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• W2015277059 abstract "Maspin is a non-inhibitory serine protease inhibitor (serpin) that influences many cellular functions including adhesion, migration, and invasion. The underlying molecular mechanisms that facilitate these actions are still being elucidated. In this study we determined the mechanism by which maspin mediates increased MCF10A cell adhesion. Utilizing competition peptides and mutation analyses, we discovered two unique regions (amino acid residues 190–202 and 260–275) involved in facilitating the increased adhesion function of maspin. In addition, we demonstrate that the urokinase-type plasminogen activator (uPA)/uPA receptor (uPAR) complex is required for the localization and adhesion function of maspin. Finally, we showed that maspin, uPAR, and β1 integrin co-immunoprecipitate, suggesting a novel maspin-uPA-uPAR-β1 integrin mega-complex that regulates mammary epithelial cell adhesion. Maspin is a non-inhibitory serine protease inhibitor (serpin) that influences many cellular functions including adhesion, migration, and invasion. The underlying molecular mechanisms that facilitate these actions are still being elucidated. In this study we determined the mechanism by which maspin mediates increased MCF10A cell adhesion. Utilizing competition peptides and mutation analyses, we discovered two unique regions (amino acid residues 190–202 and 260–275) involved in facilitating the increased adhesion function of maspin. In addition, we demonstrate that the urokinase-type plasminogen activator (uPA)/uPA receptor (uPAR) complex is required for the localization and adhesion function of maspin. Finally, we showed that maspin, uPAR, and β1 integrin co-immunoprecipitate, suggesting a novel maspin-uPA-uPAR-β1 integrin mega-complex that regulates mammary epithelial cell adhesion. Maspin is a non-inhibitory serine protease inhibitor (serpin) 4The abbreviations used are: serpinserine protease inhibitorECMextracellular matrixMEFmouse embryo fibroblastsRCLreactive center loopuPAurokinase-type plasminogen activatoruPARurokinase-type plasminogen activator receptor. that was originally identified as a type II tumor suppressor protein in mammary epithelial cells (1Zou Z. Anisowicz A. Hendrix M.J. Thor A. Neveu M. Sheng S. Rafidi K. Seftor E. Sager R. Science. 1994; 263: 526-529Crossref PubMed Scopus (830) Google Scholar). One major tumor suppressor function of maspin is suppression of tumor cell motility, as it inhibits tumor cell migration/invasion in vitro and suppresses metastasis in mouse models (1Zou Z. Anisowicz A. Hendrix M.J. Thor A. Neveu M. Sheng S. Rafidi K. Seftor E. Sager R. Science. 1994; 263: 526-529Crossref PubMed Scopus (830) Google Scholar, 2Abraham S. Zhang W. Greenberg N. Zhang M. J. Urol. 2003; 169: 1157-1161Crossref PubMed Scopus (72) Google Scholar, 3Seftor R.E. Seftor E.A. Sheng S. Pemberton P.A. Sager R. Hendrix M.J. Cancer Res. 1998; 58: 5681-5685PubMed Google Scholar, 4Shi H.Y. Zhang W. Liang R. Abraham S. Kittrell F.S. Medina D. Zhang M. Cancer Res. 2001; 61: 6945-6951PubMed Google Scholar, 5Shi H.Y. Liang R. Templeton N.S. Zhang M. Mol. Ther. 2002; 5: 755-761Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 6Zhang M. Sheng S. Maass N. Sager R. Mol. Med. 1997; 3: 49-59Crossref PubMed Google Scholar, 7Zhang M. Shi Y. Magit D. Furth P.A. Sager R. Oncogene. 