Cooperation between Thrombospondin-1 Type 1 Repeat Peptides and αvβ3 Integrin Ligands to Promote Melanoma Cell Spreading and Focal Adhesion Kinase Phosphorylation
1999; Elsevier BV; Volume: 274; Issue: 32 Linguagem: Inglês
10.1074/jbc.274.32.22755
ISSN1083-351X
AutoresJohn M. Sipes, Henry C. Krutzsch, Jack Lawler, David D. Roberts,
Tópico(s)Protease and Inhibitor Mechanisms
ResumoCD47-binding sequences from the carboxyl-terminal domain of thrombospondin-1 (TSP1) are known to regulate activity of the αvβ3 integrin (Gao, G., Lindberg, F. P., Dimitry, J. M., Brown, E. J., and Frazier, W. A. (1996) J. Cell Biol. 135, 533–544). Here we show that peptides from the type 1 repeats of TSP1 also stimulate αvβ3 integrin function in melanoma cells. Addition of soluble peptide 246 (KRFKQDGGWSHWSPWSS) enhances spreading of A2058 melanoma cells on several αvβ3integrin ligands, including vitronectin, recombinant TSP1 fragments containing the Arg-Gly-Asp sequence, and native TSP1. This activity requires the Trp residues and is independent of CD36-binding sequences in the type 1 repeats. Recombinant type 1 repeats expressed as a glutathione S-transferase fusion protein also enhance spreading on vitronectin and TSP1. Activation of αvβ3 integrin by the soluble peptide 246 stimulates organization of F-actin and increases tyrosine phosphorylation of focal adhesion kinase. In contrast, direct adhesion of melanoma cells on immobilized peptide 246 inhibits tyrosine phosphorylation of focal adhesion kinase. Stimulation of αvβ3 integrin function by the type 1 repeat peptide differs from that induced by CD47-binding TSP1 peptides in that heparan sulfate proteoglycans are required and pertussis toxin does not inhibit the former activity. Thus, the type 1 repeats contain a second sequence of TSP1 that can enhance αvβ3integrin signaling, and these two sequences stimulate recognition of both vitronectin and TSP1 by the αvβ3integrin. CD47-binding sequences from the carboxyl-terminal domain of thrombospondin-1 (TSP1) are known to regulate activity of the αvβ3 integrin (Gao, G., Lindberg, F. P., Dimitry, J. M., Brown, E. J., and Frazier, W. A. (1996) J. Cell Biol. 135, 533–544). Here we show that peptides from the type 1 repeats of TSP1 also stimulate αvβ3 integrin function in melanoma cells. Addition of soluble peptide 246 (KRFKQDGGWSHWSPWSS) enhances spreading of A2058 melanoma cells on several αvβ3integrin ligands, including vitronectin, recombinant TSP1 fragments containing the Arg-Gly-Asp sequence, and native TSP1. This activity requires the Trp residues and is independent of CD36-binding sequences in the type 1 repeats. Recombinant type 1 repeats expressed as a glutathione S-transferase fusion protein also enhance spreading on vitronectin and TSP1. Activation of αvβ3 integrin by the soluble peptide 246 stimulates organization of F-actin and increases tyrosine phosphorylation of focal adhesion kinase. In contrast, direct adhesion of melanoma cells on immobilized peptide 246 inhibits tyrosine phosphorylation of focal adhesion kinase. Stimulation of αvβ3 integrin function by the type 1 repeat peptide differs from that induced by CD47-binding TSP1 peptides in that heparan sulfate proteoglycans are required and pertussis toxin does not inhibit the former activity. Thus, the type 1 repeats contain a second sequence of TSP1 that can enhance αvβ3integrin signaling, and these two sequences stimulate recognition of both vitronectin and TSP1 by the αvβ3integrin. Thrombospondin-1 (TSP1) 1The abbreviations used are: TSP1, human thrombospondin-1; FAK, focal adhesion kinase; GST, glutathioneS-transferase; HSPG, heparan sulfate proteoglycan; peptide 246, KRFKQDGGWSHWSPWSS; peptide 388, KRFKQDGGASHASPASS; peptide 7N3, FIRVVMYEGKK; peptide 604, FIRGGMYEGKK; peptide 605, FIRVAIYEGKK; RGD, Arg-Gly-Asp; WSXW, Trp-Ser-Xaa-Trp; PBS, phosphate-buffered saline; BSA, bovine serum albumin is an extracellular matrix protein that directly mediates adhesion of some cell types. A more important function of TSP1, however, may be to regulate interactions of cells with other extracellular matrix molecules (reviewed in Ref. 1Bornstein P. J. Cell Biol. 1995; 130: 503-506Crossref PubMed Scopus (582) Google Scholar). This regulation includes stimulation or inhibition of adhesion to other extracellular matrix ligands (2Murphy-Ullrich J.E. Gurusiddappa S. Frazier W.A. Höök M. J. Biol. Chem. 1993; 268: 26784-26789Abstract Full Text PDF PubMed Google Scholar, 3Gao A.G. Lindberg F.P. Dimitry J.M. Brown E.J. Frazier W.A. J. Cell Biol. 1996; 135: 533-544Crossref PubMed Scopus (185) Google Scholar), stimulation or inhibition of proteolytic degradation of extracellular matrix components (4Qian X. Wang T.N. Rothman V.L. Nicosia R.F. Tuszynski G.P. Exp. Cell Res. 1997; 235: 403-412Crossref PubMed Scopus (139) Google Scholar, 5Hogg P.J. Thromb. Haemostasis. 