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• W2095199846 abstract "Up-regulation of urokinase receptors is common during tumor progression and thought to promote invasion and metastasis. Urokinase receptors bind urokinase and a set of β1 integrins, but it remains unclear to what degree urokinase receptor/integrin binding is important to β1 integrin signaling. Using site-directed mutagenesis, single amino acid mutants of the urokinase receptor were identified that fail to associate with either α3β1 (D262A) or α5β1 (H249A) but associate normally with urokinase. To study the effects of these mutations on β1 integrin function, endogenous urokinase receptors were first stably silenced in tumor cell lines HT1080 and H1299, and then wild type or mutant receptors were expressed. Knockdown of urokinase receptors resulted in markedly reduced fibronectin and α5β1-dependent ERK activation and metalloproteinase MMP-9 expression. Re-expression of wild type or D262A mutant receptors but not the α5β1 binding-deficient H249A mutant reconstituted fibronectin responses. Because urokinase receptor·α5β1 complexes bind in the fibronectin heparin-binding domain (Type III 12–14) whereas α5β1 primarily binds in the RGD-containing domain (Type III 7–10), signaling pathways leading to ERK and MMP-9 responses were dissected. Binding to III 7–10 led to Src/focal adhesion kinase activation, whereas binding to III 7–14 caused Rac 1 activation. Tumor cells engaging fibronectin required both Type III 7–10- and 12–14-initiated signals to activate ERK and up-regulate MMP-9. Thus urokinase receptor binding to α5β1 is required for maximal responses to fibronectin and tumor cell invasion, and this operates through an enhanced Src/Rac/ERK signaling pathway. Up-regulation of urokinase receptors is common during tumor progression and thought to promote invasion and metastasis. Urokinase receptors bind urokinase and a set of β1 integrins, but it remains unclear to what degree urokinase receptor/integrin binding is important to β1 integrin signaling. Using site-directed mutagenesis, single amino acid mutants of the urokinase receptor were identified that fail to associate with either α3β1 (D262A) or α5β1 (H249A) but associate normally with urokinase. To study the effects of these mutations on β1 integrin function, endogenous urokinase receptors were first stably silenced in tumor cell lines HT1080 and H1299, and then wild type or mutant receptors were expressed. Knockdown of urokinase receptors resulted in markedly reduced fibronectin and α5β1-dependent ERK activation and metalloproteinase MMP-9 expression. Re-expression of wild type or D262A mutant receptors but not the α5β1 binding-deficient H249A mutant reconstituted fibronectin responses. Because urokinase receptor·α5β1 complexes bind in the fibronectin heparin-binding domain (Type III 12–14) whereas α5β1 primarily binds in the RGD-containing domain (Type III 7–10), signaling pathways leading to ERK and MMP-9 responses were dissected. Binding to III 7–10 led to Src/focal adhesion kinase activation, whereas binding to III 7–14 caused Rac 1 activation. Tumor cells engaging fibronectin required both Type III 7–10- and 12–14-initiated signals to activate ERK and up-regulate MMP-9. Thus urokinase receptor binding to α5β1 is required for maximal responses to fibronectin and tumor cell invasion, and this operates through an enhanced Src/Rac/ERK signaling pathway. The urokinase receptor (uPAR), 3The abbreviations used are: uPAR, urokinase receptor; uPA, urokinase-type plasminogen activator; Fn, fibronectin; Vn, vitronectin; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; FAK, focal adhesion kinase; MMP, matrix metalloproteinase; CBD, central