ascertain the relative contribution of each agonist. The effects promoted by uPA are catalytic-independent, as its ATF retains the same ability (not shown). As expected, preincubation of HT1080 cells with 399 anti-uPAR antibodies prevented most of the uPA-dependent cytoskeletal modifications, whereas it did not significantly affect Vn-induced filamentous structures (not shown). In keeping with the previous data, anti-v3 mAb prevents neither the uPA nor the uPA/Vn-induced F-actin redistribution (not shown). In control samples, preincubation of unstimulated HT1080 cells with anti-v5 or anti-v antibodies did not produce any effect on the cytoskeletal organization. On the contrary, preincubation with anti-v5 and anti-v chain mAbs prevented the uPA-dependent effects, indicating that v5 is required by uPAR signaling (Fig. 3A)
. Anti-v5 and anti-v mAbs also inhibited the effects of Vn, indicating that despite the markedly different rearrangements observed, v5 is a mediator of both Vn-dependent and uPA-dependent cell responses. The finding that v5 supports uPAR signaling is confirmed by the results of parallel experiments carried out in MCF-7 cells (Fig. 3B)
. Exposure of MCF-7 cells to uPA, in the presence of Vn, produced filipodia and lamellipodia-type structures similar to those observed in HT1080 cells, which were prevented by the addition of anti-v anti-5 or anti-v5 antibodies, whereas anti-v3 mAb was ineffective.
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v5-dependent, these pathways are somehow divergent. To gain insights in the functional interaction of uPAR and v5, we investigated the possibility that uPAR signaling in HT1080 and MCF-7 cell lines is mediated by PKC, as in HEp3 and in U937 cell lines (10
, 13)
. Therefore, urokinase-dependent cytoskeletal rearrangements were analyzed in cells previously treated with specific PKC inhibitors, such as calphostin C or bisindolylmaleimide (GF109203X). Although to a different extent, both inhibitors are effective at preventing uPA-induced rearrangements in both cell lines examined (Fig. 3A
and Table 2
). On the contrary, Vn-induced microspike-type structures were affected neither by calphostin C nor by bisindolylmaleimide treatments, suggesting that Vn acts in a PKC-independent manner. Similar data were obtained by analyzing cells pretreated with the protein synthesis inhibitor cycloheximide, which selectively prevented uPA-dependent and not Vn-dependent effects. This finding suggests that uPAR-dependent signaling requires a short-lived factor that is dispensable for the effects of Vn on cytoskeleton. Quantitative assessment of inhibition was attempted considering the number of cells with uPA-dependent filamentous structures, Vn-dependent microspikes, or uPA- and Vn-dependent combined rearrangements as 100%. The percentage of inhibition of each series of samples is reported as relative to each internal positive control (Table 2)
. Each experiment was repeated at least three times with the same results. In particular, the finding that PKC inhibitors and cycloheximide selectively block uPA-dependent and not Vn-dependent effects on cytoskeleton indicates the existence of two different activation modes of v5.
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v5.
and MCF-7/uPAR4 (Fig. 4B)
cells were carried out in Boyden chambers. As expected, preincubation of MCF-7/uPAR4 cells with 399 anti-uPAR antibodies caused a dramatic inhibition of uPA-dependent cell migration. Interestingly, preincubation of HT1080 cells with anti-v, anti-5, or anti-v5 antibodies caused a 66, 51, or 49% inhibition of the uPA-dependent cell migration onto Vn-coated filters, respectively. A 78, 81, or 89% inhibition of uPA-dependent migration of MCF-7/uPAR4 cells onto Vn was found following cell exposure to VNR147 anti-v, anti-5, or anti-v5 antibodies, respectively. These results provide a functional support to the previously observed uPAR-v5 physical association and indicate that this integrin is required for ligand-activated, uPAR-dependent cell migration. Similar results were obtained, in the absence of Vn, using collagen-coated filters. In this case, anti-v5 mAb caused a 47 and 67% inhibition of HT1080 and MCF-7/uPAR4 cell migration, respectively. Similar results were obtained using ATF as a chemoattractant (not shown). In keeping with previous findings, anti-v3 and anti-3 mAbs did not significantly modify uPA-dependent cell migration of both cell lines, again suggesting that v3 is not involved in uPAR signaling.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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v3 and v5 in 10 human breast carcinomas. First, we found that uPAR and v5 can be copurified in human carcinomas. Then, we show that this association also occurs in HT1080 fibrosarcoma and MCF-7 breast carcinoma cell lines. The functional interaction between uPAR and v5 VnR is shown by the inhibitory effect of anti-v, anti-5, and anti-v5 antibodies exerted on uPAR-dependent effects such as cytoskeletal arrangement and cell migration. The data also suggest that the uPA-dependent physical association between uPAR and v5 leads to the acquisition of new signaling properties by v5.
