Correspondence to Harold A. Chapman: halchap{at}itsa.ucsf.edu; or Ying Wei: yingwei{at}itsa.ucsf.edu
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Introduction |
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Previous reports have indicated that uPAR can physically interact with multiple integrins including ß1 and ß2 integrins (Xue et al., 1997; Pluskota et al., 2003). We previously identified uPAR as an integrin ligand because a binding site for uPAR on the integrin Mß2 mapped to the ligand-binding region of its ß-propeller (Simon et al., 2000). Among ß1 integrins, uPAR directly associates with
3ß1 via a surface loop within the ß-propeller (W4 BC loop), but outside the laminin-5 (Ln-5) binding region. uPAR is also able to associate with
5ß1 (Aguirre-Ghiso et al., 1999; Wei et al., 2001) and to regulate
5ß1-mediated cell migration to Fn (Yebra et al., 1999),
5ß1 signaling (Aguirre-Ghiso et al., 2003; Tarui et al., 2003), and Fn matrix assembly (Monaghan et al., 2004).
Integrin 5ß1 is among many members of the integrin family that recognize an Arg-Gly-Asp (RGD) motif within their ligands (Takagi et al., 2003). Peptides containing this motif can efficiently block these integrinligand interactions (Arnaout et al., 2002).
5ß1 integrin and Fn form a prototypic integrin/ligand pair (Takagi et al., 2003), functionally important because it mediates Fn adhesion and Fn matrix assembly, which is vital to many cell functions in vivo (Cukierman et al., 2001). This integrin is also shown to play a key role in promoting tumor angiogenesis and tumor metastasis (Jin and Varner, 2004). In addition to the RGD sequence present in Fn type III module 10, a set of residues present in the Fn type III module 9 (synergy site) contribute to high affinity recognition by
5ß1 (Redick et al., 2000). The COOH-terminal heparin-binding site (HepII) of Fn also plays an important role in regulating cell adhesion, migration, Fn fibrillogenesis, signal transduction, and organization of focal adhesions and cytoskeleton (Huang et al., 2001; Kim et al., 2001). The interaction of cells to the HepII domain is currently thought to operate through proteoglycans such as syndecan 4 (Kim et al., 2001) and integrin
4ß1 (Mould and Humphries, 1991).
To date, little is known of the molecular mechanism by which uPAR regulates 5ß1-mediated function. In this report, the effect of uPAR on
5ß1-mediated adhesion, migration, and Fn matrix assembly was investigated. Surprisingly, using recombinant proteins, we found that direct binding of uPAR to
5ß1 does not change overall integrin binding to Fn, but changes integrin conformation, subsequently forming an additional binding site on Fn, which is RGD independent. In the course of this work a ß1 peptide sequence was discovered that blocks all uPAR/ß1 function. Mapping of this peptide near the known
-chain site of uPARß1 interaction confirms this region of ß1 integrins as an important regulatory site and suggests a molecular basis for PAI-1mediated cell detachment. Positioning of the uPAR-binding site near the Fn-binding site of
5ß1 not only promotes
5ß1 interactions with Fn, but allows PAI-1 to reverse Fn binding, empowering a mechanism of cell migration on Fn in either a protease-rich or protease inhibitorrich milieu.
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Results |
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These observations imply that uPAR can interact with at least two and possibly multiple ß1 integrins, suggesting the involvement of the common ß1-chain itself in uPAR binding. Moreover, the direct effect of uPAPAI-1 on Fn binding to uPAR5ß1 suggests the uPAR-binding site may be positioned close to the Fn-binding site. As demonstrated in Fig. 2 A, an energy-minimized model of integrin
5ß1 structure was generated based on the atomic coordinates of the
vß3 crystal structure (Xiong et al., 2001). Recently, we have found that the blade 4 BC loop of the proposed ß-propeller structure of integrin
3 is important for uPAR association (Wei et al., 2001). The corresponding BC loop in the
5ß1 model is highlighted in red. Inspection of the model reveals two loops on the ß1-chain (224NLDSPEGGF232 in yellow, 262FHFAGDGKL270 in purple) that are very close to the blade 4 BC loop of the ß-propeller (in red). We hypothesized that the two ß1-chain loops may also be involved in integrinuPAR association. The alignment of these ß1-chain sequences with that of other integrin ß-chains is shown in Fig. 2 B. Interestingly, the NH2-terminal Asn 224 in ß1P1 has been implicated in bonding to the Asp (D) of RGD in the
vß3 crystal, placing these loops very close to the putative RGD-binding pocket of
5ß1. This could potentially explain the direct effect of uPAPAI-1 on Fn binding to uPAR
5ß1.
