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• W2046571883 abstract "Thrombospondin (TSP) 1 is a trimeric multidomain protein that contains motifs that recognize distinct host cell receptors coupled to multiple signaling pathways. Selected TSP1-induced cellular responses are tyrosine kinase-dependent, and TSP1 contains epidermal growth factor (EGF)-like repeats. Specific receptor interactions or functions for the EGF-like repeats have not been identified. We asked whether one or more biological responses to TSP1 might be explained through EGF receptor (EGFR) activation. In A431 cells, TSP1 increased autophosphorylation of Tyr-1068 of EGFR in a dose- and time-dependent manner. The ability of TSP1 to activate EGFR was replicated by the tandem EGF-like repeats as a recombinant protein. The three EGF-like repeats alone produced a high level of Tyr-1068 phosphorylation. EGF-like repeats from TSP2 and TSP4 also activated EGFR. Tyr-1068 phosphorylation was less when individual EGF-like repeats were tested or flanking sequences were added to the three EGF-like repeats. TSP1 and its EGF-like repeats also increased phosphorylation of EGFR Tyr-845, Tyr-992, Tyr-1045, Tyr-1086, and Tyr-1173, activated phospholipase Cγ, and increased cell migration. No evidence was found for binding of the EGF-like repeats to EGFR. Instead, EGFR activation in response to TSP1 or its EGF-like repeats required matrix metalloprotease activity, including activity of matrix metalloprotease 9. Access to the ligand-binding portion of the EGFR ectodomain was also required. These findings suggest release of an endogenous EGFR ligand in response to ligation of a second unknown receptor by the TSPs. Thrombospondin (TSP) 1 is a trimeric multidomain protein that contains motifs that recognize distinct host cell receptors coupled to multiple signaling pathways. Selected TSP1-induced cellular responses are tyrosine kinase-dependent, and TSP1 contains epidermal growth factor (EGF)-like repeats. Specific receptor interactions or functions for the EGF-like repeats have not been identified. We asked whether one or more biological responses to TSP1 might be explained through EGF receptor (EGFR) activation. In A431 cells, TSP1 increased autophosphorylation of Tyr-1068 of EGFR in a dose- and time-dependent manner. The ability of TSP1 to activate EGFR was replicated by the tandem EGF-like repeats as a recombinant protein. The three EGF-like repeats alone produced a high level of Tyr-1068 phosphorylation. EGF-like repeats from TSP2 and TSP4 also activated EGFR. Tyr-1068 phosphorylation was less when individual EGF-like repeats were tested or flanking sequences were added to the three EGF-like repeats. TSP1 and its EGF-like repeats also increased phosphorylation of EGFR Tyr-845, Tyr-992, Tyr-1045, Tyr-1086, and Tyr-1173, activated phospholipase Cγ, and increased cell migration. No evidence was found for binding of the EGF-like repeats to EGFR. Instead, EGFR activation in response to TSP1 or its EGF-like repeats required matrix metalloprotease activity, including activity of matrix metalloprotease 9. Access to the ligand-binding portion of the EGFR ectodomain was also required. These findings suggest release of an endogenous EGFR ligand in response to ligation of a second unknown receptor by the TSPs. Thrombospondin (TSP) 2The abbreviations used are: TSP, thrombospondin; BS3, bis(sulfosuccinimidyl)suberate; E123, epidermal growth factor-like repeats 1-3; ECL, enhanced chemiluminescence; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; FITC, fluorescein isothiocyanate; HRP, horseradish peroxidase; MMP, matrix metalloprotease; PLCγ, phospholipase Cγ; PTK, protein-tyrosine kinase; PVDF, polyvinylidene difluoride; siRNA, small interfering RNA; TSR, thrombospondin type 1 repeat; aa, amino acid; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline. 1 is an ∼420-kDa trimeric glycoprotein composed of three identical 145-kDa polypeptide chains linked by disulfide bonds (1Lawler J. Derick L.H. Connolly J.E. Chen J.