1994; 72: 787-792Crossref PubMed Scopus (64) Google Scholar), and stimulation or antagonism of growth or survival signals from the extracellular matrix (6Schultz-Cherry S. Murphy-Ullrich J.E. J. Cell Biol. 1993; 122: 923-932Crossref PubMed Scopus (401) Google Scholar, 7Taraboletti G. Roberts D. Liotta L.A. Giavazzi R. J. Cell Biol. 1990; 111: 765-772Crossref PubMed Scopus (349) Google Scholar, 8Guo N. Krutzsch H.C. Inman J.K. Roberts D.D. Cancer Res. 1997; 57: 1735-1742PubMed Google Scholar). Domains or peptide sequences from TSP1 that express some of these activities have recently been defined, and receptors have been identified that interact with some of these TSP1 sequences. An RGD sequence in the last type 3 repeat module promotes cell adhesion and binds to the integrin αvβ3 (9Lawler J. Weinstein R. Hynes R.O. J. Cell Biol. 1988; 107: 2351-2361Crossref PubMed Scopus (336) Google Scholar). Two sequences from the carboxyl-terminal domain that contain a VVM motif bind to CD47 and regulate activity of the integrins αIIbβ3, αvβ3, and α2β1 in specific cell types (3Gao A.G. Lindberg F.P. Dimitry J.M. Brown E.J. Frazier W.A. J. Cell Biol. 1996; 135: 533-544Crossref PubMed Scopus (185) Google Scholar, 10Chung J. Gao A.G. Frazier W.A. J. Biol. Chem. 1997; 272: 14740-14746Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar,11Wang X.Q. Frazier W.A.t. Mol. Biol. Cell. 1998; 9: 865-874Crossref PubMed Scopus (135) Google Scholar). Peptides from the type 1 repeats and procollagen domains that bind to CD36 inhibit endothelial cell motility (12Tolsma S.S. Volpert O.V. Good D.J. Frazier W.A. Polverini P.J. Bouck N. J. Cell Biol. 1993; 122: 497-511Crossref PubMed Scopus (510) Google Scholar, 13Dawson 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). Heparin-binding peptides from the type I repeats promote cell adhesion and chemotaxis (14Guo N.H. Krutzsch H.C. Nègre E. Zabrenetzky V.S. Roberts D.D. J. Biol. Chem. 1992; 267: 19349-19355Abstract Full Text PDF PubMed Google Scholar) but also inhibit growth, motility, and survival of endothelial cells (8Guo N. Krutzsch H.C. Inman J.K. Roberts D.D. Cancer Res. 1997; 57: 1735-1742PubMed Google Scholar, 15Vogel T. Guo N.H. Krutzsch H.C. Blake D.A. Hartman J. Mendelovitz S. Panet A. Roberts D.D. J. Cell. Biochem. 1993; 53: 74-84Crossref PubMed Scopus (139) Google Scholar). The RFK sequence in the second type 1 repeat activates latent transforming growth factor-β1 (16Schultz-Cherry S. Chen H. Mosher D.F. Misenheimer T.M. Krutzsch H.C. Roberts D.D. Murphy-Ullrich J.E. J. Biol. Chem. 1995; 270: 7304-7310Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar). Additional peptide sequences have been identified that disrupt focal adhesion contacts in endothelial cells (2Murphy-Ullrich J.E. Gurusiddappa S. Frazier W.A. Höök M. J. Biol. Chem. 1993; 268: 26784-26789Abstract Full Text PDF PubMed Google Scholar) or promote adhesion of breast carcinoma cells (17Clezardin P. Lawler J. Amiral J. Quentin G. Delmas P. Biochem. J. 1997; 321: 819-827Crossref PubMed Scopus (24) Google Scholar), but the cell receptors interacting with these sequences are not known. While examining the ability of TSP1 to regulate adhesion of melanoma cells, we found that some type 1 repeat peptides have a similar ability to stimulate melanoma cell spreading on vitronectin as that reported for the CD47-binding sequences from the carboxyl terminus of TSP1 (3Gao A.G. Lindberg F.P. Dimitry J.M. Brown E.J. Frazier W.A. J. Cell Biol. 1996; 135: 533-544Crossref PubMed Scopus (185) Google Scholar). We report here that recombinant type 1 repeats and soluble peptides from these modules containing the WSXW motif specifically stimulate αvβ3 integrin-mediated melanoma cell adhesion and tyrosine kinase signaling when added as soluble peptides but not when immobilized on the substratum. Stimulation of αvβ3 integrin is mediated by binding of this peptide to cell surface HSPGs. Calcium-replete TSP1 was purified from human platelets as described previously (18Roberts D.D. Cashel J. Guo N. J. Tissue Cult. Methods. 1994; 16: 217-222Crossref Scopus (44) Google Scholar). Recombinant fragments and GST or T7 fusion proteins expressing fragments of TSP1 were prepared as described (15Vogel T. Guo N.H. Krutzsch H.C. Blake D.A. Hartman J. Mendelovitz S. Panet A. Roberts D.D. J. Cell. Biochem. 1993; 53: 74-84Crossref PubMed Scopus (139) Google Scholar, 19Legrand C. Thibert V. Dubernard V. Bégault B. Lawler J. Blood. 1992; 79: 1995-2003Crossref PubMed Google Scholar, 20Guo N. Zabrenetzky V.S. Chandrasekaran L. Sipes J.M. Lawler J. Krutzsch H.C. Roberts D.D. Cancer Res. 1998; 58: 3154-3162PubMed Google Scholar). Synthetic peptides containing TSP1 sequences were prepared as described previously (2Murphy-Ullrich J.E. Gurusiddappa S. Frazier W.A. Höök M. J. Biol. Chem. 1993; 268: 26784-26789Abstract Full Text PDF PubMed Google Scholar, 14Guo N.H. Krutzsch H.C. Nègre E. Zabrenetzky V.S. Roberts D.D. J. Biol. Chem. 1992; 267: 19349-19355Abstract Full Text PDF PubMed Google Scholar, 21Guo N.H. Krutzsch H.C. Nègre E. Vogel T. Blake D.A. Roberts D.D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3040-3044Crossref PubMed Scopus (141) Google Scholar, 22Guo N. Krutzsch H.C. Inman J.K. Roberts D.D. J. Peptide Res. 1997; 50: 210-221Crossref PubMed Scopus (63) Google Scholar, 23Prater C.A. Plotkin J. Jaye D. Frazier W.A. J. Cell Biol. 1991; 112: 1031-1040Crossref PubMed Scopus (188) Google Scholar, 24Gao 