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uPAR binding to 5ß1 induces an additional binding site for Fn
Next, we examined whether the apparent switch in the mechanism of 5ß1 adhesion to Fn initiated by the presence of uPAR (Fig. 3 A) was evident with purified proteins. Fn was immobilized and
5ß1-Fc binding to Fn was measured by protein AHRP as before. Fn
5ß1 binding in the presence or absence of suPAR is similar (Fig. 4 A), and both can be blocked by an
5-blocking antibody (unpublished data). The binding of
5ß1 to Fn in the absence of suPAR was abolished by RGD peptides, as expected, whereas binding in the presence of suPAR was vice versa. Interestingly, the binding of mutant integrin (
5ß1SA) to Fn remained RGD sensitive in the presence of suPAR (Fig. 4 A). These findings completely recapitulate the pattern seen with live cells and indicate that the presence of uPAR markedly changes the matrix-binding properties of
5ß1. This raises two possibilities: uPAR binding switches the integrin-binding site from the central RGD binding domain (III 10) to a different site on Fn. Or, uPAR binding to
5ß1 creates an additional binding site for Fn. To determine which possibility is more likely, we made biotinylated RGD peptides and performed binding assays with purified proteins. Binding of biotin-RGD (closed bars) and
5ß1-Fc (open bars) to Fn were measured separately and graphed together (Fig. 4 B). In the absence of uPAR, biotin-RGD (0.5 mM) competed with the RGD-binding site on immobilized Fn, blocking
5ß1 binding to the plate. However, in the presence of suPAR, biotin-RGD robustly bound to uPAR
5ß1Fn unless 10-fold excess unlabeled RGD was added (Fig. 4 B), strongly suggesting the existence of an additional RGD-independent binding site for Fn. Nevertheless, the RGD-binding site on
5ß1 must still be intact in the presence of suPAR, otherwise biotin-RGD would not be able to bind
5ß1.
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uPAR alters 5ß1 conformation and changes
5ß1 integrindependent adhesion and detachment in tumor cells
The above data indicate that binding of uPAR to 5ß1 alters integrin conformation and changes its matrix ligand binding properties. To probe this idea further and determine whether uPAR-mediated changes in
5ß1 function are observable in nontransfected cells, several tumor cell lines expressing various amounts of uPAR were evaluated. HT1080 (fibrosarcoma), MDA-MB-231 (breast carcinoma), and Skov-3 (ovarian carcinoma) cells were transfected with a small interfering RNA (siRNA) previously shown to suppress uPAR mRNA (Vial et al., 2003) or control and suppression of surface uPAR expression verified 48 h later by FACS analysis (Fig. 5 A). Suppression of surface uPAR had no effect on total ß1 integrin expression (JB1A). However, suppression of uPAR had clear effects on integrin conformation as judged by altered binding of the conformation-sensitive mAbs, HUTS-21, and 9EG7, in all of the cell lines examined. Suppression of surface uPAR was accompanied by increased binding of both HUTS-21 and 9EG7 antibodies (Fig. 5 A), confirming that endogenous uPAR expression modifies integrin ß1-chain conformation.