-H. Chao F.C. J. Biol. Chem. 1985; 260: 3762-3772Abstract Full Text PDF PubMed Google Scholar). TSP1 is one of five TSP family members (2Carlson C.B. Lawler J. Mosher D.F. Cell. Mol. Life Sci. 2008; 65: 672-686Crossref PubMed Scopus (143) Google Scholar). Each subunit of TSP1 and TSP2 contains the following structural elements: an NH2-terminal globular domain of the laminin G domain and concanavalin A-like lectin/glucanase superfamily; an α-helical region that presumably forms a parallel homotrimeric coiled coil as in matrilin-1; a von Willebrand factor type C module; three TSP type 1 (TSR) or properdin repeats, each of which is elongated and consists of a novel, antiparallel three-stranded fold; and the TSP “signature piece” that is made up of three EGF-like TSP type 2 repeats, 13 calcium-binding repeats, and a COOH-terminal domain that forms a lectin-like β-sandwich (2Carlson C.B. Lawler J. Mosher D.F. Cell. Mol. Life Sci. 2008; 65: 672-686Crossref PubMed Scopus (143) Google Scholar). The elements of the “signature piece” interact extensively to form three structural regions termed the stalk, wire, and globe, and are further stabilized by disulfide bonds and bound calcium (3Carlson C.B. Bernstein D.A. Annis D.S. Misenheimer T.M. Hannah B.L. Mosher D.F. Keck J.L. Nat. Struct. Mol. Biol. 2005; 12: 910-914Crossref PubMed Scopus (72) Google Scholar). All five vertebrate TSPs contain the signature piece. TSP3, TSP4, and TSP5 have a pentameric coiled coil (4Malashkevich V.N. Kammerer R.A. Efimov V.P. Schulthess T. Engel J. Science. 1996; 274: 761-765Crossref PubMed Scopus (269) Google Scholar), lack the von Willebrand factor C domain and TSRs, and have an extra EGF-like repeat (5Bornstein P. J. Cell Biol. 1995; 130: 503-506Crossref PubMed Scopus (582) Google Scholar, 6Tan K. Duquette M. Liu J.H. Zhang R. Joachimiak A. Wang J.H. Lawler J. Structure (Lond.). 2006; 14: 33-42Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). TSP1 recognizes multiple cell surface receptors coupled to specific signaling pathways that evoke distinct, and sometimes opposing, biological responses (5Bornstein P. J. Cell Biol. 1995; 130: 503-506Crossref PubMed Scopus (582) Google Scholar, 7Streit M. Velasco P. Riccardi L. Spencer L. Brown L.F. Janes L. Lange-Asschenfeldt B. Yano K. Hawighorst T. Iruela-Arispe L. Detmar M. EMBO J. 2000; 19: 3272-3282Crossref PubMed Scopus (182) Google Scholar, 8Uno K. Hayashi H. Kuroki M. Uchida H. Yamauchi Y. Kuroki M. Oshima K. Biochem. Biophys. Res. Commun. 2004; 315: 928-934Crossref PubMed Scopus (46) Google Scholar). The NH2-terminal globular domain recognizes heparan sulfate proteoglycans (6Tan K. Duquette M. Liu J.H. Zhang R. Joachimiak A. Wang J.H. Lawler J. Structure (Lond.). 2006; 14: 33-42Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar), low density lipoprotein receptor-related protein 1 (9Orr A.W. Pedraza C.E. Pallero M.A. Elzie C.A. Goicoechea S. Strickland D.K. Murphy-Ullrich J.E. J. Cell Biol. 2003; 161: 1179-1189Crossref PubMed Scopus (130) Google Scholar), sulfated glycolipids (10Roberts D.D. Haverstick D.M. Dixit V.M. Frazier W.A. Santoro S.A. Ginsburg V.J. J. Biol. Chem. 1985; 260: 9405-9411Abstract Full Text PDF PubMed Google Scholar), calreticulin (9Orr A.W. Pedraza C.E. Pallero M.A. Elzie C.A. Goicoechea S. Strickland D.K. Murphy-Ullrich J.E. J. Cell Biol. 2003; 161: 1179-1189Crossref PubMed Scopus (130) Google Scholar), and integrins (11Calzada M.J. Sipes J.M. Krutzsch H.C. Yurchenco P.D. Annis D.S. Mosher D.F. Roberts D.D. J. Biol. Chem. 2003; 278: 