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). Bovine type I collagen was obtained from Collaborative Research, and human vitronectin was from Sigma. Fibronectin was purified from human plasma (National Institutes of Health Blood Bank) as described (25Akiyama S.K. Yamada K.M. J. Biol. Chem. 1985; 260: 4492-4500Abstract Full Text PDF PubMed Google Scholar). Adhesion was measured on polystyrene or glass substrates (for immunofluorescence) coated with peptides or proteins as described previously (14Guo N.H. Krutzsch H.C. Nègre E. Zabrenetzky V.S. Roberts D.D. J. Biol. Chem. 1992; 267: 19349-19355Abstract Full Text PDF PubMed Google Scholar). Stimulation of melanoma cell spreading on vitronectin immobilized at limiting concentrations was used to detect activation of the αvβ3integrin as described previously (3Gao A.G. Lindberg F.P. Dimitry J.M. Brown E.J. Frazier W.A. J. Cell Biol. 1996; 135: 533-544Crossref PubMed Scopus (185) Google Scholar). For some experiments, cells were treated with 1 μg/ml pertussis toxin (Sigma) as described previously (20Guo N. Zabrenetzky V.S. Chandrasekaran L. Sipes J.M. Lawler J. Krutzsch H.C. Roberts D.D. Cancer Res. 1998; 58: 3154-3162PubMed Google Scholar). Nonpeptide antagonists of αIIbβ3(SB208651) and αvβ3 integrins (SB223245) were provided by Dr. William H. Miller (SmithKline Beecham, King of Prussia, PA) (26Keenan R.M. Miller W.H. Kwon C. Ali F.E. Callahan J.F. Calvo R.R. Hwang S.M. Kopple K.D. Peishoff C.E. Samanen J.M. Wong A.S. Yuan C.K. Huffman W.F. J. Med. Chem. 1997; 40: 2289-2292Crossref PubMed Scopus (129) Google Scholar, 27Bondinell W.E. Keenan R.M. Miller W.H. Ali F.E. Allen A.C. de Brosse C.W. Eggleston D.S. Erhard K.F. Haltiwanger R.C. Huffman W.F. Hwang S.-M. Jakas D.R. Koster P.F. Ku T.W. Lee C.P. Nichols A.J. Ross S.T. Samanen J.M. Valocik R.E. Vasko-Moser J.A. Venslavsky J.W. Wong A.S. Yuan C.-K. Bioorg. Med. Chem. 1994; 2: 897-908Crossref PubMed Scopus (26) Google Scholar). Cell spreading was assessed microscopically by counting >100 cells per experimental point using a calibrated reticle to determine the cells/unit area. Cell spreading on vitronectin or TSP1 was quantified by counting cells with a typical polygonal or spindle morphology. Cells spreading on peptides 246 and 7N3 typically did not organize actin filaments but were scored as spread if they increased their diameter more than 3-fold. Statistical significance of pairwise comparisons was assessed using a two-tailed Student's ttest. Inhibition of glycosaminoglycan sulfation was achieved by growth of the cells in a sulfate-free Ham's F12 medium containing sodium chlorate as described previously (28Guo N.H. Krutzsch H.C. Vogel T. Roberts D.D. J. Biol. Chem. 1992; 267: 17743-17747Abstract Full Text PDF PubMed Google Scholar). Briefly, A2058 cells were plated in four dishes using complete RPMI medium without antibiotics and containing 10% dialyzed fetal calf serum. After 3 h, media in three of the dishes were changed to Ham's F12 containing 10% dialyzed fetal calf serum with no antibiotics. After 24 h, the medium in the control dish was changed to fresh RPMI medium with 10% dialyzed fetal calf serum, and the media in the remaining three dishes were replaced with Ham's F12, 10% dialyzed fetal calf serum, the same medium with 10 mm sodium chlorate, or with 10 mm sodium chlorate and 1 mm sodium sulfate. Cells were harvested 24 h later by dissociation with EDTA and used for adhesion assays. Involvement of heparan sulfate and chondroitin sulfate proteoglycans in peptide responses was examined by pretreatment of melanoma cells with heparitinase or chondroitinase ABC (Seikagaku America, Ijamsville, MD) as described previously (29Roberts D.D. Cancer Res. 1988; 48: 6785-6793PubMed Google Scholar). Briefly, Falcon 1008 bacteriological dishes were coated overnight at 4 oC with 10 μg/ml vitronectin or 5 μg/ml type I collagen. The solution was aspirated, and the dishes were blocked using 1% BSA in Dulbecco's PBS for 1 h. A suspension of A2058 cells, detached using EDTA, was diluted to 2 × 106/ml in RPMI, 0.1% BSA and treated with 0.015 units/ml heparitinase, 0.1 units/ml chondroitinase ABC, or medium alone for 30 min at 25 oC with rocking. The cells were collected by centrifugation, washed twice with Dulbecco's PBS, and tested for adhesion to both proteins. Adhesion to collagen was used as a control for viable cell number and did not differ significantly among the samples. 