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uPAR expression promotes 5ß1-mediated cell adhesion, migration, and Fn matrix assembly
When cells are plated on an Fn-coated surface (5 µg/ml) for 1 h, the difference in Fn adhesion between cells with and without surface uPAR is marginal (Fig. 3 A, Fig. 4 C, Fig. 5 B). However, when cells are seeded onto lower amounts of Fn (0.25 µg/ml) for shorter periods of time (20 min), adhesion of uPAR-expressing HT1080 cells to Fn was obviously more robust (Fig. 6 A). We repeated similar experiments using MDA-MB-231 (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200404112/DC1) and Skov-3 cells with or without uPAR suppression and found similar results. These findings indicate that uPAR expression not only changes the conformation of 5ß1 and how it engages Fn, but together these changes might promote cell adhesion and migration.
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To test whether uPAR association with 5ß1 affects cell migration, we performed wound assays on tumor cells (HT1080 and MDA-MB-231) and the Tet-responsive cells with or without uPAR induction using RGD and ß1 peptides (ß1P1). The cells were seeded onto Fn-coated wells and allowed to form a monolayer before wounding. Preliminary experiments indicated that as little as 20 µM of either the RGD-containing or the ß1P1 peptide suppressed migration. As shown in Fig. 7, both RGD and ß1P1 alone blocked wound closure of HT1080 cells, and the combination of both peptides had a statistically significant greater effect. Similar results were obtained from MDA-MB-231 cells. More importantly, Tet-inducible cells with uPAR expression (+Tet) migrated faster in this assay and the migration was blocked by both RGD and ß1 peptides, whereas cells without uPAR (Tet) had little migration (unpublished data). Together, these data confirm our findings that uPAR, through its interaction with ß1 integrin(s), promotes cell motility and that this function can be specifically blocked by the ß1-chain peptides identified here.
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Discussion |
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The consequences of uPAR5ß1 complex formation contrast with that of other pathways of integrin activation. Available evidence indicates that integrin activation involves a global change in integrin conformation, at least part of which is a change in the orientation of the
and ß head domains to better accommodate ligand binding. Several lines of evidence also support a model in which the very "bent" integrin conformation found in the
Vß3 crystal structure extends to point the head domains away from the cell under activating conditions (Takagi et al., 2002). However, the full range of conformational changes a ligand-bound integrin may assume is uncertain (Mould and Humphries, 2004). It is especially difficult to envision a fully extended conformation of activated integrins acting in cis to engage the much smaller GPI-anchored uPAR at the integrin upper surface. Rather, our findings suggest uPAR
5ß1 complexes exhibit an activation state involving a modified bent integrin with distinct functional properties. Similarities and differences between models of "extended" integrin activation and a model consistent with results reported here are summarized in Fig. 8. The model raises the more general possibility that some version of an angled integrin configuration, rather than being inactive, actually functions to promote integrin binding to cis-acting membrane ligands, such as uPAR, which coordinate integrin function with specific cellular needs.
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This hypothesis is supported by our results, which reveal that suppressing uPAR expression induces ligand-induced binding site (LIBS) epitopes in HT1080, MDA-MB-231, and Skov-3 cells (Fig. 5 A). A recent report also documented increased ß1-chain LIBS epitopes on human skin fibroblasts exposed to a peptide that disrupts uPARintegrin interactions (Wei et al., 1996; Monaghan et al., 2004). The LIBS antibodies (HUTS-21, 9EG7) used here map to sites near the hinge region of the integrin but far away from the uPAR interaction site. Although these LIBS antibodies are thought to recognize the "active" conformational state of the ß1 subunit that can be induced by ligand binding (e.g., Fn, RGD peptides, by activating antibody TS2/16, or Mn2+), their binding is more sensitive to conformational changes in the hinge, knee, or leg domains than changes near the ligand-binding pocket (Bazzoni et al., 1995; Luque et al., 1996; Mould and Humphries, 2004). We postulate that in uPAR-expressing cells the lower LIBS antibody binding reflects integrin angulation resulting from uPAR5ß1 complex formation. This occurs in spite of "activation" of the integrin as judged by enhanced adhesion and Fn matrix assembly, further supporting the idea that activated integrins could exist in grossly different conformational states depending on the nature of the ligand (Fig. 8).