40679-40687Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). In the TSRs, the CSVTCG and GVQXR motifs recognize CD36 (12Dawson D.W. Pearce S.F. Zhong R. Silverstein R.L. Frazier W.A. Bouck N.P. J. Cell Biol. 1997; 138: 707-717Crossref PubMed Scopus (546) Google Scholar), and in the calcium-binding wire an RGD-containing sequence binds to αvβ3 and other integrins (13Lawler J. Weinstein R. Hynes R.O. J. Cell Biol. 1998; 107: 2351-2361Crossref Scopus (336) Google Scholar). β1 integrins also have been shown to interact with TSRs (14Calzada M.J. Annis D.S. Zeng B. Marcinkiewicz C. Banas B. Lawler J. Mosher D.F. Roberts D.D. J. Biol. Chem. 2004; 279: 41734-41743Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Finally, two cell-binding motifs in the COOH-terminal lectin-like domains, RFYVVM and FIRVVM (15Kosfeld M.D. Frazier W.A. J. Biol. Chem. 1993; 268: 8808-8814Abstract Full Text PDF PubMed Google Scholar), interact with CD47 or integrin-associated protein (16Gao A.-G. Lindberg F.P. Finn M.B. Blystone S.D. Brown E.J. Frazier W.A. J. Biol. Chem. 1996; 271: 21-24Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar). Therefore, the NH2-terminal globular domain, the TSRs and calcium-binding repeats, and the COOH-terminal domain of TSP1 all exhibit receptor binding activities that elicit distinct host cell responses. Aside from minimal recognition by integrins (14Calzada M.J. Annis D.S. Zeng B. Marcinkiewicz C. Banas B. Lawler J. Mosher D.F. Roberts D.D. J. Biol. Chem. 2004; 279: 41734-41743Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar), no such receptor binding activity or biological function has been ascribed to the type 2 EGF-like repeats of TSP1. TSP1 is secreted by numerous cell types and is present in the extracellular matrix (17Lahav J. Biochim. Biophys. Acta. 1993; 1182: 1-14Crossref PubMed Scopus (111) Google Scholar). TSP1 was first demonstrated in the releasate of thrombin-stimulated platelets (18Lawler J. Blood. 1986; 67: 1197-1209Crossref PubMed Google Scholar). It is expressed by endothelial cells, smooth muscle cells, fibroblasts, keratinocytes, cells of monocytes/macrophage lineage, and tumor cells (17Lahav J. Biochim. Biophys. Acta. 1993; 1182: 1-14Crossref PubMed Scopus (111) Google Scholar). Much of our understanding of TSP1 biology has been established in these cell systems. TSP1 is also expressed in epithelia, is abundant in the basement membranes underlying these cells (17Lahav J. Biochim. Biophys. Acta. 1993; 1182: 1-14Crossref PubMed Scopus (111) Google Scholar), and participates in epithelial cell responses, including re-epithelialization during wound healing (8Uno K. Hayashi H. Kuroki M. Uchida H. Yamauchi Y. Kuroki M. Oshima K. Biochem. Biophys. Res. Commun. 2004; 315: 928-934Crossref PubMed Scopus (46) Google Scholar), bronchial epithelial cell morphogenesis and development (19O'Shea K.S. Dixit V.M. J. Cell Biol. 1988; 107: 2737-2748Crossref PubMed Scopus (168) Google Scholar), and migration of epithelium-derived tumor cells (20Taraboletti G. Roberts D.D. Liotta L.A. J. Cell Biol. 1987; 105: 2409-2415Crossref PubMed Scopus (174) Google Scholar). In human epithelium-derived cancer cells, EGF increases TSP1 expression (21Soula-Rothut M. Coissard C. Sartelet H. Boudot C. Bellon G. Martiny L. Rothhut B. Exp. Cell Res. 2005; 304: 187-201Crossref PubMed Scopus (35) Google Scholar). TSP1 null mice display epithelial cell alterations (22Crawford S.E. Stellmach V. Murphy-Ullrich J.E. Ribeiro S.M. Lawler J. Hynes R.O. Bolvin G.P. Bouck N. Cell. 1998; 93: 1159-1170Abstract Full Text Full Text PDF PubMed Scopus (985) Google Scholar, 23Lawler J. Sunday M. Thibert V. Duquette M. George E.L. Rayburn H. Hynes R.O. J. Clin. Investig. 