2000; 19: 6053-6058Crossref PubMed Scopus (68) Google Scholar). Several studies show that pericellular maspin inhibits cell motility by enhancing cell adhesion (2Abraham S. Zhang W. Greenberg N. Zhang M. J. Urol. 2003; 169: 1157-1161Crossref PubMed Scopus (72) Google Scholar, 3Seftor R.E. Seftor E.A. Sheng S. Pemberton P.A. Sager R. Hendrix M.J. Cancer Res. 1998; 58: 5681-5685PubMed Google Scholar, 8Ngamkitidechakul C. Burke J.M. O'Brien W.J. Twining S.S. Invest. Ophthalmol. Vis. Sci. 2001; 42: 3135-3141PubMed Google Scholar, 9Cella N. Contreras A. Latha K. Rosen J.M. Zhang M. FASEB J. 2006; 20: 1510-1512Crossref PubMed Scopus (57) Google Scholar). In addition to its tumor suppressing functions, our laboratory showed that maspin is also essential for normal fetal development as maspin knock-out mice are embryonic lethal during the peri-implantation stage partially due to disrupted visceral endodermal cell adhesion (10Gao F. Shi H.Y. Daughty C. Cella N. Zhang M. Development. 2004; 131: 1479-1489Crossref PubMed Scopus (69) Google Scholar). The underlying molecular mechanism by which maspin regulates cell adhesion is currently unknown and under intense investigation. To date, there are two proposed pathways utilized by maspin to increase cell-extracellular matrix (ECM) adhesion; that is, the plasminogen activation system and β1 integrin signaling (9Cella N. Contreras A. Latha K. Rosen J.M. Zhang M. FASEB J. 2006; 20: 1510-1512Crossref PubMed Scopus (57) Google Scholar, 11Al-Ayyoubi M. Schwartz B.S. Gettins P.G. J. Biol. Chem. 2007; 282: 19502-19509Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar, 12Yin S. Lockett J. Meng Y. Biliran Jr., H. Blouse G.E. Li X. Reddy N. Zhao Z. Lin X. Anagli J. Cher M.L. Sheng S. Cancer Res. 2006; 66: 4173-4181Crossref PubMed Scopus (70) Google Scholar, 13Bass R. Wagstaff L. Ravenhill L. Ellis V. J. Biol. Chem. 2009; 284: 27712-27720Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). serine protease inhibitor extracellular matrix mouse embryo fibroblasts reactive center loop urokinase-type plasminogen activator urokinase-type plasminogen activator receptor. The plasminogen activation system is believed to be a central player in several different processes important for tumor progression and metastasis (14Andreasen P.A. Egelund R. Petersen H.H. Cell. Mol. Life Sci. 2000; 57: 25-40Crossref PubMed Scopus (845) Google Scholar, 15Blasi F. Carmeliet P. Nat. Rev. Mol. Cell Biol. 2002; 3: 932-943Crossref PubMed Scopus (1073) Google Scholar, 16Mazar A.P. Henkin J. Goldfarb R.H. Angiogenesis. 1999; 3: 15-32Crossref PubMed Scopus (155) Google Scholar). In this system urokinase-type plasminogen activator (uPA), a serine protease, binds to its glycosylphosphatidylinositol-anchored receptor (uPAR) and readily activates plasminogen to initiate a protease cascade resulting in localized ECM degradation for the purpose of cell migration (17Ellis V. Behrendt N. Danø K. J. Biol. Chem. 1991; 266: 12752-12758Abstract Full Text PDF PubMed Google Scholar, 18Jo M. Thomas K.S. Wu L. Gonias S.L. J. Biol. Chem. 2003; 278: 46692-46698Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). It has been suggested that maspin integrates into the plasminogen activation system. Maspin inhibits prostate carcinoma cell migration and invasion by strengthening mature focal adhesion contacts, reducing uPA activity by internalizing the maspin-uPA-uPAR complex and by binding to pro-uPA, thus blocking its activation (12Yin S. Lockett J. Meng Y. Biliran Jr., H. Blouse G.E. Li X. Reddy N. Zhao Z. Lin X. Anagli J. Cher M.L. Sheng S. Cancer Res. 2006; 66: 4173-4181Crossref PubMed Scopus (70) Google Scholar). Although maspin is classified as a serpin and decreases pericellular uPA activity, maspin does not directly inhibit uPA proteolytic activity (19McGowen R. Biliran Jr., H. Sager R. Sheng S. Cancer Res. 2000; 60: 4771-4778PubMed Google Scholar, 20Biliran Jr., H. Sheng S. Cancer Res. 2001; 61: 8676-8682PubMed Google Scholar, 21Bass R. Fernández A.M. Ellis V. J. Biol. Chem. 2002; 277: 