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The GPIIb-IIIa-like complex may function as a human melanoma cell adhesion receptor for thrombospondin
Experimental Cell Research 182 (1989) 473-481
The GPllb-llla-like Complex May Function as a Human Melanoma Cell Adhesion Receptor for Thrombospondin GEORGE P. TUSZYNSKI,’ JERZY KARCZEWSKI, LINDA ANDREW MURPHY, VICKI L. ROTHMAN, and KAREN A. KNUDSEN Lankenau
Medical
SMITH,
Research Center, Lancaster Avenue West of City Line Avenue, Philadelphia, Pennsylvania 19151
The purpose of this study was to determine whether a heterodimeric complex immunologically related to the fibrinogen receptor could function as a thrombospondin (TSP) receptor in TSP-mediated cell-substratum adhesion of human melanoma cells. We found that polyclonal antibodies to the platelet GPIIb-IIIa complex, GPIIIa, and the human vitronectin receptor inhibited TSP-mediated cell adhesion by 6348 %. Immunoprecipitation of detergent extracts of ‘251-surface-labeled melanoma cells using either anti-human platelet GPIIb-IIIa or anti-human vitronectin receptor antibody revealed the presence of a single heterodimeric complex, suggesting that both antisera recognize the same integrin receptor, GPIIb-IIIa-like antigen, Adhesion of cells to TSP is likely mediated through a region of the TSP molecule containing the arginine-glycine-aspartic (RGD) peptide sequence, since cell attachment to TSP was inhibited 5066% in the presence of peptides containing RGD. These results strongly suggest that a GPIIb-IIIa-like/vitronectin receptor can serve as a cell binding site for TSP in mediating cell-substratum adhesion. @ 19139 Academic
Press, Inc.
Thrombospondin (TSP) was first isolated and characterized from proteins secreted by activated platelets [ 13.Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed that TSP has three identical disulfide-linked polypeptide chains, each with an apparent molecular weight of 180,000 [2, 31. TSP binds specifically to a number of macromolecules, including fibrinogen [4], fibronectin [5], type V collagen [6], plasminogen [7], and heparin
HI. More recently, TSP has been found to be produced by other cells. For example, endothelial cells [8-lo], fibroblasts [ll, 121, and smooth cells [13, 141 synthesize and release TSP and incorporate it into their extracellular matrix. The presence of TSP as a component of the extracellular matrix has stimulated interest in its physiological role and has led to studies investigating the ability of TSP to promote cell-cell and cell-matrix adhesion. To date, TSP isolated from human platelets has been shown to promote platelet aggregation [ 151,adhesion of malaria-parasitized erythrocytes [ 161, adhesion of human melanoma and squamous cell carcinoma [17, 181, and the in vitro cell-substratum adhesion of many ’ To whom correspondence should be addressed at the present address: Department of Medicine, Medical College of Pennsylvania, Philadelphia, PA 19129. 473
Copyright 0 1989 by Academic Press, Inc. All rights nf~epmduction in any form resewed CO14-4827/89 603.00