1008 or 1029 Falcon bacteriological dishes were coated with vitronectin, fibronectin, T7-RGD fusion protein (expressing TSP1 amino acids 879–947) or TSP1 peptides (388, 7N3, or 246) in Dulbecco's PBS with divalent cations, pH 7.4, overnight at 4 °C. The dishes were aspirated and blocked with Dulbecco's PBS, 1% BSA for 60 min at room temperature. Cells were detached with Dulbecco's PBS containing 2.5 mm EDTA and added to coated dishes in RPMI, 0.1% BSA with or without TSP1 peptides 388, 7N3, or 246 at the indicated concentrations and incubated 20 to 60 min at 37 °C with 5% CO2. The plates were chilled on ice for 5 min, and all cells were recovered for phosphotyrosine assays. The cell medium was pipetted into chilled tubes and centrifuged for 3 min to pellet any unbound cells. Modified RIPA buffer (50 mmTris/HCl, pH 7.4, 0.15 m NaCl, 1% (w/v) Nonidet P-40, 0.5% (w/v) sodium deoxycholate, 1 mm EGTA, 1 mm Na3VO4, and protease inhibitors at 10 μg/ml antipain, pepstatin A, chymostatin, leupeptin, soybean trypsin inhibitor, aprotinin, and 1 mmphenylmethanesulfonyl fluoride) was added to the pellets and returned to the cells bound to the dishes. After a 30-min incubation at 4 °C with modified RIPA buffer, lysed cells were scraped from the dishes and centrifuged at 13,000 × g for 30 min at 4 °C. The supernatant was removed from the cell pellet, and 30 μl of the sample was heated at 95 °C with 2× β-mercaptoethanol/SDS sample buffer and held for Western blotting. For immunoprecipitation, the lysates were incubated with monoclonal anti-FAK antibody (mouse IgG, Transduction Laboratories) for 3 h at 4 °C. 2.5 μl of goat anti-mouse IgG-agarose was added, and the samples were briefly vortexed and incubated overnight at 4 °C. The beads were washed and centrifuged three times for 5 s in 1× modified RIPA buffer before resuspending the pellets in 30 μl of 2× β-mercaptoethanol/SDS sample buffer and boiling. Samples were run on 4–15% gradient Tris glycine SDS-polyacrylamide gels. Proteins were transferred to nitrocellulose membranes in a semi-dry Western blot apparatus. The membrane was blocked with 1% BSA in TBST (0.1% Tween 20, 10 mm Tris/HCl, pH 7.5, 0.10 m NaCl) for 1 h. Horseradish peroxidase-conjugated RC-20 anti-phosphotyrosine antibody was added and incubated in TBST-0.1% BSA for 1 h at room temperature. The membrane was washed in TBST, 0.1% BSA for 30 min, changing the TBST, 0.1% BSA every 5 min. Antibody binding was detected using ECL reagent (Amersham Pharmacia Biotech) as directed by the manufacturer. Lab-Tek 177402 glass chamber slides were coated with human vitronectin, fibronectin, TSP1, or TSP1 peptides in Dulbecco's PBS with divalent cations and incubated overnight at 4 °C. A2058 cells, grown to 65% confluence, were preincubated for 3 h at 37 °C with complete RPMI (10% fetal bovine serum, penicillin/streptomycin, glutamine) with or without 1 μg/ml pertussis toxin (Sigma). The slides were aspirated and blocked with Dulbecco's PBS, 1% BSA for 1 h at room temperature. Cells were detached with PBS containing 2.5 mm EDTA and added to the slides at 2 × 105 cells per ml in 200 μl of RPMI, 0.1% BSA, with or without 1 μg/ml pertussis toxin, and with or without TSP1 peptides at 3 μm final concentration. After incubation for 60 min at 37 °C with 5% CO2, the slides were gently aspirated, washed twice in warm Dulbecco's PBS, and fixed in Dulbecco's PBS with 4% formaldehyde for 15 min at room temperature. After washing twice in Dulbecco's PBS, the slides were placed in Dulbecco's PBS with 0.2% Triton X-100 for 4 min at room temperature. The slides were washed twice in Dulbecco's PBS and stained with BODIPY TR-X Phallacidin (Molecular Probes) according to the manufacturer's protocol. Slides were photographed with a Zeiss fluorescence microscope using a × 63 oil immersion objective. Peptides containing the two VVM sequences from the carboxyl-terminal domain of TSP1 that bind to CD47 stimulate spreading of melanoma cells on substrates coated with low densities of vitronectin (3Gao A.G. Lindberg F.P. Dimitry J.M. Brown E.J. Frazier W.A. J. Cell Biol. 1996; 135: 533-544Crossref PubMed Scopus (185) Google Scholar). This stimulation was concluded to be specific based on lack of activity of the Mal III and Hep III peptides derived from two other domains of TSP1. Although the CD36-binding Mal III peptide from the type 1 repeats was inactive in the previous study (3Gao A.G. Lindberg F.P. Dimitry J.M. Brown E.J. Frazier W.A. J. Cell Biol. 1996; 135: 533-544Crossref PubMed Scopus (185) Google Scholar), a second peptide from the type 1 repeats, peptide 246, strongly induced spreading of melanoma cells on vitronectin (Fig.1 A). The stimulation of spreading was comparable for peptide 246 and the CD47-binding peptide 7N3, and both peptides were active at low micromolar concentrations (Fig. 1 B). The activity of peptide 246 was maximal at 3 μm but reproducibly decreased at higher concentrations. The stimulation of spreading on vitronectin by peptide 246 required the Trp residues, as peptide 388, with the three Trp residues replaced by Ala residues, was inactive at all concentrations tested (Fig. 1,A and B). Two control peptides for 7N3 with the VVM sequence altered to GGM (604) or VAI (605) were also inactive. The known cell surface ligands for the type 1 repeat peptides containing the WSXW motif are HSPGs and sulfated glycolipids (21Guo N.H. Krutzsch H.C. Nègre E. Vogel T. Blake D.A. Roberts D.D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3040-3044Crossref PubMed Scopus (141) Google Scholar). However, at least two adjacent sequences in the type 1 repeats have been reported to bind to CD36 (13Dawson 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, 30Asch A.S. Silbiger S. Heimer E. Nachman R.L. Biochem. Biophys. Res. Commun. 