46845-46848Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Together, these studies demonstrated that maspin can reduce prostate carcinoma cell migration and invasion by internalization of cell surface maspin-uPA-uPAR complexes. The second proposed cell adhesion pathway involves maspin associating with β1 integrin, thus, altering integrin-mediated signaling. Initial studies investigating the anti-invasive function of maspin showed that MDA-MB-435 breast carcinoma cells treated with exogenous maspin had increased expression of α5- and α3-integrins. In addition to altered integrin expression profile, maspin stimulated focal adhesion and stress fiber formation in MDA-MB-231 breast carcinoma cells to a fibronectin matrix acting through the α5β1 integrin receptor (3Seftor R.E. Seftor E.A. Sheng S. Pemberton P.A. Sager R. Hendrix M.J. Cancer Res. 1998; 58: 5681-5685PubMed Google Scholar, 22Odero-Marah V.A. Khalkhali-Ellis Z. Chunthapong J. Amir S. Seftor R.E. Seftor E.A. Hendrix M.J. Cancer Biol. Ther. 2003; 2: 398-403Crossref PubMed Scopus (73) Google Scholar). We have suggested that cell surface maspin co-localizes with β1 integrin to increase MCF10A cell adhesion. This increased adhesion was facilitated by amino acids residues 139–225 in the maspin molecule (9Cella N. Contreras A. Latha K. Rosen J.M. Zhang M. FASEB J. 2006; 20: 1510-1512Crossref PubMed Scopus (57) Google Scholar). Another study demonstrated that maspin inactivation of β1 reduces vascular smooth muscle cell migration on laminin or fibronectin matrices (13Bass R. Wagstaff L. Ravenhill L. Ellis V. J. Biol. Chem. 2009; 284: 27712-27720Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). A peptide mimicking the G α-helix (G-helix, amino acids 237–251) region of maspin was both essential and sufficient for inhibiting cell migration, but it had no effect on cell adhesion (13Bass R. Wagstaff L. Ravenhill L. Ellis V. J. Biol. Chem. 2009; 284: 27712-27720Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 23Ravenhill L. Wagstaff L. Edwards D.R. Ellis V. Bass R. J. Biol. Chem. 2010; 285: 36285-36292Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). These discoveries showed that the G-helix of maspin regulates cell migration, but another region (amino acids 139–225) is involved in regulating cell adhesion. Initially thought to simply localize uPA for ECM degradation, recent evidence indicates that uPAR also initiates intracellular signaling cascades that regulate cell adhesion, migration, and proliferation independent of protease activity (24Smith H.W. Marshall C.J. Nat. Rev. Mol. Cell Biol. 2010; 11: 23-36Crossref PubMed Scopus (722) Google Scholar). In fact, it is now becoming clear that uPAR can specifically modify integrin functions to regulate ECM binding, cell adhesion, and migration (24Smith H.W. Marshall C.J. Nat. Rev. Mol. Cell Biol. 2010; 11: 23-36Crossref PubMed Scopus (722) Google Scholar, 25Tarui T. Andronicos N. Czekay R.P. Mazar A.P. Bdeir K. Parry G.C. Kuo A. Loskutoff D.J. Cines D.B. Takada Y. J. Biol. Chem. 2003; 278: 29863-29872Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 26Tarui T. Mazar A.P. Cines D.B. Takada Y. J. Biol. Chem. 2001; 276: 3983-3990Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). We speculate that maspin acts as an integrator of these two systems, ultimately leading to decreased cell migration and increased cell adhesion. Therefore, the objective of this study was to determine the intramolecular region(s) of maspin necessary for its pro-adhesive function and decipher its mechanism of action. We demonstrate two different regions proximal to the reactive center loop (RCL) of maspin that are responsible for maspin-mediated MCF10A cell adhesion. Importantly, this enhanced adhesion is dependant on