1998; 101: 982-992Crossref PubMed Scopus (387) Google Scholar). In previous studies, we demonstrated that TSP1 increases tyrosine phosphorylation of the zonula adherens proteins γ-catenin and p120ctn (24Goldblum S.E. Young B.A. Wang P. Murphy-Ullrich J.E. Mol. Biol. Cell. 1999; 10: 1537-1551Crossref PubMed Scopus (33) Google Scholar), an event that can occur downstream of the EGF receptor (EGFR) (25Hoschuetzky H. Aberle H. Kemler R. J. Cell Biol. 1994; 127: 1375-1380Crossref PubMed Scopus (673) Google Scholar, 26Mariner D.J. Davis M.A. Reynolds A.B. J. Cell Sci. 2003; 117: 1339-1350Crossref Scopus (82) Google Scholar), also referred to as HER1 or ErbB1 (27Olayioye M.A. Neve R.M. Lane H.A. Hynes N.E. EMBO J. 2000; 19: 3159-3167Crossref PubMed Google Scholar). EGFR contains an NH2-terminal, ligand-binding ectodomain that is coupled to an intracellular catalytic domain and its tyrosine autophosphorylation sites (27Olayioye M.A. Neve R.M. Lane H.A. Hynes N.E. EMBO J. 2000; 19: 3159-3167Crossref PubMed Google Scholar). Ligand binding to the EGFR ectodomain induces receptor homodimerization and heterodimerization with other ErbB family members, intrinsic kinase activity, and autophosphorylation of specific tyrosine residues which, in turn, serve as docking sites within the cytoplasmic domain for signaling molecules (27Olayioye M.A. Neve R.M. Lane H.A. Hynes N.E. EMBO J. 2000; 19: 3159-3167Crossref PubMed Google Scholar). High affinity EGFR ligands share a 45-55-aa EGF motif with six spatially conserved cysteine residues that form three intramolecular disulfide bonds that dictate their tertiary conformation (28Harris R.C. Chung E. Coffey R.J. Exp. Cell Res. 2003; 284: 2-13Crossref PubMed Scopus (613) Google Scholar). These ligands are synthesized as transmembrane precursor proteins that are cleaved by cell surface matrix metalloproteases (MMP) (28Harris R.C. Chung E. Coffey R.J. Exp. Cell Res. 2003; 284: 2-13Crossref PubMed Scopus (613) Google Scholar) and ADAMs (a disintegrin and metalloproteinases) (29Sahin U. Weskamp G. Kelly K. Zhou H.M. Higashiyama S. Peschon J. Hartmann D. Saftig P. Blobel C.P. J. Cell Biol. 2004; 164: 769-779Crossref PubMed Scopus (784) Google Scholar, 30Izumi Y. Hirata M. Hasuwa H. Iwamoto R. Umata T. Miyado K. Tamai Y. Kurisaki T. Sehara-Fujisawa A. Ohno S. Mekada E. EMBO J. 1998; 17: 7260-7272Crossref PubMed Scopus (474) Google Scholar, 31Asakura M. Kitakaze M. Takashima S. Liao Y. Ishikura F. Yoshinaka T. Ohmoto H. Node K. Yoshino K. Ishiguro H. Asanuma H. Sanada S. Matsumura Y. Takeda H. Beppu S. Tada M. Hori M. Higashiyama S. Nat. Med. 2002; 8: 35-40Crossref PubMed Scopus (640) Google Scholar, 32Kurisaki T. Masuda A. Sudo K. Sakagami J. Higashiyama S. Matsuda Y. Nagabukuro A. Tsuji A. Nabeshima Y. Asano M. Iwakura Y. Sehara-Fujisawa A. Mol. Cell. Biol. 2003; 23: 55-61Crossref PubMed Scopus (126) Google Scholar) to release mature growth factors for auto-crine/paracrine stimulation. EGFR ligands that specifically activate EGFR include EGF, transforming growth factor α, amphiregulin, and others that activate both EGFR and ErbB4, including heparin-binding EGF, betacellulin, and epiregulin (28Harris R.C. Chung E. Coffey R.J. Exp. Cell Res. 2003; 284: 2-13Crossref PubMed Scopus (613) Google Scholar). In addition to these “authentic” ErbB ligands, EGF-like sequences are present in many other proteins, including TSP1 (33Apella E. Weber I.T. Blasi F. FEB. Lett. 1988; 231: 1-4Crossref PubMed Scopus (239) Google Scholar, 34Schenk S. Hintermann E. Bilban M. Koshikawa N. Hojilla C. Khokha R. Quaranta V. J. Cell Biol. 2003; 161: 197-209Crossref PubMed Scopus (248) Google Scholar, 35Swindle C.S. Tran K.T. Johnson T.D. Banerjee P. Mayes A.M. Griffith L. Wells A. J. Cell Biol. 2001; 154: 459-468Crossref PubMed Scopus (228) Google Scholar). EGF-like repeats in the γ2 chain of laminin-5 (34Schenk S. Hintermann