Changes of integrin expression and subcellular distribution have been reported in mammary tumor cell lines and in tissue sections (44, 45, 46, 47)
. These alterations have been shown to be associated with tissue disorganization, loss of polarity, increased tumor aggressiveness, and metastasis (45, 46, 47)
. Recently, Weaver et al. have reported that modification of cell surface 1 and 4 integrins influences mammary morphogenesis and regulates cell growth and signal transduction in malignant breast cells (48)
. These findings raise the hypothesis that integrins may play a crucial role in the expression of malignant phenotype in human epithelial breast cells. v3 VnR is considered an endothelial cell marker with significant prognostic value in breast cancer (49)
, and it promotes cell migration on Vn independently of growth factors or uPA-uPAR expression (31)
. On the contrary, v5 is expressed in a number of breast cancer cell lines (50)
and requires exogenous activation of PKC to mediate cell spreading and migration (34)
. Our data show the occurrence of a physical association between uPAR and v chain of VnR in malignant epithelial cells of breast carcinomas expressing high levels of uPAR and v5 but not v3.
Association of uPAR with v, as well as with 1 and 3 integrin receptor subunits in HT1080 cells plated on ECM-coated surfaces, has been reported (25)
. Here, we report the physical and functional association of uPAR with v5 in nonadherent cells, to avoid any interference by integrin activation. In addition, we selectively dissected the molecular and cellular effects following uPAR ligation with uPA. Although uPAR may be located in the close proximity of different integrins, the data presented here demonstrate that uPAR-dependent cell responses, such as cytoskeletal rearrangements and migration, are specifically mediated by v5. The functional coupling between v5 and ligand-activated uPAR is instrumental to gain insights into the role of integrins in uPAR-dependent signaling. Unlike v3, v5 is a Vn-dependent integrin receptor that can direct cell migration and stimulate a redistribution of talin, vinculin, and v-actinin only in the presence of PKC activators (31)
. In a variety of cases, it has been described the ability of uPAR to activate PKC and DAG formation (10
, 11) . Our data show that the ligation of v5 with Vn leads to the formation of microspike-type structures that do not require PKC activation. However, the finding that PKC is required for uPAR-dependent, v5-mediated effects, raises a question about the mechanism of PKC activation. One possibility is that uPAR triggers PKC activation by influencing the membrane level of diacylglycerol, via a presently unknown mechanism (11)
. Alternatively, uPAR ligation with uPA may lead to PKC activation through the recruitment of a specific partner in a signaling complex. The occurrence of a functional coupling between uPAR and v5 is in agreement with previous findings showing the ability of uPA-uPAR complexes to inhibit the adhesive function of 1 integrin (16)
. Here, we show that anti-v5 antibodies inhibit cell migration on Vn and collagen-coated filters, suggesting that uPAR signaling is not based on the normal adhesive function of v5. On the other hand, uPA does not simply activate v5 in a Vn-like manner but triggers unique effects that are PKC-dependent. These data definitely highlight an alternative activation mode of v5, triggered by uPAR.
The molecular mechanisms underlying the signaling ability of uPAR are intriguing, as this receptor lacks a transmembrane domain, suggesting the occurrence of a specific "adaptor." In this paper, we provide such evidence: uPA modulates cytoskeleton and cell migration via v5, although additional mediators, associated to this signaling complex may be required to fully support these effects. We could hypothesize that uPAR and integrin-containing signaling units recruit other components, such as caveolin, Src kinases, and PKC, thereby triggering a different pathway than integrins stimulated by their cognate ligand. On the other hand, it has been reported that a functional unit of uPAR, integrin, and caveolin regulates integrin function (14)
. Further work is required to address these issues.