the presence of both uPA and uPAR and is present in a complex with uPA-uPAR-β1 integrin on the cell surface. Together, we suggest that maspin coordinates both uPA-uPAR and β1 integrin receptors to regulate both mammary epithelial cell-ECM adhesion and migration. For immunoprecipitation and functional blocking experiments, we used the rabbit anti-human uPAR and anti-human uPA (American Diagnostics). We used mouse monoclonal anti-maspin (BD Pharmingen) and rabbit polyclonal anti-β1 integrin (Chemicon) for both immunoprecipitation and immunoblot probing. An affinity-purified rabbit polyclonal antibody raised against maspin RCL peptide (AbS4A) was used from previous studies (1Zou Z. Anisowicz A. Hendrix M.J. Thor A. Neveu M. Sheng S. Rafidi K. Seftor E. Sager R. Science. 1994; 263: 526-529Crossref PubMed Scopus (830) Google Scholar). Both the horseradish peroxidase-conjugated secondary antibodies and maspin peptides were obtained from Sigma. Human uPA was purchased from Chemicon. MCF10A, immortalized human mammary luminal epithelial cells (CRL-10317; American Type Culture Collection), were cultured in Dulbecco's modified Eagle's medium (DMEM)/F-12 (Invitrogen) containing 5% donor horse serum, 20 μg/ml epidermal growth factor, 100 μg/ml cholera toxin, 10 μg/ml insulin, 500 μg/ml hydrocortisone, 50 units/ml penicillin, and 50 μg/ml streptomycin at 37 °C and 5% CO2. All growth factors and hormones were purchased from Sigma. For maspin secretion studies, MCF10A cells either directly ordered from ATCC (low passage) or carried in our laboratory for 2 years (high passage) were maintained in defined keratinocyte serum-free medium (Invitrogen) supplemented with ciprofloxacin and MITO+ (BD Biosciences). Cells were passaged weekly and fed three times per week. Conditioned medium was collected on the day of passage, centrifuged to remove cell debris, and concentrated 24× using a 30-kDa spin filter (Millipore). Harvested protein was then used for Western blot analysis, as described later. Control (uPAR+/+) and uPAR-deficient (uPAR−/−) murine embryonic fibroblasts (MEFs) were isolated from embryos as previously described (27Ma Z. Thomas K.S. Webb D.J. Moravec R. Salicioni A.M. Mars W.M. Gonias S.L. J. Cell Biol. 2002; 159: 1061-1070Crossref PubMed Scopus (94) Google Scholar). We recently demonstrated that amino acid residues 139–225 within maspin are important for increased MCF10A cell adhesion to its self-deposited matrix (9Cella N. Contreras A. Latha K. Rosen J.M. Zhang M. FASEB J. 2006; 20: 1510-1512Crossref PubMed Scopus (57) Google Scholar). To determine the surface-exposed amino acid sequences that may regulate cell adhesion, we analyzed the three-dimensional structure of maspin by Molsoft ICM-pro software. Subsequently, we purchased peptides (Sigma) composed of 16–23 amino acids (depending on the hydrophilicity and molecular weight) to test in adhesion assays. GST-tagged maspin (GST-maspin) was produced as previously described (28Zhang M. Volpert O. Shi Y.H. Bouck N. Nat. Med. 2000; 6: 196-199Crossref PubMed Scopus (410) Google Scholar). Point mutants were constructed with a QuikChange multisite-directed mutagenesis kit (Stratagene) according to the manufacturer's instruction. The primers are as follows: control mutant D177A, S178L, T180P (forward, 5′-GGATGAAGAAATTTCCGGCATTAGAACCAAAAGAATGTCC; reverse, 5′-GGACATTCTTTTGGTTCTAATGCCGGAAATTTCTTCATCC); single (E201K) mutant (forward, 5′-GATGAATCTTAAGGCCACTTTCTGCTTGGG; reverse, 5′-CCCAAGCAGAAAGTGGCCTTAAGATTCATC); double (K268E, K270E) mutant (forward, 5′-GGCCAATGCCGAAGTCGAACTTTCCCTCCC; reverse, 5′-GGGAGGGAAAGTTCGACTTCGGCATTGGCC). To develop the triple mutant (E201K,K268E,K270E), primers of mutant K268E, K270E, and E201K were used as the template. To