E. Bilban M. Koshikawa N. Hojilla C. Khokha R. Quaranta V. J. Cell Biol. 2003; 161: 197-209Crossref PubMed Scopus (248) Google Scholar) and in the counter-adhesive protein, tenascin-C (35Swindle C.S. Tran K.T. Johnson T.D. Banerjee P. Mayes A.M. Griffith L. Wells A. J. Cell Biol. 2001; 154: 459-468Crossref PubMed Scopus (228) Google Scholar), have been demonstrated to activate EGFR. EGFR not only responds to direct binding of EGF motif-containing ligands, but it can be transactivated by heterologous receptors, including G protein-coupled receptors (36Luttrell L.M. Daaka Y. Lefkowitz R.J. Curr. Opin. Cell Biol. 1999; 11: 177-183Crossref PubMed Scopus (608) Google Scholar) and integrins (37Moro L. Venturino M. Bozzo C. Silengo L. Altruda F. Beguinot L. Tarone G. Defilippi P. EMBO J. 1998; 17: 6622-6632Crossref PubMed Scopus (504) Google Scholar). Whether TSP1 elicits biological responses through EGFR and/or other ErbB receptors, either through direct binding or transactivation, is not known. Here, we provide evidence that the EGF-like repeats of TSP1 and other TSP family members, likely through an MMP-mediated indirect process, activate EGFR and that this activation is coupled to downstream signaling events and cellular responses that can explain aspects of TSP1 bioactivity. Human Intact TSP1 Preparation-Human platelet TSP1 was purified as described (24Goldblum S.E. Young B.A. Wang P. Murphy-Ullrich J.E. Mol. Biol. Cell. 1999; 10: 1537-1551Crossref PubMed Scopus (33) Google Scholar). Briefly, fresh human platelets (Birmingham American Red Cross, Birmingham, AL) were thrombin-stimulated, and the platelet releasate was applied to a heparin-Sepharose CL-6B (Pharmacia, Piscataway, NJ) affinity column preequilibrated with Tris-buffered saline (TBS-C: 0.01 m Tris-HCl, 0.15 m NaCl, 0.1 mm CaCl2, pH 7.4). The bound TSP1 was eluted and applied to an A0.5 M gel filtration column (Bio-Rad) pre-equilibrated with TBS-C, pH 7.4. Preparation of Recombinant TSP Proteins-Baculovirus-expressed recombinant human TSP1 domains were purified after secretion as described (38Misenheimer T.M. Huwiler K.G. Annis D.S. Mosher D.F. J. Biol. Chem. 2000; 275: 40938-40945Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 39Misenheimer T.M. Hannah B.A. Annis D.S. Mosher D.F. Biochemistry. 2003; 42: 5125-5132Crossref PubMed Scopus (19) Google Scholar, 40Misenheimer T.M. Mosher D.F. J. Biol. Chem. 2005; 280: 41229-41235Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). These recombinant proteins (numbered from the initiating methionine of the full-length subunit) include the following: 1) the NH2-terminal heparin-binding domain + oligomerization domain + von Willebrand factor-C domain (aa 19-374) (NoC); 2) the von Willebrand factor-C domain + TSRs 1-3 (aa 312-548) (CP123); 3) TSR repeat 3 + EGF-like repeats 1-3 (aa 491-691) (P3E123); 4) EGF-like repeats 1-3 (aa 549-691) (E123); 5) EGF-like repeats 1 and 2 (aa 549-647) (E12); 6) EGF-like repeat 2 (aa 590-647) (E2); 7) EGF-like repeat 3 (aa 648-691) (E3); 8) EGF-like repeats 1-3 to the COOH terminus (aa 549-1170) (E123CaG); 9) the third EGF repeat to the COOH terminus (aa 648-1170) (E3CaG); and 10) the wire and COOH-terminal lectin-like domain (aa 692-1170) (CaG). In addition, two baculovirus-expressed TSP2 constructs, P3E123 (aa 493-693) and E123CaG (aa 551-1172), and two TSP-4 constructs, E1234 (aa 286-462) and E1234CaG, were prepared (40Misenheimer T.M. Mosher D.F. J. Biol. Chem. 2005; 280: 41229-41235Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Protein concentration and purity were determined by absorbance at 280 nm using an extinction coefficient based on the amino acid composition and by PAGE in SDS with and without prior reduction. Cell Culture-Human epidermoid carcinoma A431 (American Type Culture