The results presented here extend beyond the uPAR field, as they have important implications for the spatial and temporal regulation of integrin function by other receptor pathways that may be important in adhesion and migration. In this paper, we show the existence of two alternative activation modes of v5, either by Vn or by activated uPAR. It is tempting to speculate that the first mode is more appropriate for stable adhesion to the ECM and the latter for a reversible activation required during cell locomotion. The latter possibility is in agreement with previous findings showing a reversible association of uPAR with CR3 during granulocyte locomotion (51)
. Another example of "lateral" activation of integrins by proteases is provided by the activation of v3 by the PEX domain of the matrix metalloprotease 2 on the surface of angiogenic blood vessels (52)
. Cell migration implies the dynamic formation of adhesive contacts at the leading edge of the cell and disruption at the cell rear: in particular, at the cell front, the formation of cytoskeletal and catalytic signaling protein complexes promotes PKC activation (53)
. It is possible that during directional migration of HT1080 and MCF-7 cells, uPAR cycles between the cell front and rear, locally regulating adhesion through the local activation/disruption of its interaction with v5. The v5-uPAR interaction may be activated under both physiological and pathological conditions, due to an altered cell migration: we and others have previously shown a coordinate overexpression of uPA and uPAR on the cell surface of human breast carcinoma cells (35
, 54
, 55)
. Other authors claim that epithelial tumor cells bear uPARs and bind uPA produced by fibroblast-like stromal cells in a paracrine manner (2)
. Overexpression of v, in human mammary carcinomas has been reported (23)
. The finding that uPAR and v5 physically associate in breast carcinomas raises the possibility that they may functionally cooperate in vivo, thereby favoring the metastatic process. Our results show that epithelial tumor cells of breast carcinomas express considerable amount of v5 whereas v3 is expressed exclusively by endothelial cells. Blocking uPAR is a major goal of antimetastatic therapy (56
, 57)
. Molecular antagonists include peptides, antibodies, and antisense, as well as the naturally occurring phosphorylated uPA and the relative phosphorylation-like variants, which bind to uPAR but fail to activate receptor signaling (56
, 58) . In the emerging picture, the tumor cell metastasis can be regulated by a functional cooperation between uPA-uPAR complexes and VnR type v5. A detailed molecular analysis of the uPAR and v5 domains involved in the interaction may lead to the development of novel drugs, blocking this association and thereby inhibiting tumor invasion.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by the Associazione Italiana per la Ricerca sul Cancro. A. Z. is a recipient of a fellowship from Fondazione Italiana per la Ricerca sul Cancro. 
2 To whom requests for reprints should be addressed, at National Cancer Institute, Via M. Semmola, 80131 Naples, Italy. Phone: 39-081-5903569; Fax: 39-081-5461688; E-mail: stoppelli{at}iigbna.iigb.na.cnr.it
3 The abbreviations used are: ECM, extracellular matrix; uPA, urokinase-type plasminogen activator; uPAR, urokinase receptor; Vn, vitronectin; VnR, vitronectin receptor; ATF, amino-terminal fragment of urokinase; PKC, protein kinase C; mAb, monoclonal antibody.
Received 4/12/99. Accepted 8/19/99.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2-integrins, and Src kinases within a single receptor complex of human monocytes. J. Exp. Med., 181: 1381-1390, 1995.1 and 3 integrins of fibrosarcoma cells: dependence on extracellular matrix components. Cancer Res., 57: 1682-1689, 1997.v3 binding to vitronectin. Nature (Lond.), 383: 441-443, 1996.[Medline]
v5-mediated adhesion of a human keratynocyte cell line (HaCaT) to vitronectin. Exp. Cell Res., 220: 274-282, 1995.[Medline]
v5-directed cell migration. J. Biol. Chem., 271: 29393-29399, 1996.v3)-ligand interaction. J. Biol. Chem., 265: 2168-2172, 1990.v integrins. Science (Washington DC), 270: 1500-1502, 1995.v5-dependent cytoskeletal associations and focal adhesion kinase phosphorylation. J. Cell Biol., 134: 1323-1332, 1996.v3 and v5 contribute to cell attachment to vitronectin but differentially distribute on the cell surface. J. Cell Biol., 113: 919-929, 1991. subunit is associated with the vitronectin receptor subunit (v) in a human osteosarcoma cell line and is a substrate for protein kinase C. EMBO J., 8: 2955-2965, 1989.[Medline]
v3 integrin: a unique epitope of v3 integrin is present on human osteoclasts. Exp. Cell Res., 205: 345-352, 1993.[Medline]
3 subunit and defective activation of platelet integrin IIb3 (glycoprotein IIb-IIIa) in a variant of Glanzmann thrombasthenia. Proc. Natl. Acad. Sci. USA, 89: 10169-10173, 1992.21 integrin abrogates the malignant phenotype of breast carcinoma cells. Proc. Natl. Acad. Sci. USA, 92: 7411-7415, 1995.64 integrin expression in breast carcinomas is affected by laminin production from tumor cells. Clin. Cancer Res., 4: 407-410, 1998.v3: a new prognostic indicator in breast cancer. Clin. Cancer Res., 4: 2625-2634, 1998.[Abstract]
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