cell-binding domain of fibronectin; HepII, COOH-terminal heparin-binding domain of fibronectin; EGFR, epidermal growth factor receptor; WT, wild type; mut, mutant; mAb, monoclonal antibody; pAb, polyclonal antibody; RNAi, RNA interference; FACS, fluorescence-activated cell sorting; HRP, horseradish peroxidase; GFP, green fluorescent protein; siRNA, short interfering RNA; shRNA, short hairpin RNA. a glycosylphosphatidylinositol-anchored membrane protein, has been shown to initiate signal transduction and regulate cell proliferation, adhesion, migration, and invasion (1Blasi F. Carmeliet P. Nat. Rev. Mol. Cell. Biol. 2002; 3: 932-943Crossref PubMed Scopus (1073) Google Scholar, 2Nusrat A.R. Chapman Jr., H.A. J. Clin. Investig. 1991; 87: 1091-1097Crossref PubMed Scopus (121) Google Scholar, 3Ossowski L. Aguirre-Ghiso J.A. Curr. Opin. Cell Biol. 2000; 12: 613-620Crossref PubMed Scopus (359) Google Scholar, 4Andreasen P.A. Kjoller L. Christensen L. Duffy M.J. Int. J. Cancer. 1997; 72: 1-22Crossref PubMed Scopus (1449) Google Scholar). The expression of uPAR on tumor cells strongly correlates with their migratory and invasive phenotype (5Wang Y. Med. Res. Rev. 2001; 21: 146-170Crossref PubMed Scopus (118) Google Scholar, 6Aguirre Ghiso J.A. Alonso D.F. Farias E.F. Gomez D.E. de Kier Joffe E.B. Eur. J. Biochem. 1999; 263: 295-304Crossref PubMed Scopus (186) Google Scholar, 7Ossowski L. Clunie G. Masucci M.T. Blasi F. J. Cell Biol. 1991; 115: 1107-1112Crossref PubMed Scopus (201) Google Scholar, 8Kook Y.H. Adamski J. Zelent A. Ossowski L. EMBO J. 1994; 13: 3983-3991Crossref PubMed Scopus (174) Google Scholar). Down-regulation of uPAR expression by antisense or RNAi strategies inhibits tumor invasion and metastasis of various cancer types (8Kook Y.H. Adamski J. Zelent A. Ossowski L. EMBO J. 1994; 13: 3983-3991Crossref PubMed Scopus (174) Google Scholar, 9Gondi C.S. Lakka S.S. Yanamandra N. Siddique K. Dinh D.H. Olivero W.C. Gujrati M. Rao J.S. Oncogene. 2003; 22: 5967-5975Crossref PubMed Scopus (88) Google Scholar, 10Ahmed N. Oliva K. Wang Y. Quinn M. Rice G. Br. J. Cancer. 2003; 89: 374-384Crossref PubMed Scopus (70) Google Scholar, 11Lakka S.S. Rajagopal R. Rajan M.K. Mohan P.M. Adachi Y. Dinh D.H. Olivero W.C. Gujrati M. Ali-Osman F. Roth J.A. Yung W.K. Kyritsis A.P. Rao J.S. Clin. Cancer Res. 2001; 7: 1087-1093PubMed Google Scholar, 12Wang Y. Liang X. Wu S. Murrell G.A. Doe W.F. Int. J. Cancer. 2001; 92: 257-262Crossref PubMed Scopus (45) Google Scholar). But because uPAR has multiple functions, the mechanisms underlying its influence on tumor cell invasion remain incompletely defined. One mechanism by which uPAR is reported to influence cellular behavior is by associating with signaling molecules and initiating signal transduction (3Ossowski L. Aguirre-Ghiso J.A. Curr. Opin. Cell Biol. 2000; 12: 613-620Crossref PubMed Scopus (359) Google Scholar, 13Dumler I. Weis A. Mayboroda O.A. Maasch C. Jerke U. Haller H. Gulba D.C. J. Biol. Chem. 1998; 273: 315-321Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 14Yebra M. Goretzki L. Pfeifer M. Mueller B.M. Exp. Cell Res. 1999; 250: 231-240Crossref PubMed Scopus (106) Google Scholar, 15Wei Y. Yang X. Liu Q. Wilkins J.A. Chapman H.A. J. Cell Biol. 1999; 144: 1285-1294Crossref PubMed Scopus (369) Google Scholar). As uPAR lacks both transmembrane and cytoplasmic domains, uPAR-mediated signaling is thought to require transmembrane partners, particularly integrins (3Ossowski L. Aguirre-Ghiso J.A. Curr. Opin. Cell Biol. 2000; 12: 613-620Crossref PubMed Scopus (359) Google Scholar, 16Kugler M.C. Wei Y. Chapman H.A. Curr. Pharm. Des. 2003; 9: 1565-1574Crossref PubMed Scopus (96) Google Scholar, 17Wei Y. Lukashev M. Simon D.I. Bodary S.C. Rosenberg S. Doyle M.V. Chapman H.A. Science. 