474 Tuszynski et al.
cell types regardless of species [19]. These studies suggest that TSP, like tibronectin, is an extracellular protein involved in cell-matrix attachment. The ability of cells to adhere to a matrix is a basic property of most cell types. A number of extracellular proteins, including fibronectin, vitronectin, fibrinogen, and thrombospondin, have been shown to promote cellular adhesion. TSP, like fibronectin, vitronectin, and fibrinogen, contains in its primary amino acid structure the amino acid sequence, RGD [20], a sequence that has been shown to be critical to a cell surface recognition domain of fibronectin, vitronectin, and fibrinogen [21]. The RGD domains of these proteins have recently been shown to interact with members of a family of cell surface, heterodimeric glycoprotein receptors, including the platelet fibrinogen receptor GPIIb-IIIa [22], the vitronectin receptor (VnR) [23], the mammalian fibronectin receptor (FnR) [24], and the avian fibronectin receptor, CSAT antigen [25]. The identification of the cell binding site(s) for TSP is of great interest. Recent observations in our laboratories suggested that human melanoma cells possess GPIIb-IIIa-like adhesion receptors (26). In this paper we explore the role of the GPIIb-IIIa-like complex in TSP-promoted cell-substratum adhesion. MATERIALS AND METHODS Materials. The peptides, GRGDS, GRGDSP, and GRGESP were from Penninsula. TSP was purified from the secreted proteins of ionophore A23187-activated human platelets as previously described [4]. Purified TSP preparations contained undetectable levels of libronectin, vitronectin, PF,, von Willebrand factor, and less than 0.1% fibrinogen as previously described [4, 191. Cell culture. The human melanoma cell line, C32 (ATCC CRL 1585)was purchased from American Type Culture Collection and grown in Dulbecco’s modified essential medium (DMEM) (Flow) supplemented with glutamine and 10% fetal calf serum (Hyclone). Antibodies. The monoclonal antibody, A2A9 [27], to human platelet GPIIb-IIIa was a gift from Dr. J. Bennett, University of Pennsylvania School of Medicine. The specific polyclonal antiserum to GPIIb-IIIa was generated to the complex purified from human platelets by Lens culinaris lectin affinity chromatography, anion exchange, and monoclonal antibody aftinity chromatography using A& coupled to CnBr-Sepharose 4B (Pharmacia) [26]. This antisemm recognizes both GPIIb and GPIIIa by immunoblot analysis [26]. Anti-GPIIIa was generated by injecting a rabbit with GPIIIa, purified as above and eluted from nonreduced SDS-polyacrylamide gels as previously described 1261. This antiserum immunoblots only GPIIIa but immunoprecipitates the complex [26]. Immunoglobulin fractions (IgG) were prepared by protein A-Sepharose 4B affinity chromatography (Pharmacia). Antisera to the vitronectin receptor (anti-VnR) isolated from human placenta [23] was a gift from Dr. E. Ruoslahti, La Jolla Cancer Research Foundation. Anti-VnR cross-reacts with GPIIb-IIIa because it recognizes the /I subunit of the highly conserved integrin family of adhesion receptors. SDS-polyacrylamide gel electrophoresis. SDS-PAGE was performed according to the procedure of Laemmli [28], except that reducing agents were sometimes omitted. Molecular weight markers included myosin (200 kDa), ,!I-galactosidase (116 kDa), phosphorylase b (94 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), and carbonic anhydrase (30 kDa). Cell adhesion assays. Cell binding to TSP was assayed essentially as previously described 1191, except that 0.5% BSA was included in the assay. Briefly, cells were harvested with trypsin/EDTA and washed to remove any serum. Wells (1 cm’) of eight-well glass tissue culture slides were treated overnight (refrigerated in a humidified atmosphere) with a S-pl drop of TSP (50-200 &ml). Cells were added and incubated for 60 min at 37°C. Nonadherent cells were washed away; adherent cells were fmed with 2% glutaraldehyde for 10 min, stained with Giemsa for 20 min, viewed microscopically at 100x, and counted using a l-mm’ reticle in the 10x eyepiece. For each well of the eight-chamber slide, cells in one representative area circumscribed by the grid were counted and recorded. Antibody and peptide concentrations yielding maximal effects were used in these studies. The absolute number of cells in a l-mm’ field under control conditions varied by as much as 60% from day to day. Therefore, experiments done on different days cannot be compared directly.
GPIIb-IIIa-like
complex as a thrombospondin receptor
475
Fig. I. Effect of anti-GPIIb-IIIa IgG on C32 melanoma cell attachment to TSP. The cell adlheziion assay \vas performed as described under Materials and Methods. (A) Preimmune IgG, 400 1d ml. @I Anti-G IIb-IIIa IgG, 400 &ml. Cells were tied and photographed at a final magnification of lo(OX. Back@pund debris is due to dye adhering to the adsorbed TSP.