verify their fidelity, the constructs were sequenced. The constructs were transformed into Escherichia coli BL21 cells and expressed and purified according to the manufacturer's instructions (GE Healthcare). Assays utilizing endogenous ECM proteins generated by MCF10A cells were performed as previously described (29Langhofer M. Hopkinson S.B. Jones J.C. J. Cell Sci. 1993; 105: 753-764Crossref PubMed Google Scholar). MCF10A cells were plated in 96-well dishes and allowed to reach confluence. Cells were washed with PBS and treated for 5 min with fresh sterile 20 mm NH4OH followed by extensive water washes. Wells were blocked with heat-denatured BSA (10 mg/ml) for 1 h at room temperature. Subconfluent cultures were trypsinized, washed with 37 °C serum-free DMEM/F-12 medium, and incubated with either antibodies or recombinant proteins (500 nm) for 30 min at 37 °C (when assaying endogenous maspin, enzyme-free cell dissociation solution was used instead of trypsin). In all assays 2.0 × 104 cells were plated in triplicate and allowed to adhere for 30 min at 37 °C. The GST protein was used as a control. Wells were washed with 37 °C serum-free DMEM/F-12, and adhered cells were fixed with 5% glutaraldehyde and stained with crystal violet dye. Cell adhesion was determined by the reading at 590 nm subtracted by the blank value (determined by BSA-coated wells, 5% of maximal cell adhesion). Cell adhesion was plotted as the percentage of the corresponding control value. Cell surface stripping of uPA from uPAR was conducted as previously described (30Stoppelli M.P. Tacchetti C. Cubellis M.V. Corti A. Hearing V.J. Cassani G. Appella E. Blasi F. Cell. 1986; 45: 675-684Abstract Full Text PDF PubMed Scopus (275) Google Scholar). In brief, subconfluent cultures were washed twice with DMEM/F-12 supplemented with 20 mm HEPES and 1 mg/ml BSA. Then cells were incubated with 50 mm glycine-HCl (pH 3.0) with 100 mm NaCl at 25 °C for 2 min, and the reaction was halted by neutralizing with 500 mm HEPES (pH 7.4). In the function-blocking studies, cells were first incubated with the function blocking anti-uPAR or control rabbit IgG for 10 min; uPA and recombinant GST-maspin was added for another 20 min. Bacterial recombinant GST-maspin was labeled with 125I as described previously (31Conlon M. Walker J.M. The Protein Protocols Handbook. Humana Press Inc, Totowa, NJ2002: 971-977Google Scholar). Wild type (WT) and uPAR−/− MEFs (5.0 × 104 cells) were cultured in DMEM with 10% FBS on 96-well plates overnight. Cells were chilled on ice for 30 min, washed 3 times with ice-cold DMEM, then blocked on ice for 60 min using blocking buffer (DMEM containing 5% heat-inactivated BSA). MEFs were incubated with increasing concentrations of 125I-GST-maspin in blocking buffer at 4 °C for 90 min. After incubation, unbound 125I-GST-maspin protein was removed by washing 3 times with blocking buffer. Specific 125I-GST-maspin binding was determined by subtracting the detected radioactivity by the nonspecific binding (determined in the presence of a 50-fold excess of non-labeled GST-maspin). In the studies evaluating the uPA-uPAR interactions, 125I-GST-maspin was treated with ABS4A and S-20 (Santa Cruz) antibodies, which block the RCL and N-terminal domains, before adding to WT MEF cells. In one set, WT MEF cells were treated with an anti-mouse uPAR antibody (R&D), which blocks uPA binding to uPAR. In the other set, WT MEF cells were stripped of uPA, then treated with the anti-mouse uPAR antibody and then administered uPA (800 nm) and 125I-GST-maspin. Results were reported as 125I-GST-maspin binding to WT MEF cell surface as a percentage of control (IgG) antibody treatment. Because MCF10A cells express uPAR at low levels, we