1996; 273: 1551-1555Crossref PubMed Scopus (698) Google Scholar) and tyrosine kinase receptors such as platelet-derived growth factor receptor and EGFR (18Mazzieri R. D'Alessio S. Kenmoe R.K. Ossowski L. Blasi F. Mol. Biol. Cell. 2006; 17: 367-378Crossref PubMed Scopus (68) Google Scholar, 19Liu D. Aguirre Ghiso J. Estrada Y. Ossowski L. Cancer Cell. 2002; 1: 445-457Abstract Full Text Full Text PDF PubMed Scopus (358) Google Scholar, 20Jo M. Thomas K.S. Marozkina N. Amin T.J. Silva C.M. Parsons S.J. Gonias S.L. J. Biol. Chem. 2005; 280: 17449-17457Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 21Kiyan J. Kiyan R. Haller H. Dumler I. EMBO J. 2005; 24: 1787-1797Crossref PubMed Scopus (82) Google Scholar). uPAR has been shown to associate with β1, β2, β3, and β5 integrins (17Wei Y. Lukashev M. Simon D.I. Bodary S.C. Rosenberg S. Doyle M.V. Chapman H.A. Science. 1996; 273: 1551-1555Crossref PubMed Scopus (698) Google Scholar, 22Sitrin R.G. Todd R.F. II I Albrecht E. Gyetko M.R. J. Clin. Investig. 1996; 97: 1942-1951Crossref PubMed Scopus (193) Google Scholar, 23Xue W. Mizukami I. Todd R.F. II I Petty H.R. Cancer Res. 1997; 57: 1682-1689PubMed Google Scholar, 24Carriero M.V. Del Vecchio S. Capozzoli M. Franco P. Fontana L. Zannetti A. Botti G. D'Aiuto G. Salvatore M. Stoppelli M.P. Cancer Res. 1999; 59: 5307-5314PubMed Google Scholar). The uPAR binding sites on β1 integrins have been identified (25Wei Y. Czekay R.P. Robillard L. Kugler M.C. Zhang F. Kim K.K. Xiong J.P. Humphries M.J. Chapman H.A. J. Cell Biol. 2005; 168: 501-511Crossref PubMed Scopus (115) Google Scholar, 26Wei Y. Eble J.A. Wang Z. Kreidberg J.A. Chapman H.A. Mol. Biol. Cell. 2001; 12: 2975-2986Crossref PubMed Scopus (223) Google Scholar, 27Simon D.I. Wei Y. Zhang L. Rao N.K. Xu H. Chen Z. Liu Q. Rosenberg S. Chapman H.A. J. Biol. Chem. 2000; 275: 10228-10234Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar); both domains II and III of uPAR are implicated in the integrin interaction (28Degryse B. Resnati M. Czekay R.P. Loskutoff D.J. Blasi F. J. Biol. Chem. 2005; 280: 24792-24803Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 29Chaurasia P. Aguirre-Ghiso J.A. Liang O.D. Gardsvoll H. Ploug M. Ossowski L. J. Biol. Chem. 2006; 281: 14852-14863Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Recently we have shown that uPAR directly binds integrin α5β1 and regulates its conformation and function (25Wei Y. Czekay R.P. Robillard L. Kugler M.C. Zhang F. Kim K.K. Xiong J.P. Humphries M.J. Chapman H.A. J. Cell Biol. 2005; 168: 501-511Crossref PubMed Scopus (115) Google Scholar); however, the exact signaling pathway(s) initiated by uPAR/α5β1 integrin interactions remains unclear. Ossowski and co-workers (30Aguirre-Ghiso J.A. Liu D. Mignatti A. Kovalski K. Ossowski L. Mol. Biol. Cell. 2001; 12: 863-879Crossref PubMed Scopus (383) Google Scholar, 31Aguirre-Ghiso J.A. Estrada Y. Liu D. Ossowski L. Cancer Res. 2003; 63: 1684-1695PubMed Google Scholar) have found that high levels of uPAR expression and its interaction with α5β1 enhanced the basal level of activated ERK favoring tumor growth in vivo, possibly through FAK and Src signaling pathways downstream of the integrin as well as EGFR (19Liu D. Aguirre Ghiso J. Estrada Y. Ossowski L. Cancer Cell. 2002; 1: 445-457Abstract Full Text Full Text PDF PubMed Scopus (358) Google Scholar, 32Aguirre Ghiso J.A. Oncogene. 