416 Tuszynski et al.
O-
control Sor”m
antiIlb-llla
antIllla
antlVnR
Fig. 2. Effect of anti-GPIIb-IIIa, anti-GPIIIa, and anti-VnR on C32 melanoma cell attachment to TSP. The adhesion assays were performed as described under Materials and Methods. The bars represents the mean number of cells attached f the standard deviation. The assays were done in the presence of a 1: 5 dilution of serum. The number of replicates are as follows: control=7, anti-GPIIbIIIa=7, anti-GPIIIa=4, anti-VnR=2.
Zmmunoprecipitarion.Immunoprecipitation studies were performed as previously described [291. Briefly, cells were surface labeled with ‘*‘I using Iodogen (Pierce) and extracted with 1.O% NP-40 in 20 mM ‘B-is acetate, pH 8.0, containing 2 mkf PMSF. The extract was incubated with antisera and then exposed to fured Staphylococcusaureus. The immune complex was eluted with 2% SDS and analyzed by SDS-PAGE followed by autoradiography.
RESULTS The efSect of anti-GPIIb-IIIa, anti-GPIIIa, and anti-vitronectin receptor on attachment of cells to TSP. The adhesion of C32 melanoma cells to TSP was perturbed by the addition of polyclonal antisera to GPIIb-IIIa, GPIIIa, or the vitronectin receptor. The antisera to GPIIb-IIIa and GPIIIa are monospecific and have been previously characterized by us [26], while the antiserum to the vitronectin receptor was a gift of E. Ruoslahti and has also been previously characterized (23). In the adhesion assay, 63-86% fewer cells attached to TSP in the presence of anti-GPIIb-IIIa IgG (Fig. 1) or antisera to GPIIb-IIIa, GPIIIa, or VnR (Fig. 2) as compared to cells in the presence of control IgG (Fig. 1) or preimmune serum (Fig. 2). In addition to decreasing the number of cells attached to TSP, each immune antiserum or purified immune IgG visibly inhibited cell spreading (Fig. 1). Thus, it appeared that TSP-promoted cell-substratum adhesion of C32 melanoma cells proceeded by a mechanism involving GPIIb-IIIa-like receptors and/or the melanoma receptor. Immunoprecipitation of ‘251-labeled cells. We previously showed that our antisera to purified platelet GPIIb-IIIa and GPIIIa both immunoprecipitated a heterodimeric complex from ‘251-surface-labeled C32 melanoma cells (26), indicating that the antisera recognized a GPIIb-IIIa-like complex on the surface of the melanoma cells. Previous immunoblotting studies revealed that the band with lower apparent molecular weight (~5subunit) was recognized by both monoclonal and polyclonal antibodies to GPIIIa, while the band with the higher apparent molecular weight (a subunit) was not recognized by either polyclonal or mono-
GPZZb-ZZZa-likecomplex as a thrombospondin receptor
477
MW x lo-3
100 -
Fig. 3. SDS-PAGE analysis of ‘%labeled material immunoprecipitated by anti-GPIIb-IIIa and antiVnR. Cells were surface labeled with radioactive iodine and extracted, and the extract was immunoprecipitated with the antisera as described under Materials and Methods. Lane 1, preimmune serum. Lane 2, anti-IIb-IIIa serum. Lane 3, anti-VnR serum. Lane 4, whole extract.