1992; 182: 1208-1217Crossref PubMed Scopus (162) Google Scholar, 31Li W.X. Howard R.J. Leung L.L. J. Biol. Chem. 1993; 268: 16179-16184Abstract Full Text PDF PubMed Google Scholar). To verify that the activity of peptide 246 is independent of CD36 binding, we compared the response of A2058 melanoma cells to that of two other melanoma cell lines that are known to express (C32) or lack expression (G361) of CD36 (32Barnwell J.W. Ockenhouse C.F. Knowles D.M. J. Immunol. 1985; 135: 3494-3497PubMed Google Scholar, 33Panton L.J. Leech J.H. Miller L.H. Howard R.J. Infect. Immun. 1987; 55: 2754-2758Crossref PubMed Google Scholar). The VVM and WSXW peptides both stimulated spreading of the melanoma cells on vitronectin, regardless of their CD36 phenotype (Fig. 1 C, p < 0.003 for each peptide treatment compared with control for all three cell lines). The type 1 repeat peptide induced cells plated on a limiting concentration of vitronectin to adopt a similar morphology (Fig.2 C) as induced by the CD47-binding peptide 7N3 (Fig. 2 B). Both peptides stimulated spreading on vitronectin and increased the organization of F-actin. The spreading response induced by CD47-binding peptides was reported to be pertussis toxin-sensitive (3Gao A.G. Lindberg F.P. Dimitry J.M. Brown E.J. Frazier W.A. J. Cell Biol. 1996; 135: 533-544Crossref PubMed Scopus (185) Google Scholar). We observed a 63 ± 10% inhibition of the A2058 cell spreading induced by the VVM peptide 7N3 in the presence of pertussis toxin (Fig. 2 E and data not shown). However, stimulation by the type 1 repeat peptide 246 was not inhibited (104 ± 3% of control) in the presence of pertussis toxin (Fig.2 F and data not shown). The differential sensitivity to pertussis toxin of the responses to peptide 246 and 7N3 combined with the known ligand binding specificities of these peptides (14Guo N.H. Krutzsch H.C. Nègre E. Zabrenetzky V.S. Roberts D.D. J. Biol. Chem. 1992; 267: 19349-19355Abstract Full Text PDF PubMed Google Scholar, 24Gao 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) suggested that different receptors mediate their effects on αvβ3integrin function. Because 246 is a heparin-binding peptide (14Guo N.H. Krutzsch H.C. Nègre E. Zabrenetzky V.S. Roberts D.D. J. Biol. Chem. 1992; 267: 19349-19355Abstract Full Text PDF PubMed Google Scholar) and TSP1 binds to HSPGs expressed by melanoma cells (29Roberts D.D. Cancer Res. 1988; 48: 6785-6793PubMed Google Scholar), we examined the role of glycosaminoglycans in mediating the stimulatory activity of peptide 246. Inhibition of sulfation by preincubating A2058 melanoma cells with the inhibitor of 3′-phosphoadenosine 5′-phosphosulfate synthesis sodium chlorate at 10 mm ablated the stimulatory activity of peptide 246 but not that of peptide 7N3 (Fig.3 A). This concentration of chlorate inhibited glycosaminoglycan sulfation by 95% in A2058 cells (28Guo N.H. Krutzsch H.C. Vogel T. Roberts D.D. J. Biol. Chem. 1992; 267: 17743-17747Abstract Full Text PDF PubMed Google Scholar). A partial inhibition of the response to peptide 246 but not of the response to peptide 7N3 was also observed by simply growing the cells in a sulfate-free Ham's F12 medium (Fig. 3 A). The inhibitory activity of chlorate was verified to be mediated through sulfation inhibition by restoring the stimulating activity of peptide 246 in the presence of both chlorate and 1 mm sulfate (Fig.3 A). Melanoma cells express both chondroitin sulfate proteoglycans and HSPGs. To determine which mediated the response to peptide 246, A2058 melanoma cells were treated with heparitinase or chondroitinase ABC, and their spreading on vitronectin was assessed in the absence and presence of peptides (Fig. 3 B). Chondroitinase treatment did not significantly alter responses to peptide 246 or 7N3 (p > 0.1 for both peptides versus treatment control), whereas heparitinase strongly and selectively inhibited the enhancement of spreading simulated by peptide 246 (p = 0.0004 versus treatment control). Therefore, HSPGs are required for the response to peptide 246 but not for the response to the CD47-binding peptide 7N3. Because TSP1 is also an αvβ3 integrin ligand (9Lawler J. Weinstein R. Hynes R.O. J. Cell Biol. 1988; 107: 2351-2361Crossref PubMed Scopus (336) Google Scholar), we examined whether these TSP1 peptides modulated A2058 cell adhesion to TSP1. A2058 melanoma cells express αvβ3 (34Aznavoorian S. Stracke M.L. Parsons J. McClanahan J. Liotta L.A. J. Biol. Chem. 1996; 271: 3247-3254Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar), and their adhesion on TSP1, a recombinant T7 fusion protein expressing the type 3 repeat modules of TSP1 containing the RGD sequence, or vitronectin was inhibited by an αvβ3 integrin antagonist SB223245 but not by the αIIbβ3 integrin antagonist SB208651 (Fig. 4 A). The partial (32%) resistance of TSP1 adhesion to the αvβ3antagonist suggested that additional TSP1 receptors may contribute to adhesion to the intact protein. We observed induction of spreading on TSP1 by both the VVM peptide 7N3 and the type 1 repeat peptide 246 (Fig. 4 B). This induction was specific for the αvβ3 integrin ligands in A2058 melanoma cells, however, because spreading on the α2β1 integrin ligand type I collagen was not stimulated