2002; 21: 2513-2524Crossref PubMed Scopus (206) Google Scholar). Classical studies by Werb et al. (33Werb Z. Tremble P.M. Behrendtsen O. Crowley E. Damsky C.H. J. Cell Biol. 1989; 109: 877-889Crossref PubMed Scopus (903) Google Scholar) established that signaling through the fibronectin receptor induces up-regulation of several MMPs. More recently, signaling mechanisms that underlie these effects and a role for the urokinase-type plasminogen activator (uPA) system in MMP expression have begun to emerge. Cell adhesion on fibronectin (Fn) induces MMP-9 expression, and this induction requires α5β1 integrin (34Xie B. Laouar A. Huberman E. J. Biol. Chem. 1998; 273: 11576-11582Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 35Shibata K. Kikkawa F. Nawa A. Suganuma N. Hamaguchi M. Cancer Res. 1997; 57: 5416-5420PubMed Google Scholar, 36Esparza J. Vilardell C. Calvo J. Juan M. Vives J. Urbano-Marquez A. Yague J. Cid M.C. Blood. 1999; 94: 2754-2766Crossref PubMed Google Scholar). Src and Src/FAK interactions, as well as Rac activation, have been implicated in integrin-mediated MMP-9 secretion (37Hsia D.A. Mitra S.K. Hauck C.R. Streblow D.N. Nelson J.A. Ilic D. Huang S. Li E. Nemerow G.R. Leng J. Spencer K.S. Cheresh D.A. Schlaepfer D.D. J. Cell Biol. 2003; 160: 753-767Crossref PubMed Scopus (461) Google Scholar, 38Segarra M. Vilardell C. Matsumoto K. Esparza J. Lozano E. Serra-Pages C. Urbano-Marquez A. Yamada K.M. Cid M.C. FASEB J. 2005; 19: 1875-1877Crossref PubMed Scopus (56) Google Scholar). It is also reported that MEK1-MAPK is required for the Fn-dependent activation of MMP-9 secretion (39Thant A.A. Nawa A. Kikkawa F. Ichigotani Y. Zhang Y. Sein T.T. Amin A.R. Hamaguchi M. Clin. Exp. Metastasis. 2000; 18: 423-428Crossref PubMed Scopus (132) Google Scholar), and Rac activation could enhance the association of ERK2 with MEK1, promoting MAPK activity (40Eblen S.T. Slack J.K. Weber M.J. Catling A.D. Mol. Cell. Biol. 2002; 22: 6023-6033Crossref PubMed Scopus (199) Google Scholar). The uPA system and MMP-9 are both overexpressed in malignant tumors and highly correlated with cancer metastasis (41Mazzieri R. Masiero L. Zanetta L. Monea S. Onisto M. Garbisa S. Mignatti P. EMBO J. 1997; 16: 2319-2332Crossref PubMed Scopus (373) Google Scholar, 42Ahmed N. Pansino F. Baker M. Rice G. Quinn M. J. Cell. Biochem. 2002; 84: 675-686Crossref PubMed Scopus (55) Google Scholar, 43Inuzuka K. Ogata Y. Nagase H. Shirouzu K. J. Surg. Res. 2000; 93: 211-218Abstract Full Text PDF PubMed Scopus (39) Google Scholar). uPA/uPAR is not only important in MMP-9 activation through the uPA-plasmin-MMP-3 cascade (44Hahn-Dantona E. Ramos-DeSimone N. Sipley J. Nagase H. French D.L. Quigley J.P. Ann. N. Y. Acad. Sci. 1999; 878: 372-387Crossref PubMed Scopus (86) Google Scholar) but also appears to be involved in regulating MMP-9 production. Suppression of uPAR by an antisense approach in a colon cancer cell line or disruption of uPAR·β1 integrin complex by a uPAR binding peptide, P25, resulted in complete inhibition of pro-MMP-9 secretion and decrease of basal or uPA-induced ERK activation (10Ahmed N. Oliva K. Wang Y. Quinn M. Rice G. Br. J. Cancer. 2003; 89: 374-384Crossref PubMed Scopus (70) Google Scholar). Although uPAR expression is reported to activate Rac and promote cell motility (45Vial E. Sahai E. Marshall C.J. Cancer Cell. 2003; 4: 67-79Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar), the role of uPAR or uPAR/α5β1 in Fn-induced MMP-9 production has not been explored, and mechanisms underlying Rac activation via uPAR are unknown. Fn has been shown to regulate multiple cellular functions including gene expression, survival, and cytoskeleton organization through interaction with its principal cell surface receptor, α5β1 (46Mao Y. Schwarzbauer J.E. Matrix Biol. 2005; 24: 389-399Crossref PubMed Scopus (618) Google Scholar). Fn contains NH2-, gelatin-, cell-, and COOH-terminal heparin-binding domains. The central cell-binding domain of fibronectin (CBD) has an RGD sequence in