Previous studies have shown that initial cell attachment and spreading on Fn is mediated by the interaction of the RGD-containing Fn cell-binding domain (type III repeats 910) with 5ß1 (Mould et al., 2000; Redick et al., 2000; Takagi et al., 2003), but that further progression of the cytoskeletal response requires additional signals (Hocking et al., 1998; Tarui et al., 2003). Additional binding sites for cells on Fn provide the necessary signals. For example, interaction of cells with the Fn NH2-terminal region can trigger integrin-mediated intracellular signals that are distinct from those generated in response to ligation with the RGD sequence (Forsyth et al., 2002). However, in our assays, adhesion of epithelial cells to this 70-kD fragment was not influenced by uPAR expression and not inhibited by
5ß1 blocking antibodies (unpublished data). Signals for cytoskeletal reorganization may also be provided by the interaction of Fn fragments containing the heparin-binding domain (Hep II) (type III repeats 1214) with cell surface proteoglycans (Huang et al., 2001). In fibroblasts, this response requires two cooperative signals provided by interactions of the RGD sequence with
5ß1 integrin and the heparin-binding domain with syndecan-4 (Kim et al., 2001). Our data show that both cells with uPAR or without uPAR adhere to Fn III 9-11 in an RGD-dependent manner, whereas only cells bearing uPAR adhere to Fn III 12-15. The latter cannot be blocked by RGD peptides, but can be blocked by ß1 peptides that disrupt uPARß1 integrin interaction (Fig. 4 C). In most uPAR-expressing cells there are likely to be pools of
5ß1 both free and bound to uPAR, suggesting that the incorporation of the heparin binding domain into the uPAR
5ß1 complex results in distinct signals that lead to enhanced integrin function, as our data show (Fig. 6). We cannot be sure whether uPAR
5ß1 complexes possess both Fn-binding sites or binding to both sites in Fn requires free and uPAR-complexed integrin. Future studies may distinguish between these possibilities.
We have previously reported that uPAR expression in kidney embryonic 293 cells both promotes Vn adhesion through association of uPAR with 3ß1 and impairs Fn adhesion mediated by
5ß1 (Wei et al., 1996, 2001). Impairment of Fn adhesion in 293 cells appears anomalous with respect to all other cells expressing uPAR examined here and by others (Aguirre-Ghiso et al., 1999). Consistent with this difference, expression of uPAR in 293 cells did not decrease binding of HUTS-21 and 9EG7 antibodies (unpublished data), implying that for some reason uPAR interacts, but not in the same manner, with
5ß1 in 293 cells as that seen in other transformed cells. The molecular basis for the anomalous behavior of 293 cells remains to be defined.
The discovery of the capacity of 5ß1 to undergo a phenotypic switch (i.e., RGD vs. ß1P1 dependent; Fig. 8), in Fn attachment may be relevant to attempts to regulate inflammation or tumor progression through integrin inhibition in vivo. uPAR is up-regulated in both inflammatory cells and many tumor cells with a metastatic phenotype. Indeed, uPAR expression is an independent risk factor for tumor metastasis in several clinical studies. RGD-based compounds or peptides have been shown to inhibit integrin function in vivo, but our data imply that one limitation in their use is the complete resistance of ß1 integrins complexed with uPAR from RGD-dependent ligand binding. Vn adhesion mediated by uPAR
3ß1 complexes is also RGD-resistant (Wei et al., 1994). Instead, uPAR-bound ß1 integrins are sensitive to ß1 peptides that map to the region of uPARintegrin interaction. As these ß1 peptides block cell adhesion (Fig. 5 B) and migration of various tumor cells (Fig. 7), it is possible that these reagents, perhaps coupled with RGD-based compounds, have therapeutic potential for suppression of tumor progression.