clonal antibodies to GPIIb [26]. These results indicated that the heterodimeric complex on the surface of melanoma cells shares immunological similarity to platelet GPIIb-IIIa by virtue of having a similar, and perhaps identical, fi subunit, but is not GPIIb-IIIa. Here we show that a complex apparently identical to the GPIIb-IIIa-like complex was immunoprecipitated from extracts of surface-labeled melanoma cells by an antiserum to the vitronectin receptor isolated from human placenta (Fig. 3), suggesting that anti-GPIIb-IIIa and anti-VnR antisera recognize the same glycoprotein complex expressed on the surface of C32 cells. The effect of RGD-containing peptides on cell attachment to TSP. If a GPIIbIIIa-like integrin receptor was serving as a cell binding site for TSP, one would expect that the adhesion of C32 melanoma cells to TSP would be perturbed by addition of RGD-containing peptides. We found 50-66% fewer cells attached to TSP in the presence of 200 p,M GRGDS or GRGDSP, two RGD-containing peptides, than in the presence of 200 @4 GRGESP, an inactive peptide having glutamic acid (E) substituted for aspartic acid (D) (Figs. 4 and 5). In addition to decreasing the number of cells attached to TSP, the RGD-containing peptides visibly inhibited cell spreading (Fig. 4). Thus, it appeared that TSP-promoted cellsubstratum adhesion proceeds at least in part through a mechanism involving the RGD sequence. DISCUSSION Recent studies using a variety of cell types including melanoma cells 1171, squamouscell carcinoma cells [18], and endothelial cells [193have indicated that TSP can function to support cell-substratum adhesion. The identity of the cellular
478 Tuszynski et al.
Fit :. 4. Effect of RGD-containing peptide on C32 melanoma cell adhesion 1to, and spraading on, TSP. The cell-TSP ’ adhesion assay was performed as described under Materi als andI Mc:thods. (4 Cells on TSP in the absence of added peptides. (B) Cells on TSP in the presence:of200 w GRGE SP. d at (c)C :ells on TSP in the presence of 200 @%fGRGDSP. Cells were fixed, stained , and pl1oto 100x
GPIIb-Ma-like
E f m
complex as a thrombospondin receptor
479
6ool 400
f
300
6 &
200
'i z
100
i
500 0 I
El 8 i3 2 8 l3-d-l
w Y 8
Fig. 5. Effect of RGD-containing peptides on C32 melanoma cell attachment to TSP. Adhesion assays were performed as described under Materials and Methods. Peptides were added at a concentration of 200 t&f. The bar denotes the average of six replicates + the standard deviation. The number of cells binding to TSP in the presence of GRGDS and GRGDSP differed significantly (P
TSP receptor mediating TSP-promoted cell adhesion has yet to be determined. Recent work by Asch et al. [30] indicated that an 88,000-Da membrane glycoprotein present in platelets, endothelial cells, and various tumor cells is a TSP membrane binding site. However, a functional role for this glycoprotein in cellsubstratum adhesion has not been described. GPIIb-IIIa is a surface heterodimeric glycoprotein complex that mediates platelet aggregation and other platelet adhesive functions by its interaction with fibrinogen, fibronectin, and von Willebrand factor [21]. Our recent studies suggest that GPIIb-IIIa mediates TSP-promoted thrombin-induced platelet aggregation [ 151.We have also shown that TSP binds GPIIb-IIIa in vitro [3 11.Briefly, we found that polystyrene tubes coated with TSP bound radiolabeled GPIIb-IIIa in a dose-dependent and saturable manner. The binding was divalent-cation dependent and specific as assessed by anti-GPIIb-IIIa antibodies. The association of TSP with GPIIb-IIIa has also been suggested from electron microscopic studies showing that TSP colocalizes with GPIIb-IIIa on the surface of thrombin-activated platelets [32]. In recent years numerous adhesion receptors have been identified in eukaryotic cells that show biochemical properties similar to the platelet GPIIb-IIIa complex. These receptors belong to a superfamily of heterodimeric complexes composed of noncovalently-linked protein subunits termed a subunits (140-200 kDa) and /3 subunits (95-120 kDa) [21]. The receptors recognize the RGD-containing sequences in such extracellular