Collection, Manassas, VA) were cultured in Dulbecco’s modified Eagle’s medium (ATCC) enriched with 10% fetal bovine serum (FBS; Hyclone Laboratories, Logan, UT), 5 mm l-glutamine, nonessential amino acids, and vitamins in the presence of penicillin (50 units/ml) and streptomycin (50 μg/ml) (Sigma). Knockdown of EGFR and MMP9 through RNA Interference-Small interfering RNA (siRNA) duplex products designed to target EGFR and MMP9, as well as an irrelevant control duplex siRNA not corresponding to any known sequence in the human genome, were introduced into A431 cells (Dharmacon, Lafayette, CO) (41Gong P. Angelini D.J. Yang S. Xia G. Cross A.S. Mann D. Bannerman D.D. Vogel S.N. Goldblum S.E. J. Biol. Chem. 2008; 283: 13437-13449Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). First, 5 × 105 A431 cells were centrifuged (200 × g, 10 min), after which the pellet was resuspended in 100 μl of A431 Nucleofector solution (Amaxa Biosystems) and incubated with 4.0 μg of siRNA duplexes. The A431 cell/siRNA mixture was transferred to an Amaxa-certified cuvette and subjected to programmed electroporation (program X-001) (Amaxa Biosystems). The MMP9 siRNA-transfected cells were cultured for increasing times, and the supernatants were concentrated and assayed for MMP9 in an MMP9 ELISA kit (Calbiochem). At these same time points, the EGFR siRNA-transfected cells were lysed and processed for immunoblotting with anti-EGFR antibody (BD Biosciences). To confirm equivalent protein loading and transfer, blots were stripped with 100 mm 2-mercaptoethanol, 2% SDS, 62.5 mmol/liter Tris-HCl, pH 6.7, and reprobed with 0.5 ng/ml murine anti-physarum β-tubulin IgG2b (Roche Applied Science) followed by HRP-conjugated anti-mouse IgG (BD Transduction Laboratories) and again developed with enhanced chemiluminescence (ECL). EGFR Activation-To determine whether TSP1 activates EGFR, A431 cells were serum-starved for 6 h, after which they were exposed for 0.5 h to increasing concentrations of recombinant human EGF (R & D Systems, Inc), TSP1, or media alone, or they were exposed for increasing times to a fixed concentration of EGF (100 ng/ml or 16.7 nm), TSP1 (30 μg/ml or 214 nm), or media alone. In selected experiments, cells were pretreated for 2 h with the EGFR-selective tyrphostin, AG1478 (5 μm) (Calbiochem) (42Yaish P. Gazit A. Gilon C. Levitzki A. Science. 1988; 242: 933-935Crossref PubMed Scopus (542) Google Scholar), the MMP2/MMP9 inhibitor IV, SB-3CT (1 μm) (Calbiochem) (43Krüger A. Arlt M.J.E. Gerg M. Kopitz C. Bernardo M.M. Chang M. Mobashery S. Fridman R. Cancer Res. 2005; 65: 3523-3526Crossref PubMed Scopus (114) Google Scholar), the EGFR ectodomain-blocking antibody, GR13L (Calbiochem) (44Gill G.N. Kawamoto T. Cochet C. Le A. Sato J.D. Masui H. McLeod C. Mendelsohn J. J. Biol. Chem. 1984; 259: 7755-7760Abstract Full Text PDF PubMed Google Scholar), a murine monoclonal anti-human MMP9 neutralizing antibody (Calbiochem) (45O-charoenrat P. Modjtahedi H. Rhys-Evans P. Court W.J. Box G.M. Eccles S.A. Cancer Res. 2000; 60: 1121-1128PubMed Google Scholar), or a species- and isotype-matched antibody control, B7-1/CD80 (R & D Systems, Inc.). In other experiments, A431 cells were transfected with EGFR or MMP9 targeting or control siRNAs. In still other experiments, A431 cells were exposed for 0.5 h to increasing concentrations of recombinant TSP1 domains or media alone or were exposed for increasing times to a fixed concentration (3.8 μg/ml or 214 nm) of TSP1 EGF-like repeats, i.e. E123 (aa 549-691) equimolar to native TSP1 at 30 μg/ml (214 nm). Finally, in one set of experiments, TSP1 (214 nm) was pre-incubated with either of two monoclonal antibodies raised against the EGF-like repeats, C6.7 and HB8432 (46Annis D.S. Gunderson K.A. Mosher D.F. J. Biol. Chem. 2007; 282: 27067-27075Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 47Annis D.S. Murphy-Ullrich J.E. Mosher D.F. J. Thromb. Haemostasis. 