by either peptide 246 or 7N3 (Fig. 4 B). Enhancement of spreading on TSP1 was also specific for peptides 246 and 7N3 in that the TSP1 peptides Mal II from the second type 1 repeat, peptide Hep1 from the amino-terminal heparin-binding domain, and two analogs of peptide 7N3 with the VVM sequence disrupted (peptides 604 and 605) failed to stimulate spreading on TSP1 (Fig. 4 B). Peptide 246 was more potent than peptide 7N3 for inducing spreading of A2058 melanoma cells on TSP1 (Fig. 4 C). The activity of peptide 246 to enhance melanoma cell spreading on TSP1 also required the Trp residues, as peptide 388 was inactive at all concentrations examined. Based on the activities of recombinant TSP1 fragments, the RGD sequence in the type 3 repeats of TSP1 are primarily responsible for adhesion of A2058 melanoma cells on this protein (Fig.5 A). The entire type 3 repeats are not required, as a GST fusion protein expressing only the last type 3 repeat and the carboxyl-terminal domain (residues 877–1152) promoted strong adhesion, whereas a GST fusion protein expressing a part of the type 3 repeats without the RGD sequence (residues 784–824) was inactive. The RGD sequence in the type 3 repeats also mediated enhancement of spreading induced by the TSP1 peptides (Fig.5 B). On an intact TSP1 substrate, enhancement of spreading by soluble TSP1 peptides was observed only at limiting concentrations of TSP1. At higher concentrations of TSP1, additional TSP1 receptors may permit αvβ3 integrin-independent spreading. The amino-terminal heparin binding domain may mediate this peptide-independent spreading, because melanoma cells spread on a proteolytic 140-kDa fragment of TSP1 only in the presence of the activating peptides. The CD47-binding site in the carboxyl terminus may not contribute to this response, because the GST-type 3 fusion protein (residues 674–925) and the RGD + carboxyl-terminal domain fusion protein (residues 877–1152) both promoted spreading only in the presence of the activating peptides. Although the RGD + carboxyl-terminal domain fusion protein contains both VVM sequences, these immobilized VVM sequences did not enhance recognition of the RGD in the same protein. Enhancement of spreading on TSP1 and vitronectin are mediated by αvβ3 integrin, because responses to both peptides were reversed by the αvβ3antagonist SB223245 (Fig. 6,p < 0.05 for all conditions treated with SB223245versus the respective controls). Thus, both the type 1 repeat peptide containing the WSXW motif and the CD47-binding peptide from the carboxyl-terminal domains of TSP1 can synergize with the RGD domain of TSP1 to promote αvβ3-dependent spreading of melanoma cells. A soluble GST fusion protein that expressed the type 1 repeat modules containing the TSP1 peptide 246 sequence (residues 385–522) also significantly stimulated αvβ3 activity (Fig. 7). The stimulating activity of the recombinant type 1 repeats was observed using either vitronectin (Fig.7 A) or TSP1 as substrates (Fig. 7 B) and was specific in that GST alone or a GST fusion protein expressing the type 2 repeats of TSP1 (residues 559–669) at the same concentration was inactive. Therefore, the activity of the peptide 246 sequence is replicated by a recombinant fusion protein containing this TSP1 sequence. As reported previously for the CD47-binding sequences of TSP1 using C32 melanoma cells (3Gao A.G. Lindberg F.P. Dimitry J.M. Brown E.J. Frazier W.A. J. Cell Biol. 1996; 135: 533-544Crossref PubMed Scopus (185) Google Scholar), addition of soluble peptide 246 induced tyrosine phosphorylation of a protein comigrating with FAK in A2058 melanoma cells plated on a vitronectin substrate (Fig.8 A). The control peptide 388 did not significantly enhance phosphorylation. The response to peptide 246 was maximal at 15 μm and decreased at higher concentrations in parallel with the decrease in spreading (Fig.1 B). Soluble peptide 246 stimulated a similar level of tyrosine phosphorylation in A2058 cells on a TSP1 substrate (Fig.8 B) that was maximal after 40–60 min. The phosphorylated protein was confirmed to be FAK by immunoprecipitation using a specific antibody (Fig. 8 B). Because the type 1 repeat peptides displayed a strong adhesive activity for melanoma cells (14Guo N.H. Krutzsch H.C. Nègre E. Zabrenetzky V.S. Roberts D.D. J. Biol. Chem. 1992; 267: 19349-19355Abstract Full Text PDF PubMed Google Scholar, 21Guo N.H. Krutzsch H.C. Nègre E. Vogel T. Blake D.A. Roberts D.D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3040-3044Crossref PubMed Scopus (141) Google Scholar), we considered the possibility that the peptide 246 response results from a pro-adhesive activity of the soluble peptide following its adsorption onto the substrate. If so, then direct coating of the peptides should induce the same cell spreading and tyrosine phosphorylation responses as the soluble peptide. Co-immobilization of the peptides with vitronectin was compared with addition of the same peptides in solution for induction of FAK phosphorylation (Fig. 8 C). Immobilized peptide 7N3 was more active than the soluble peptide for inducing FAK phosphorylation, but peptide 246 was more active when