overexpressed uPAR in MCF10A cells. Lysates from MCF10A cells overexpressing uPAR were prepared in lysis buffer: 50 mm Tris (pH 7.4), 1% Triton X-100, 1% sodium deoxycolate, 150 mm NaCl, 5 mm EDTA (pH 8.0), 5 mm PMSF, and protease inhibitor mixture (Thermo Scientific). Cellular debris was cleared from lysates by centrifugation, and protein concentration was determined by the BCA Protein Assay (Pierce). Whole cell extracts (500 μg) were incubated overnight (constant rocking) with 5 μg of specific antisera or control rabbit irrelevant antiserum (Ki67) at 4 °C. Protein A-Sepharose-coupled beads (Amersham Biosciences) were added and incubated for 2 h at 4 °C under constant agitation. Beads were centrifuged, washed 3 times with ice-cold lysis buffer, and boiled for 5 min in sample buffer containing 5% β-mercaptoethanol. Samples were separated on SDS-PAGE gels, transferred to a PVDF membrane (GE Healthcare), and probed for β1 integrin, uPAR, and maspin. Appropriate secondary antibodies were added, and proteins were visualized with enhanced chemiluminescence substrate (Pierce). Statistical differences between two individual groups were determined using an unpaired t test. Statistical significance was considered when the p value was less than 0.05. One key function of maspin is the regulation of cell motility and adhesion. After its discovery, researchers determined that maspin was ubiquitous in the cellular cytoplasm but was also localized to secretory vesicles and the cell surface (32Pemberton P.A. Tipton A.R. Pavloff N. Smith J. Erickson J.R. Mouchabeck Z.M. Kiefer M.C. J. Histochem. Cytochem. 1997; 45: 1697-1706Crossref PubMed Scopus (136) Google Scholar). Additional studies by our laboratory and others further confirmed that maspin can be secreted or localized to the cell surface (8Ngamkitidechakul C. Burke J.M. O'Brien W.J. Twining S.S. Invest. Ophthalmol. Vis. Sci. 2001; 42: 3135-3141PubMed Google Scholar, 9Cella N. Contreras A. Latha K. Rosen J.M. Zhang M. FASEB J. 2006; 20: 1510-1512Crossref PubMed Scopus (57) Google Scholar, 33Khalkhali-Ellis Z. Hendrix M.J. Cancer Res. 2007; 67: 3535-3539Crossref PubMed Scopus (41) Google Scholar). Despite these findings, a recent study has suggested that maspin is an obligate intracellular protein that is not associated with the cytoskeleton or present on the cell surface (34Teoh S.S. Whisstock J.C. Bird P.I. J. Biol. Chem. 2010; 285: 10862-10869Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Therefore, we tested whether MCF10A cells from our laboratory can export maspin into the culturing medium. In these experiments, we utilized human corneal epithelial cells as a positive control for maspin secretion. Studies have shown that maspin is expressed and secreted from corneal epithelial cells, which seems to regulate stromal wound healing and maintaining avascularity (8Ngamkitidechakul C. Burke J.M. O'Brien W.J. Twining S.S. Invest. Ophthalmol. Vis. Sci. 2001; 42: 3135-3141PubMed Google Scholar). Additionally, human corneal epithelial cells provide further evidence that maspin can be secreted from cells. Because the existence of extracellular maspin is crucial for the findings presented in this manuscript, we sought to verify that the MCF10A cells in our laboratory can export maspin. The culturing media was obtained from two different MCF10A populations; (i) cells obtained directly from ATCC or (ii) cells that have been cultured in our laboratory for 2 years and analyzed by Western immunoblot for maspin protein expression. Maspin was detected in all media and cells tested (Fig. 1A). Interestingly, using densitometry analysis, we discovered that the detection of extracellular maspin is significantly reduced in high passage MCF10A cells