2006; 4: 459-468Crossref PubMed Scopus (37) Google Scholar), or a species- and isotype-matched antibody control, B7-1/CD80. Cells were thoroughly rinsed with ice-cold HEPES buffer and solubilized with ice-cold lysis buffer containing 50 mm Tris-HCl, pH 7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mm NaCl, 1 mm EGTA, 1 mm phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, 1 μg/ml pepstatin, 1 μg/ml aprotinin, 1 mm Na2VO4, 1 mm NaF, 10 mm pyrophosphate, 500 μm paranitrophenol, and 1 mm phenylarsine oxide (all purchased from Sigma). The EC lysates were resolved by electrophoresis on a 4-12% SDS-polyacrylamide gel (Invitrogen) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA). The blots were probed with murine monoclonal anti-phospho-EGFR (Tyr-1068) followed by HRP-conjugated goat anti-mouse IgG (Pierce) and developed with ECL (Amersham Biosciences). To confirm equivalent protein loading, blots were stripped and reprobed for β-tubulin. For each immunoblot, densitometric quantification of phospho-EGFR Tyr-1068 signal was normalized to the β-tubulin signal for the same lane on the same stripped and reprobed blot. Patterns of EGFR Tyrosine Phosphorylation in Response to EGF, TSP1, and E123-A431 cells were serum-starved for 6 h after which they were exposed to media alone, EGF (10 ng/ml or 1.67 nm, 10 min), native TSP1 (30 μg/ml or 214 nm, 0.5 h), or an equimolar concentration (3.8 μg/ml or 214 nm, 1 h) of TSP1 E123 (aa 549-691). The cells were lysed and the lysates processed for immunoblotting with a series of defined, epitope-mapped, rabbit polyclonal anti-phospho-EGFR antibodies that recognize Tyr-845, Tyr-992, Tyr-1045, and Tyr-1173 (Cell Signaling Technology, Inc., Beverly, MA) and Tyr-1086 (Zymed Laboratories Inc.) all contained within the cytoplasmic domain of EGFR (27Olayioye M.A. Neve R.M. Lane H.A. Hynes N.E. EMBO J. 2000; 19: 3159-3167Crossref PubMed Google Scholar). As stated above, a murine monoclonal antibody was use to probe for phospho-EGFR Tyr-1068. The blots were stripped and reprobed with anti-β-tubulin antibodies. Activation of PLCγ-A431 cells were cultured to ∼80% confluence in 6-well plates, serum-starved for 4 h, and incubated for increasing times with increasing concentrations of TSP1 or media alone. In selected experiments, cells were pretreated with the EGFR-selective tyrphostin, AG1478 (5 μm). In other experiments, cells were transfected with EGFR targeting or control siRNAs. In still other experiments, cells were exposed for 0.5 h to E123 (214 nm). Cells were lysed, and the lysates were resolved by 4-12% SDS-PAGE, transferred to PVDF, and blocked. The membranes were immunoblotted with rabbit anti-human phospho-PLCγ (Tyr-783) antibody (Cell Signaling Technology, Inc.) followed by goat anti-rabbit HRP-conjugated IgG (Pierce) and developed with ECL (48Kim J.W. Sim S.S. Kim U.-H. Nishibe S. Wohl M.I. Carpenter G. Rhee S.G. J. Biol. Chem. 1990; 265: 3940-3943Abstract Full Text PDF PubMed Google Scholar). To confirm equivalent protein loading and transfer, blots were stripped and reprobed for total PLCγ with rabbit anti-human PLCγ antibody (Cell Signaling Technology). Migration Assay-A431 cells were cultured to confluence in the wells of 24-well plates (Corning Glass, Corning, NY). Using a sterile 200-μl pipette tip, a single wound was made across the diameter of each monolayer, after which cell debris was removed by washing with HEPES as described (49Wang W. Passaniti A. J. Cell. Biochem. 