added in solution. We compared the morphology and F-actin organization in cells directly attached on the TSP1 peptides to that of cells attached on vitronectin or TSP1 substrates in the presence or absence of the same peptides. Although the cells spread on peptides 246 and 7N3 (Fig.9, A and B), their morphologies were different from that induced by the soluble peptides added to cells on vitronectin or TSP1 substrates (compare Fig. 9 with Fig. 2, B and C) or by direct adhesion on fibronectin (Fig. 9 C). Actin organized only in the periphery of cells on the peptides (Fig. 9, A and B). Although both the 7N3 and 246 substrates promoted some cell spreading, adhesion of cells on peptides 7N3 or 246 rapidly decreased FAK phosphorylation (Fig. 9 D). Thus, the direct response of A2058 melanoma cells to these immobilized peptides in the absence of an αvβ3 integrin ligand was inhibition rather than stimulation of FAK phosphorylation. We have identified the WSXW peptides in the type 1 repeats as a second TSP1 motif that stimulates αvβ3 integrin function in melanoma cells. Stimulation of αvβ3 integrin by both peptides results in similar enhancement of FAK phosphorylation. This response is sulfation-dependent and is mediated by binding of peptide 246 to HSPGs, which are expressed by the melanoma cells and were shown previously to bind TSP1 (29Roberts D.D. Cancer Res. 1988; 48: 6785-6793PubMed Google Scholar). This activity is expressed both by the synthetic peptide and a recombinant fusion protein containing the type 1 repeats of TSP1. In contrast to the CD47-mediated enhancement by the TSP1 carboxyl-terminal peptides (3Gao A.G. Lindberg F.P. Dimitry J.M. Brown E.J. Frazier W.A. J. Cell Biol. 1996; 135: 533-544Crossref PubMed Scopus (185) Google Scholar), this signal does not require pertussis toxin-sensitive G proteins. Thus, TSP1 contains two independent sequences that can stimulate function of the αvβ3 integrin to interact with ligands including TSP1 itself. This suggests that both the VVM and WSXW motifs may function as synergy sites (35Aota S. Nomizu M. Yamada K.M. J. Biol. Chem. 1994; 269: 24756-24761Abstract Full Text PDF PubMed Google Scholar) for αvβ3-mediated melanoma cell adhesion to TSP1 (Fig. 10). Although soluble TSP1 shows some stimulatory activity for melanoma cell adhesion to immobilized αvβ3 integrin ligands, it is not known which of these two sequences is/are responsible for the activity of the soluble intact protein. Interactions with other extracellular matrix components may also alter the accessibility of both sites in TSP1. The manner in which the type 1 sequences are presented to the melanoma cells appears to be critical for the resulting signal. Soluble peptide 246 stimulates tyrosine phosphorylation of FAK induced by αvβ3 ligands, but direct adhesion to the immobilized peptide inhibits the same response. Immobilized peptide 246 stimulates melanoma cell adhesion, but the resulting cell morphology differs from that induced on immobilized αvβ3 ligands by addition of the same peptide in solution. These differential responses may have biological significance, because TSP1 is found both as a soluble protein and immobilized in extracellular matrix. TSP1 effects on melanoma behaviorin vivo show a similar dichotomy. Addition of soluble TSP1 to melanoma cells prior to injection in an experimental metastasis assay stimulated lung colonization (36Tuszynski G.P. Gasic T.B. Rothman V.L. Knudsen K.A. Gasic G.J. Cancer Res. 1987; 47: 4130-4133PubMed Google Scholar), whereas subclones of a murine melanoma cell line with increased TSP1 expression, which may accumulate in an immobilized state in the matrix, were less metastatic in a spontaneous metastasis assay (37Zabrenetzky V. Harris C.C. Steeg P.S. Roberts D.D. Int. J. Cancer. 1994; 59: 191-195Crossref PubMed Scopus (202) Google Scholar). These opposing effects on melanoma tumor progression could also arise from differences in the animal models used (38Roberts D.D. FASEB J. 1996; 10: 1183-1191Crossref PubMed Scopus (244) Google Scholar) but are consistent with the differential activities of soluble versus immobilized TSP1 peptides in vitroto modulate adhesive responses of melanoma cells. Gao et al. (3Gao A.G. Lindberg F.P. Dimitry J.M. Brown E.J. Frazier W.A. J. Cell Biol. 1996; 135: 533-544Crossref PubMed Scopus (185) Google Scholar) reported that stimulation of adhesion by VVM peptides from TSP1 was not limited to vitronectin. TSP1 is another αvβ3 ligand for which recognition is enhanced by 246 and the VVM peptides. However, the α2β1 integrin is not stimulated by either the WSXW or the CD47-binding peptides in A2058 melanoma cells. This result contrasts with smooth muscle cells, in which VVM peptides enhanced α2β1 integrin function (39Wang X. Frazier W.A. Mol. Biol. Cell. 1998; 9: 865-874Crossref PubMed Scopus (139) Google Scholar). Several results indicate that adhesive activity is not sufficient for a peptide to stimulate αvβ3 integrin activity in melanoma cells. The Mal II peptide promotes adhesion of these cells (23Prater C.A. Plotkin J. Jaye D. Frazier W.A. J. Cell Biol. 1991; 112: 1031-1040Crossref PubMed Scopus (188) Google