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Materials and methods |
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Cell culture
Mouse kidney epithelial cells expressing wt 3 (a gift from Dr. Jordan A. Kreidberg, Harvard Medical School, Boston, MA) or mut
3 and their uPAR cotransfected cells were cultured in DME as described previously (Wang et al., 1999; Zhang et al., 2003). HEK293, human fibrosarcoma HT1080, breast cancer MCF-7, T47D, and MDA-MB-231 Skov-3 cell lines were obtained from American Type Culture Collection (Rockville, MD) and grown in DME. The Tet-inducible uPAR cells (Tet-uPAR) were maintained in DME supplemented with zeocin, hygromycin, and blasticidin (5 µg/ml). Additional Tet (2 µg/ml) was added to induce uPAR expression. Modified Skov-3 cell line was a gift from Dr. Ernest Lengyel (University of Chicago, Chicago, IL).
Cell detachment assay
Microtiter plates were coated with 5 µg/ml Fn or Ln-5 supernatant (1:100) for 18 h at 4°C. The cell detachment assay was performed as described previously (Czekay et al., 2003). In brief, cells attached were acid washed, resuspended in incubation buffer (RPMI, 20 mM Hepes, and 0.02% BSA), and then incubated in the absence or presence of active uPA followed by PAI-1. After wash, the remaining adherent cells were fixed and stained. The amount of extracted stain was quantified by absorbance at 590 nm.
Cell adhesion assay
The cell adhesion assay was performed as described previously (Wei et al., 2001). In brief, cells were seeded onto Fn (5 µg/ml) or Fn fragment (10 µg/ml)coated plates and incubated in DME/0.1% BSA with or without RGD or ß1 peptides for 1 h at 37°C. After washing, attached cells were fixed and stained with Giemsa. The data were quantified by measuring absorbance at 550 nm.
Biotinylation of suPAR, III 9-11, and RGD peptides
Human suPAR, Fn fragment III 9-11, or RGD peptides were biotinylated at 0.25 mg/ml using FluoReporter Biotin-XX Protein Labeling Kit (Molecular Probes, Inc.) following the manufacturer's instructions.
Purification of 5ß1-Fc and
5ß1SA-Fc integrins
Integrin 5-Fc and ß1-Fc or Ser227 to Ala mutant ß1SA-Fc constructs (
5/pEE12.2hFc and ß1/pV.16hFc or ß1SA/pV.16hFc; Coe et al., 2001) were transfected into 293 cells. Culture supernatant was harvested after 4872 h and passed through a Protein Aagarose column. Soluble integrin was eluted using 0.1 M glycine, pH 3.0, and neutralized in 1 M Tris-HCl, pH 8.0. Protein-containing fractions were dialyzed, concentrated, and identified by SDS-PAGE.
Purified protein binding assay
Nunc high binding microtiter plates were coated with 20 µg/ml Fn and blocked with 1% BSA. 20 µg/ml purified recombinant 5ß1-Fc with or without 20 nM purified suPAR was added to each well in PBS with 1 mM MnCl2, and the plates were incubated for 1 h at 25°C. For different purposes, RGD peptides or uPAPAI-1 mixture may be added together with suPAR. After washing, bound
5ß1-Fc was detected by protein AHRP and quantified by measuring absorbance at 490 nm. Data were expressed as specific binding (i.e., total binding minus the binding to wells coated with BSA alone).
To test biotinylated RGD peptides binding to Fn5ß1suPAR, Nunc microtiter plates were coated with Fn and incubated with
5ß1-Fc with or without suPAR as above. 0.5 mM biotin-RGD was then added to each well for another hour. After washing, avidin peroxidase was added and the bound biotin-RGD was quantified as described above. To test specificity of binding, 10-fold molar excess nonbiotinylated RGD peptides were added. Biotinylated suPAR binding assay was performed similarly to confirm Fn
5ß1suPAR complex formation.
uPAR RNA interference
HT1080 cells were transfected with siRNAs that specifically target the uPAR gene or nonsilencing control and used within 4872 h. siRNA duplexes were synthesized by in vitro transcription. The sequence of the DNA targeting uPAR is 5'-GGTGAAGAAGGGCGTCCAA-3'. A nonsilencing siRNA 5'-AACCTGCGGGAAGAAGTGG-3' was used as a control (Vial et al., 2003). Synthetic siRNA oligonucleotides were purified with Microspin G-25 columns from Amersham Biosciences.