1999; 73: 321-331Crossref PubMed Scopus (34) Google Scholar). The cells were then incubated for 48 h with EGF (10 ng/ml or 1.67 nm), TSP1 (30 μg/ml or 214 nm), recombinant TSP1 E123 (3.8 μg/ml or 214 nm), or media alone, each in the presence or absence of the EGFR-selective tyrphostin, AG1478 (5 μm). In selected experiments, cells were transfected with EGFR-targeting or control siRNAs. At 48 h, images of each monolayer were captured using a Nikon Eclipse TS100 microscope coupled to a Nikon Cool pix 4300 camera. Cell migration into the wound was calculated using Image J software (Rasband, WS, Image-J, National Institutes of Health, rsb.info.nih.gov). To evaluate for AG1478-induced A431 cell cytotoxicity, A431 cells cultured to confluence in 96-well plates were incubated for 48 h with AG1478 (5 μm) or media alone. At 48 h, the media were removed, and the monolayers were washed and incubated for 4 h at 37 °C with 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide dye (5 mg/ml PBS). The crystalline formazan product was solubilized for 16 h in 10% SDS, 0.01 n HCl, and A540 nm determined. Cross-competition Binding Studies-To determine whether E123 binds to the same receptor and/or same portion of the EGFR ectodomain as do high affinity EGFR ligands, binding of fluoroprobe-labeled EGF and E123 to suspended A431 cells was studied with flow cytometry. Purified recombinant E123 was dialyzed versus borate buffer (Slide-A-Lyzer Mini Dialysis Unit, Pierce), reacted with fluorescein isothiocyanate (FITC) reconstituted in dimethylformamide, and again dialyzed to remove excess unconjugated FITC dye (EZ-Label FITC protein labeling kit, Pierce). A431 cells in fluorescence-activated cell sorter tubes (0.5-1.0 × 106 cells/tube) were incubated for 10 min at 4 °C with increasing final concentrations of either FITC-EGF (Invitrogen) or FITC-E123. To define binding over time, A431 cells were incubated at 4 °C with either FITC-EGF (50 ng/ml) or FITC-E123 (2 μg/ml) for increasing times. To establish binding specificity, FITC-EGF (3 μg/ml) was incubated for 10 min at 4 °C with increasing concentrations of unlabeled EGF (up to 500-fold relative to labeled ligand), and FITC-E123 (5 μg/ml) was incubated for 10 min at 4 °C with increasing concentrations of unlabeled E123 (up to 500-fold relative to labeled ligand). Finally, cross-competition binding studies were performed using FITC-EGF with increasing concentrations of unlabeled E123 and FITC-E123 with increasing concentrations of unlabeled EGF. The cells were washed and resuspended in PBS and analyzed by FACScan (BD Biosciences). Chemical Cross-linking Experiments-To establish a direct receptor-ligand interaction between E123 and cell surface-expressed EGFR ectodomain, E123 (3.8 μg/ml or 214 nm) and cells were co-incubated in the presence of the H2O-soluble, cell-nonpermeable, chemical cross-linking reagent, bis(sulfos-uccinimidyl) suberate (BS3) (Mr = 572.43) (Pierce) as described (50Ringerike T. Blystad F.D. Levy F.O. Madshus I.H. Stang E. J. Cell Sci. 2002; 115: 1331-1340Crossref PubMed Google Scholar). The two ends of this homobifunctional reagent cross-link amine groups and are separated by a flexible 11.4D spacer arm. A431 cells were serum-starved for 6 h, washed three times with ice-cold PBS (20 mm NaPO4, 0.15 m NaCl, pH 8.0, in HEPES) to remove amine-containing media, and incubated for 0.5 h at 4 °C with E123 (3.8 μg/ml or 214 nm) or m" @default.
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• W2046571883 date "2009-03-01" @default.
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• W2046571883 title "Epidermal Growth Factor-like Repeats of Thrombospondins Activate Phospholipase Cγ and Increase Epithelial Cell Migration through Indirect Epidermal Growth Factor Receptor Activation" @default.
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• W2046571883 doi "https://doi.org/10.1074/jbc.m809198200" @default.
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