Scholar), but it does not activate αvβ3integrin. Direct adhesion to peptides 246 and 7N3 in the absence of an αvβ3 integrin ligand induces a signal that opposes that induced by the same peptide when added in solution. Adhesion to immobilized peptides results in inhibition rather than stimulation of FAK phosphorylation, and co-immobilization of peptide 246 with vitronectin did not stimulate FAK phosphorylation as well as addition of the same peptide in solution. Although the effects of pertussis toxin indicate that a different signaling pathway links peptide 246 binding to integrin activation than that utilized by the CD47-binding peptides, the details of this pathway remain unknown. Lack of pertussis toxin sensitivity for the αvβ3 integrin response to peptide 246 is consistent with our previous report (20Guo N. Zabrenetzky V.S. Chandrasekaran L. Sipes J.M. Lawler J. Krutzsch H.C. Roberts D.D. Cancer Res. 1998; 58: 3154-3162PubMed Google Scholar) that chemotactic and antiproliferative activities of the same peptide for A2058 cells were not inhibited by pertussis toxin. Several previous studies (40Yamagata M. Suzuki S. Akiyama S.K. Yamada K.M. Kimata K. J. Biol. Chem. 1989; 264: 8012-8018Abstract Full Text PDF PubMed Google Scholar, 41Makabe T. Saiki I. Murata J. Ohdate Y. Kawase Y. Taguchi Y. Shimojo T. Kimizuka F. Kato I. Azuma I. J. Biol. Chem. 1990; 165: 14270-14276Google Scholar, 42Hayashi K. Madri J.A. Yurchenco P.D. J. Cell Biol. 1992; 119: 945-959Crossref PubMed Scopus (164) Google Scholar, 43Iida J. Meijne A.M. Oegema Jr., T.R. Yednock T.A. Kovach N.L. Furcht L.T. McCarthy J.B. J. Biol. Chem. 1998; 273: 5955-5962Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 44Iida J. Meijne A.M. Knutson J.R. Furcht L.T. McCarthy J.B. Semin. Cancer Biol. 1996; 7: 155-162Crossref PubMed Scopus (71) Google Scholar) have provided precedent for modulation of integrin functions by proteoglycans. A similar mechanism for cooperation between integrins and proteoglycans has been proposed to promote interactions of melanoma cells with both fibronectin and type IV collagen (45Knutson J.R. Iida J. Fields G.B. McCarthy J.B. Mol. Biol. Cell. 1996; 7: 383-396Crossref PubMed Scopus (126) Google Scholar, 46Iida J. Meijne A.M. Spiro R.C. Roos E. Furcht L.T. McCarthy J.B. Cancer Res. 1995; 55: 2177-2185PubMed Google Scholar). Although some of these effects of proteoglycan binding may require only extracellular interactions, studies in melanoma (43Iida J. Meijne A.M. Oegema Jr., T.R. Yednock T.A. Kovach N.L. Furcht L.T. McCarthy J.B. J. Biol. Chem. 1998; 273: 5955-5962Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 45Knutson J.R. Iida J. Fields G.B. McCarthy J.B. Mol. Biol. Cell. 1996; 7: 383-396Crossref PubMed Scopus (126) Google Scholar, 46Iida J. Meijne A.M. Spiro R.C. Roos E. Furcht L.T. McCarthy J.B. Cancer Res. 1995; 55: 2177-2185PubMed Google Scholar) and other cell types (47Yoshida T. Pan Y. Hanada H. Iwata Y. Shigekawa M. J. Biol. Chem. 1998; 273: 1583-1590Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar) suggest that cell surface proteoglycans can also induce intracellular signals that modulate integrin function. Protein kinase C is implicated in signaling through syndecan 4 (48Oh E.S. Woods A. Couchman J.R. J. Biol. Chem. 1997; 272: 8133-8136Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar), regulates stimulation of melanoma cell chemotaxis by TSP1 (20Guo N. Zabrenetzky V.S. Chandrasekaran L. Sipes J.M. Lawler J. Krutzsch H.C. Roberts D.D. Cancer Res. 1998; 58: 3154-3162PubMed Google Scholar), and can increase integrin activation (reviewed in Ref. 49Kolanus W. Seed B. Curr. Opin. Cell Biol. 1997; 9: 725-731Crossref PubMed Scopus (128) Google Scholar). Activation of protein kinase C using phorbol ester also induced spreading of A2058 melanoma cells on vitronectin, low concentrations of TSP1, and recombinant fragments of TSP1 containing the RGD sequence. 2D. D. Roberts, unpublished results. Thus, the stimulatory effect of peptide 246 on αvβ3may be mediated by protein kinase C. TSP1 interacts with syndecans as well as other HSPGs on melanoma cells (29Roberts D.D. Cancer Res. 1988; 48: 6785-6793PubMed Google Scholar, 50Sun X. Mosher D.F. Rapraeger A. J. Biol. Chem. 1989; 264: 2885-2889Abstract Full Text PDF PubMed Google Scholar). Interactions of TSP1 with chondroitin sulfate proteoglycans may also promote cell spreading or formation of microspikes (51Adams J.C. Mol. Biol. Cell. 1997; 8: 2345-2363Crossref PubMed Scopus (82) Google Scholar), although TSP1 bound to HSPGs but not chondroitin sulfate proteoglycans extracted from melanoma cells (29Roberts D.D. Cancer Res. 1988; 48: 6785-6793PubMed Google Scholar), and our data demonstrate that an HSPG is required for stimulation of αvβ3integrin function by peptide 246. Based on the activity of this heparin-binding peptide, simultaneous engagement of the heparan sulfate and αvβ3 integrin-binding sites of intact TSP1 may induce synergistic signaling responses in cells that express both classes of receptors. We thank Dr. William H. Miller for providing the integrin antagonists.
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