FACS analysis
Cells with or without siRNA uPAR transfection were incubated with primary antibody to active form ß1 integrin (HUTS-21, 9EG7) or to total ß1 integrin (JB1A) and secondary FITC-conjugated antimouse IgG or antirat IgG (for 9EG7; Sigma-Aldrich) and analyzed on a flow cytometer (FACSCaliber; BD Biosciences). uPAR was detected by a mAb to uPAR.
Biotin-III 9-11 binding assay
All the procedures were done at 4°C. HT1080 cells were acid washed and incubated without or with uPA followed by PAI-1. The cells were then incubated with 50 nM biotinylated Fn fragment III 9-11 in RPMI/0.02% BSA for 1 h. After washing, the cells were lysed and the total protein separated by SDS-PAGE. The bound biotin-III 9-11 was detected by avidin-HRP. The bands were quantified and analyzed by densitometry.
Generation of inducible uPAR clones (Tet-uPAR)
Wt 3 epithelial cells were transfected with Tet repressor (pcDNA6/TR, blasticidin; Invitrogen) and Tet on an expression construct containing full-length uPAR (pcDNA5/TO, hygromycin; Invitrogen) with a ratio of 6:1. After antibiotic selection, 2 µg/ml Tet was added and uPAR-expressing clones were selected by cell sorting. Tet was removed and the cells resorted for nonexpressing clones. These cells were diluted to select single clones. The data presented in this paper were obtained from one of the representative clones.
Detection of ECM-associated Fn
Fn fibrils were detected as described previously (Aguirre-Ghiso et al., 2001). In brief, Tet-uPAR cells without or with Tet induction were lysed with 3% Triton X-100 buffer. Triton-insoluble pellets were treated with DNase and then extracted with 2% deoxycholate buffer. The insoluble and soluble fractions were mixed with sample buffer and analyzed by SDS-PAGE and Western blotting using antihuman Fn antibodies and an antibody to ß-actin.
Wound healing
HT1080 cells were grown to confluence on Fn-coated surface. Medium was replaced with DME/0.1% BSA 6 h before wounding. The wound was made using a 1-ml pipet tip. The detached cells were removed by washing and the wounded cells were incubated without or with RGD or ß1 peptides (20 µM) for 24 h. Cells were imaged at 0 and 24 h by phase-contrast videomicroscopy.
Coimmunoprecipitation
HT1080 cells were lysed in Triton lysis buffer (50 mM Hepes, pH 7.5, 150 mM NaCl, and 1% Triton X-100) supplemented with protease inhibitors and 1 mM PMSF. Clarified lysates were incubated with or without 400 µM peptide ß1P1 and immunoprecipitated with antibody to integrin 5 (P1D6). The immunoprecipitates were blotted for uPAR (R2) or integrin
5 (pAb).
Online supplemental material
The supplemental material (Figs. S1S3) is available at http://www.jcb.org/cgi/content/full/jcb.200404112/DC1. Fig. S1 shows that suppression of uPAR expression by RNA interference induces LIBS epitope and changes 5ß1-mediated Fn binding in MDA-MB-231 cells. Fig. S2 shows the effect of uPAR expression level on Fn adhesion of different breast cancer cell lines. Fig. S3 shows the RGD-resistant adhesion of uPAR-expressing cells on different concentrations of Fn.
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Acknowledgments |
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This work was supported by National Institutes of Health grants HL44712 (to H.A. Chapman) and HL 31950 (to D.J. Loskutoff).
Submitted: 20 April 2004
Accepted: 7 December 2004
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