adhesion proteins as fibronectin and vitronectin 1231.Platelet GPIIb-IIIa and the vitronectin receptor belong to a subfamily of adhesion receptors with apparently identical B subunits and somewhat similar, but not identical, a subunits 1331.Recently, a large variety of different cell types, such as melanoma cells 1261,endothelial cells [34], fibroblasts [35], and smooth muscle cells [35], have been shown to contain adhesion receptors with immunological cross-reactivity to GPIIb-IIIa. Based on studies in our laboratory indicating an in vitro interaction between TSP and platelet GPIIb-IIIa [31], it seemed reasonable to speculate that GPIIb-
480 Tuszynski et al. IIIa-like proteins might function as TSP receptors in TSP-mediated cellular adhesion. Furthermore, previous studies indicated that melanoma cells expressed a heterodimeric complex with a b subunit immunologically and biochemically similar to GPIIIa. This complex was shown to be involved in melanoma cell attachment to fibronectin, vitronectin, and fibrinogen [26]. To ascertain whether melanoma cell GPIIb-IIIa-like proteins might function as TSP adhesion receptors, human melanoma cells were evaluated for their ability to adhere to TSP in the presence of antisera (or IgG) to platelet GPIIb-IIIa, GPIIIa, or the human vitronectin receptor. In addition, the effect of RGD-containing peptides was evaluated on TSP-promoted cell adhesion. Our results suggest that TSP-promoted C32 melanoma cell adhesion is mediated by a GPIIb-IIIa-like receptor that appears similar to the vitronectin receptor. In general, TSP-promoted melanoma cell adhesion was qualitatively and quantitatively similar to tibronectin-promoted melanoma cell adhesion as previously described [26, 361. The conclusion that GPIIb-IIIa-like receptors mediate C32 melanoma cell adhesion to TSP is based on several lines of evidence. First, antibodies to GPIIb-IIIa, IIIa, or the vitronectin receptor block adhesion. Second, anti-GPIIb-IIIa and anti-VnR immunoprecipitate the same heterodimeric complex from 1251-surface-labeledmelanoma cells. Third, RGD-containing peptides block TSP-promoted adhesion, an observation consistent with the RGD recognition site being present on GPIIb-IIIa-like adhesion receptors. Our data do not exclude the possibility that other molecules may function as TSP receptors since a significant number of cells remained attached even at high concentrations of either antibody or peptide. While our work was being considered for publication, Lawler et al. [37] showed that human endothelial, smooth muscle, U937 monocyte, and normal rat kidney cell attachment to thrombospondin was RGD-dependent and mediated by an integrin complex related to GPIIb-IIIa. This research was supported by Grants HL28149 and AR37945 from the Department of Health Services, Grant PDT-287 from the American Cancer Society, and a grant from W. W. Smith Charitble -Rust.
REFERENCES 1. Lawler, J. W., Slayter, H. S., and Coligan, J. E. (1978) J. Biol. Chem. 253, 8609-8616. 2. Lawler, J., Derick, L. H., Connolly, J. E., Chen, J. H., and Chao, F. C. (1985) J. Biol. Chem. 260, 3762-3772. 3. Margossian, S., Lawler, J. W., and Slayter, H. S. (1981) J. Biol. Chem. 256, 7495-7500.
4. Tuszynski, G. P., Srivastava, S., Switalska, H. I., Holt, J. C., Ciemiewski, C. S., and Niewiarowski, S. (1985) J. Biol. Chem. 260, 12,240-12,245. 5. Lahav, J., Lawler, J., and Gimbrone, M. A. (1984) Eur. J. Biochem. 145, 151-156. 6. Galvin, N. J., Vance, P. M., Dixit, V. M., Fink, B., and Frazier, W. A. (1987) J. Cell Biol. 104, 1413-1422. 7. Silverstein, R. L., Leung, L. L. K., Harpel, P. C., and Nachman, R. L. (1984)J. C/in. Znwsf. 74, 1625-1633. 8. Mosher, D. F., Doyle, M. J., and Jatfe, E. A. (1982) J. Cell Biol. 93, 343-348. 9. McPherson, J., Sage, H., and Bomstein, P. (1981) J. Biol. Chem. 256, 11,330-11,336. 10. Vischer, P., Beeck, H., and Voss, B. (1986) Eur. J. Biochem. 153, 43543. 11. Jaffe, E. A., Ruggiero, J. T., Leung, L. L. K., Doyle, M. J., McKeown-Longo, P. J., and Mosher, D. F. (1986) Proc. N&l. Acad. Sci. USA SO,998-1002. 12. McKeown-Longo, P. J., Hanning, R., and Mosher, D. F. (1984) J. Cell Biol. 98, 22-28.