compared with the low passage cells (Fig. 1B). Additionally, intracellular maspin expression was elevated in the high passage MCF10A cells compared with the low passage cells. These results suggest that maspin is exported from MCF10A cells, and maspin secretion may be reduced by extended culturing conditions. MCF10A cells deposit ECM proteins and can nucleate adhesive complexes typical of epithelia (35Goldfinger L.E. Stack M.S. Jones J.C. J. Cell Biol. 1998; 141: 255-265Crossref PubMed Scopus (273) Google Scholar, 36Goldfinger L.E. Hopkinson S.B. deHart G.W. Collawn S. Couchman J.R. Jones J.C. J. Cell Sci. 1999; 112: 2615-2629Crossref PubMed Google Scholar). In our cell adhesion model, we previously demonstrated that the region between amino acids 139 and 225 in maspin appears to mediate cell adhesion (9Cella N. Contreras A. Latha K. Rosen J.M. Zhang M. FASEB J. 2006; 20: 1510-1512Crossref PubMed Scopus (57) Google Scholar). However, the exact region(s) involved has not been elucidated. To determine the region(s) of maspin that is involved in adhesion, we developed a variety of competitive peptides. First, we developed peptides that dissect the aforementioned region (amino acid residues 139–225) into peptides corresponding to amino acid residues 137–158, 169–189, 181–202, 190–211, and 203–225. Then, using Molsoft ICM software to analyze the tertiary structure of maspin, we developed peptides to other regions of maspin that are exposed such as the RCL-(329–343), 260–275, and the control peptide (97–112). Using these peptides, we determined which region of maspin is involved in mediating MCF10A cell adhesion. The only peptides (derived from amino acid residues 139–225) that did compete with GST-maspin were the 181–202 and 190–211 peptides (Fig. 2A). These results have identified the 190–202 region of maspin is partially responsible for mediating cell adhesion. In addition, we discovered a novel region between amino acids 260 and 275 that is also important in regulating maspin-mediated MCF10A cell adhesion (Fig. 2A). Although the 190–202 (blue) and 260–275 (green) peptides are separated by 58 amino acids in the primary structure of maspin, they are adjacent to one another in the tertiary structure (Fig. 2B). To further demonstrate that these regions are involved in maspin-mediated adhesion, we developed mutants of GST-maspin; single mutant (E201K), double mutant (K268E,K270E), triple mutant (E201K,K268E,K270E), and a control mutant (D177A, S178L, T180P). The single (E201K) mutant corresponds to peptide 181–202, whereas the double (K268E,K270E) mutant corresponds to peptide 260–275. The control (D177A, S178L, T180P) mutant failed to have any reduced cell adhesion. However, both the single and double GST-maspin mutants had significantly reduced MCF10A cell adhesion (by 16 and 17%, respectively) as compared with WT GST-maspin (Fig. 2C). The triple (E201K,K268E,K270E) mutant had an additive effect whereby MCF10A cell adhesion was decreased by ∼28% when compared with WT GST-maspin (Fig. 2C). These findings support the notion that proximal amino acid residues from 190–202 and 260–275 in maspin mediate its effect on MCF10A cell adhesion. Recent accumulating evidence suggests an important role of the uPA-uPAR complex in regulating cell adhesion (24Smith H.W. Marshall C.J. Nat. Rev. Mol. Cell Biol. 2010; 11: 23-36Crossref PubMed Scopus (722) Google Scholar, 37Dass K. Ahmad A. Azmi A.S. Sarkar S.H. Sarkar F.H. Cancer Treat. Rev. 2008; 34: 122-136Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar). Because maspin is localized with the uPA-uPAR complex at the cell surface (12Yin S. Lockett J. Meng Y. Biliran Jr., H. Blouse G.E. Li X. Reddy N. Zhao Z. Lin X. Anagli J. Cher M.L. Sheng