domain III 10 recognized by α5β1 integrin, and several sites in the COOH-terminal heparin-binding domain of fibronectin (HepII) also interact with the cell surface with varying affinities (47Mould A.P. Humphries M.J. EMBO J. 1991; 10: 4089-4095Crossref PubMed Scopus (166) Google Scholar, 48Walker A. Gallagher J.T. Biochem. J. 1996; 317: 871-877Crossref PubMed Scopus (67) Google Scholar, 49Kim J. Han I. Kim Y. Kim S. Oh E.S. Biochem. J. 2001; 360: 239-245Crossref PubMed Google Scholar). Our recent data indicate that direct binding of uPAR with α5β1 forms an additional binding site within the HepII that is RGD-independent (25Wei Y. Czekay R.P. Robillard L. Kugler M.C. Zhang F. Kim K.K. Xiong J.P. Humphries M.J. Chapman H.A. J. Cell Biol. 2005; 168: 501-511Crossref PubMed Scopus (115) Google Scholar). It has been documented that signaling events are often mediated by two different but adjacent sites within Fn. Fibroblasts plated on the HepII induced formation of filopodia and lamellipodia, whereas cells plated on the CBD require additional signals from HepII to form focal adhesions and stress fibers (50Bloom L. Ingham K.C. Hynes R.O. Mol. Biol. Cell. 1999; 10: 1521-1536Crossref PubMed Scopus (124) Google Scholar, 51Woods A. Longley R.L. Tumova S. Couchman J.R. Arch. Biochem. Biophys. 2000; 374: 66-72Crossref PubMed Scopus (195) Google Scholar). In addition, in some systems both CBD and HepII cooperatively regulate p125FAK activity, affecting cell survival (52Jeong J. Han I. Lim Y. Kim J. Park I. Woods A. Couchman J.R. Oh E.S. Biochem. J. 2001; 356: 531-537Crossref PubMed Scopus (32) Google Scholar). These observations invite the hypothesis that the second binding site on Fn created by uPAR/α5β1 association is critical for Fn signaling and enhanced protease expression and raise the possibility that invasive tumor cells are addicted to this pathway for their malignant phenotype. In this study, we tested these hypotheses by generating stable knockdown of uPAR in invasive tumor cell lines of different origins and then reintroducing point mutants of uPAR with selective defects in α5β1 binding. This allowed us to dissociate the capacity of uPAR to bind uPA from its direct binding of α5β1 and to dissect the signaling pathways initiated by direct binding of uPAR to the integrin. We demonstrate that suppression of uPAR expression in tumor cells reduces α5β1/Fn-dependent induction of ERK activation and MMP-9 secretion. Reconstitution of signaling requires expression of uPAR capable of α5β1 binding. Because uPAR/α5β1 binds Fn HepII (III 12–14) whereas α5β1 primarily binds the RGD sequence in Fn fragment III 7–10, we tested whether ERK and MMP-9 responses are binding site-dependent. Our results demonstrate that Fn adhesion-mediated ERK activation and MMP-9 up-regulation require cell engagement with both RGD and HepII sites on Fn either as a single fusion protein or as separate polypeptides. For the first time, we show that uPAR is required for maximal α5β1-dependent responses to Fn that promote tumor cell invasion, and this operates through integration of two signals initiated by cell engagement to the HepII and RGD sites on Fn by uPAR-bound and unbound α5β1. Cell Culture and Reagents—Human fibrosarcoma HT1080 and lung cancer cell lines H1299 were obtained from the American Type Culture Collection (Manassas, VA). Both cell lines were cultured in Dulbecco's modified minimum Eagle's medium supplemented with 10% fetal bovine serum and penicillin-streptomycin. Fn, an NH2-terminal 70-kDa Fn fragment, vitronectin (Vn), collagen type I, anti-β-actin mAb, peptides GRGDSPK and GRADSPK, and 0.01% polylysine solution were purchased from Sigma. Fn fragments III 7–10, III 7–14, and III 12–14 were