S. Cancer Res. 2006; 66: 4173-4181Crossref PubMed Scopus (70) Google Scholar), we investigated whether uPAR is involved in maspin binding to the cell surface. Using WT and uPAR−/− MEFs, we found that WT MEFs displayed specific 125I-GST-maspin binding, whereas uPAR−/− MEFs were absent of any specific 125I-GST-maspin binding using concentrations within the normal physiological range (<500 nm) (Fig. 3A). These results suggest that uPAR expression is necessary for maspin to bind or localize to the cell surface. In this study and others (11Al-Ayyoubi M. Schwartz B.S. Gettins P.G. J. Biol. Chem. 2007; 282: 19502-19509Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar), we have implied that a region proximal to the RCL of maspin is involved in mediating cell adhesion. Therefore, we determined the binding of 125I-GST-maspin to WT MEF cells that were incubated with either control (IgG), RCL (ABS4A), or N-terminal (S-20) blocking antibodies. We found that only the RCL (and/or surrounding area) and not the N terminus of maspin is necessary for maspin binding to the surface of WT MEFs (Fig. 3B). To determine whether the uPA-uPAR complex is required for 125I-GST-maspin binding to MEF cell surface, we used an antibody that disrupts uPA binding to uPAR. In the first set of experiments, treatment with this antibody did not change the binding of 125I-GST-maspin to WT MEF cells (Fig. 3C, first set). However, 125I-GST-maspin binding to the WT MEF cell surface is reduced when cells were stripped of uPA and incubated with the uPAR blocking antibody before reintroducing exogenous uPA and 125I-GST-maspin (Fig. 3C). These results demonstrated that cell surface localization of maspin requires not only uPAR but the uPA-uPAR complex. As stated earlier, maspin binds to both uPA and pro-uPA, causing reduced cell attachment by strengthening mature focal adhesion contacts (12Yin S. Lockett J. Meng Y. Biliran Jr., H. Blouse G.E. Li X. Reddy N. Zhao Z. Lin X. Anagli J. Cher M.L. Sheng S. Cancer Res. 2006; 66: 4173-4181Crossref PubMed Scopus (70) Google Scholar). A current model proposes that an unidentified region of maspin, in close proximity to the RCL, is responsible for maspin binding to uPA (and pro-uPA) (11Al-Ayyoubi M. Schwartz B.S. Gettins P.G. J. Biol. Chem. 2007; 282: 19502-19509Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). In this study, we have demonstrated that the amino acid residues from 190–202 and 260–275 in maspin mediate its effect on cell adhesion, and these regions are proximal to the RCL (Fig. 2). Using this information, we hypothesized that the association of maspin and uPA may mediate cell adhesion. To test this hypothesis, we acid-stripped endogenous uPA before the addition of GST-maspin and evaluated the effect on MCF10A cell adhesion. Stripping uPA substantially blocks maspin-induced MCF10A cell adhesion (Fig. 4A). To verify that this ablation of maspin-induced adhesion was a direct effect of uPA removal, we resupplied uPA-stripped MCF10A cells with exogenous uPA (800 nm). In fact, the addition of exogenous uPA rescues the maspin-mediated adhesion of uPA-stripped MCF10A cells (154.06% of GST control, Fig. 4A). These experiments demonstrate that uPA is needed for maspin-mediated adhesion. These combined studies revealed that uPAR or uPA removal from the surface of MCF10A cells ablates maspin localization to the cell surface (Fig. 3) and its ability to enhance cell adhesion (Fig. 4A). However, whether uPA and uPAR function independently or if the localization and adhesion functions of maspin require the uPA-uPAR complex is still not clear. Therefore, we utilized uPAR blocking antibody (anti-uPAR, blocks" @default.
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