gifts from Dr. Harold Erickson (Duke University, Durham, NC). An NH2-terminal fragment of uPA fused with human Fc (1–48-Fc) was a gift from Dr. Steven Rosenberg (Chiron, Emeryville, CA). Peptides β1P1 (NLDSPEGGF) and scβ1P1 (EDGLFNPSG) were synthesized at Anaspec (San Jose, CA) and purified by high pressure liquid chromatography. MEK inhibitor PD98059, EGFR inhibitor AG1478, Src kinase inhibitor PP2 and its structurally inert isomer PP3, and recombinant MMP-9 were purchased from Calbiochem. Precast 10% gelatin gels, Cy3-conjugated anti-mouse IgG, 4′,6-diamidino-2-phenylindole, Prolong antifade mounting solution, and phalloidin-Texas Red were purchased from Invitrogen. The site-directed mutagenesis kit was purchased from Stratagene (La Jolla, CA). uPAR mAb for FACS was purchased from American Diagnostica (Stamford, CT). uPAR mAb for blotting (R2) was a kind gift from Dr. Michael Ploug (Finsen Laboratory, Copenhagen, Denmark). Blocking antibodies to human integrin α3 (P1B5), human integrin α5 (P1D6), human integrin αvβ3, and α5 pAb were purchased from Chemicon International (Temecula, CA). Anti-integrin α3 pAb (D23) was a kind gift from Dr. Martin E. Hemler (Dana Farber Cancer Institute, Boston, MA). Anti-paxillin, anti-phospho-FAK (Tyr-397), anti-FAK, anti-phospho-ERK, and anti-ERK antibodies were purchased from Transduction Laboratories. Anti-human Fc-HRP was purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). Dominant negative Rac 1 construct was kindly provided by Dr. Henry Bourne (University of California, San Francisco, CA). Anti-Rac 1 mAb was purchased from Upstate (Chicago, IL). Generation of Stable uPAR Knockdown Cells—pSicoR-GFP (kindly provided by Dr. Michael McManus, University of California, San Francisco, CA) was used for the construction of a plasmid expressing siRNA for uPAR downstream of a mouse U6 promoter and GFP gene downstream of the cytomegalovirus promoter. A validated siRNA target sequence (45Vial E. Sahai E. Marshall C.J. Cancer Cell. 2003; 4: 67-79Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar) was used for uPAR RNAi: GGTGAAGAAGGGCGTCCAA. Two complementary oligonucleotides, XhoI-(target sense)-TTCAAGAGA-(target antisense)-TTTTTT-HpaI and HpaI-AAAAAA-(target sense)-TCTCTTGAA-(target antisense)-XhoI, were synthesized for each target sequence and annealed in vitro. The annealed complementary oligonucleotides for uPAR were inserted into the HpaI and XhoI sites of pSicoR-GFP vector. All constructs were verified by DNA sequencing. These plasmids produce short hairpin RNAs with the linker sequence (TTCAAGAGA) forming a loop structure, and then the linker is processed by Dicer, forming a double-stranded RNA that acts as a siRNA. Expression of Wild Type and Mutant uPARs in uPAR Knockdown Cells—To express wild type (WT) or mutant (mut) uPARs in cells already expressing uPAR RNAi, we introduced three silent mutations in the siRNA uPAR-targeting region and used this as the template uPAR cDNA for Ala point mutations. Preliminary experiments varying the number of silent bp substitutions determined that 3-bp substitutions in the targeting region produced optimal expression in the presence of uPAR RNAi. The point mutations on uPAR were generated by site-directed mutagenesis using a PCR megaprimer procedure using Pfu polymerase (Stratagene) and pcDNA-uPAR as a template as described previously (53Costa G.L. Bauer J.C. McGowan B. Angert M. Weiner M.P. Methods Mol. Biol. 1996; 57: 239-248PubMed Google Scholar). All the mutated DNAs containing the desired mutation(s) were introduced into pCEP4 for expression. The PCR-generated sequences of all constructs were verified by DNA sequencing. The uPAR knockdown cells expressing WT or mut (H249A or D262A) uPARs were stably selected and sorted. Protein expression was verified by FACS analysis and/or Western blot. Adhesion Assay—Cells were seeded onto Fn or Vn (5 μg/ml)-coated plates and incubated in Dulbecco's modified Eagle's medium, 0.1% bovine serum albumin with or without 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 as described previously (26Wei Y. Eble J.A. Wang Z. Kreidberg J.A. Chapman H.A. Mol. Biol. Cell. 2001; 12: 2975-2986Crossref PubMed Scopus (223) Google Scholar). TaqMan Quantitative PCR—Verification of transcript quantity in several selected cDNAs was performed using TaqMan real time PCR. The primer pairs and probe for each cDNA were designed using Primer Express software (Applied Biosystems). The quantification was performed using the standard protocol of ABI PRISM 7700 (Applied Biosystems). Gelatin Zymography—Cells were starved and seeded on Fn or Fn fragment-(5 μg/ml), Vn-(5 μg/ml), collagen I-(5 μg/ml), or polylysine (50 μg/ml)-coated wells in Dulbecco's modified Eagle's medium, 0.01% BSA for 24 h. The conditioned media were collected, centrifuged, and frozen until use. 10 μl of conditioned medium were separated by a 10% SDS-PAGE gel containing 1% gelatin (Invitrogen) under non-reducing conditions. The gels were rinsed in renaturing buffer (2.5% Triton X-100); developed in buffer containing 50 mm Tris-HCl (pH 7.7), 5 mm CaCl2, and 0.02% NaN3; stained with Coomassie Brilliant Blue to indicate MMP-9 clear bands; and imaged. Recombinant MMP-9 was used as positive control. In some cases, cells were treated with different inhibitors during a 24-h incubation: MEK1 inhibitor PD98059 (10 μm), EGFR inhibitor AG1478 (1 μm), and peptide β1P1 or its scrambled control scβ1P1 (0.4 mm). Flow Cytometry—Stable clones expressing siRNA uPAR and WT or mut uPAR were stained with primary antibody to uPAR and secondary allophycocyanin-conjugated anti-mouse IgG (Sigma) and analyzed on a flow cytometer (FACSCalibur®,BD Biosciences). Isolation of various uPAR-expressing cell lines was done by high throughput cell sorting (MoFlo, Dako). uPAR Ligand Binding Assay—All the procedures were done in triplicate at 4 °C. Control cells, cells with uPAR silencing, and uPAR knockdown cells reconstituted with WT or mut uPAR were plated to form a monolayer, acid-washed, and incubated with NH2-terminal fragment of uPA 1–48-Fc fusion protein (100 nm). The cells were then incubated with anti-Fc-HRP in Dulbecco's modified Eagle's medium, 0.02% BSA for 1 h. After washing, the bound 1–48-Fc was detected by HRP substrates and quantified by measuring A490 nm. Western Blot—Control cells and cells with uPAR silencing or uPAR knockdown cells reconstituted with WT or mut uPAR were lysed in RIPA buffer (150 mm NaCl, 50 mm Tris, pH 8.0, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS supplemented with protease inhibitors and 1 mm phenylmethylsulfonyl fluoride). Equal amounts of protein were loaded per lane and separated by SDS-PAGE. The protein was transferred to nitrocellulose membrane and blotted for uPAR using primary anti-uPAR mAb and secondary anti-mouse-HRP conjugated antibody. The same membrane was blotted for β-actin as loading control. Kinase and Rac Activity Assays—Cells were serum-starved for 4 h and seeded on Fn-, Vn-, or polylysine-coated 6-well plates for 20 min. After incubating with or without treatment, cells were lysed in RIPA buffer supplemented with protease inhibitors and phosphatase inhibitors. Lysates were immunoblotted for phospho-ERK or FAK and total ERK or FAK. For Rac pulldown assays, cells were lysed in cold Rac assay buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 10 mm MgCl2,1% Triton X-100, 0.5% sodium deoxycholate plus protease inhibitors). Lysates were then incubated with purified glutathione S-transferase-p21-activated kinase 1 protein-p21-binding domain (GST-PAK-PBD) beads (54Schmitz U. Thommes K. Beier I. Wagner W. Sachinidis A. Dusing R. Vetter H. J. Biol. Chem. 2001; 276: 22003-22010Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar) for 30 min and washed three times with Rac assay buffer. The bead-bound active Rac and total Rac in the lysates were analyzed by Western blotting with anti-Rac mAb. Immunoprecipitation—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 phenylmethylsulfonyl fluoride. Clarified lysates were immunoprecipitated with antibody to integrin α5 (P1D6) or α3 (P1B5). The immunoprecipitates were blotted for uPAR or integrins (pAb). Immunofluorescence Microscopy—Cells plated on Fn-coated chambered slides were fixed in 3.7% paraformaldehyde, permeabilized with 0.5% Nonidet P-40, and blocked with 10% horse serum and 1% bovine serum albumin in phosphate-buffered saline. The slides were stained with primary anti-paxillin antibody and Cy3-conjugated secondary anti-mouse IgG antibody or phalloidin-Texas Red. Slides were incubated with 4′, 6-diamidino-2-phenylindole before mounting in Prolong (Molecular Probes). uPAR has been shown previously to regulate α5β1 conformation and binding to Fn (25Wei Y. Czekay R.P. Robillard L. Kugler M.C. Zhang F. Kim K.K. Xiong J.P. Humphries M.J. Chapman H.A. J. Cell Biol. 2005; 168: 501-511Crossref PubMed Scopus (115) Google Scholar). In addition, most malignant tumor cells already express high levels of uPAR (55de Bock C.E. Wang Y. Med. Res. Rev. 2004; 24: 13-39Crossref PubMed Scopus (133) Google Scholar). Thus, we sought to test the role of uPAR on the regulation of α5β1/Fn-mediated signaling in tumor cells expressing uPAR. The expression of endogenous uPAR was knocked down using RNA interference with short hairpin (sh) RNA in HT1080 and H1299 cells by transfection with pSicoR-GFP-shRNA uPAR (uPAR knockdown) or control vector pSicoR-GFP (control). A pool of GFP-positive and low uPAR-expressing cells were sorted, and uPAR expression was inspected by Western blot (Fig. 1A) or FACS (see Fig. 4C). Because both HT1080 and H1299 cells attach to Fn via α5β1 (Fig. 1C), we tested the effect of uPAR suppression on Fn adhesion. Although uPAR expression does not affect overall Fn adhesion on high concentrations of Fn, reductions in uPAR protein levels resulted in a conversion from RGD-resistant to RGD-sensitive adhesion to Fn (Fig. 1B). These data are consistent with prior observations in tumor cells transiently transfected with synthetic siRNA uPAR (25Wei Y. Czekay R.P. Robillard L. Kugler M.C. Zhang F. Kim K.K. Xiong J.P. Humphries M.J. Chapman H.A. J. Cell Biol. 2005; 168: 501-511Crossref PubMed Scopus (115) Google Scholar). Conversely overexpression of wild type uPAR in uPAR knockdown cells restored RGD-resistant Fn adhesion (see Fig. 5B).FIGURE 4Expression of wild type and mutant uPARs that are able to escape siRNA targeting. A, point mutations on uPAR molecule. The crystal structure of uPAR is displayed as a ribbon diagram using Protein Data Bank code 1YWH (57Llinas P. Le Du M.H. Gardsvoll H. Dano K. Ploug M. Gilquin B. Stura E.A. Menez A. EMBO J. 2005; 24: 1655-1663Crossref PubMed Scopus (208) Google Scholar). The individual uPAR domains are colored blue (domain I), gray (domain II), and yellow (domain III). The uPAR binding peptide is marked red. Part of the mutations we made in domains II and III are listed. B, silent mut" @default.
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• W2095199846 title "Urokinase Receptors Are Required for α5β1 Integrin-mediated Signaling in Tumor Cells" @default.
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