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13. Raugi, G. J., Mumby, S. M., Abbott-Brown, D., and Bornstein, P. (1982) .I. Cell Biol. 95, 351-354. 14. Majack, R. A., Goodman, L. V., and Dixit, V. M. (1988) .Z. Cell Biol. 106, 415422. 15. Tuszynski, G. P., Rothman, V. L., Murphy, A., Siegler, K., and Knudsen, K. A. (1988)Blood72, lO%llS. 16. Roberts, D. R., Sherwood, J. A., Spitalnik, S. L., Panton, L. J., Howard, R. J., Dixit, V. M., Frazier, W. A., Miller, L. H., and Ginsburg, V. (1985) Nature (London) 318, 64-65. 17. Roberts, D. D., Sherwood, J. A., and Ginsburg, V. (1987) J. Cell Biol. 104, 131-139. 18. Varani, J., Dixit, V. M., Fligiel, S. E. G., McKeever, P. E., and Carey, T. E. (1986) Exp. Cell Res. 167, 376-390.
19. Tuszynski, G. P., Rothman, V. L., Murphy, A., Siegler, K., Smith, L., Smith, S., Karczewski, J., and Knudsen, K. A. (1987) Science 236, 157&1573. 20. Lawler, J., and Hynes, R. 0. (1986) J. Cell Biol. 103, 1635-1648. 21. Hynes, R. (1987) Cell 48, 549-554. 22. Gartner, T. K., and Bennett, J. S. (1985) J. Biol. Chem. 260, 11,891-11,894. 23. Pytela, R., Pierschbacher, M. D., and Ruoslahti, E. (1985) Proc. Natl. Acad. Sci. USA 82, 5765770. 24. Pytela, R., Pierschbacher, M. D., and Ruoslahti, E. (1985) Cell 40, 191-198. 25. Horwitz, A., Duggan, K., Greggs, R., Decker, C., and Buck, C. (1985) J. Cell Biol. 101, 21362144. 26. Knudsen, K. A., Smith, L., Smith, S., Karczewski, J., and Tuszynski, G. P. (1988) .I. Cell Physiol.
136, 471-478.
27. Bennett, J. S., Horde, J. A., Leitman, S. F., Vilaire, G., and Cines, D. B. (1983) Proc. N&l. Acad. Sci. USA 80, 2417-2421. 28. Laemmh, U. K. (1970) Nature (London) 227, 68O-fj85. 29. Tuszynski, G. P., Komecki, E., Ciemiewski, C., Knight, L. C., Koshy, A., Srivastava, S., Niewiarowski, S., and Walsh, P. N. (1984) J. Biol. Chem. 259, 5247-5254. 30. Asch, A. S., Barnwell, J., Silverstein, R. C., and Nachman, R. A. (1987) J. Clin. Znwst. 79, 1054-1060. 31. Karczewski, J., Knudsen, K. A., Smith, L., Murphy, A., Rothman, V. L., and Tuszynski, G. P. (1988), submitted for publication. 32. Asch, A. S., Leung, L. L. K., Polley, M. J., and Nachman, R. L. (1985) Blood 66,926-934. 33. Fitzgerald, L. A., Poncz, M., Steiner, B., Rall, S. C., Bennett, J. S., and Phillips, D. R. (1987) Biochemistry 26, 8158-8165. 34. Thiagarajan, P., Shapiro, S., Levine, E., DeMarco, L., and Yalcin, A. (1985) .I. Clin. Znwst. 75, 896-901. 35. Charo, 1. F., Fitzgerald, L. A., Steiner, B., Rall, S. C., Jr., Bekeart, L. S., and Phillips, D. R. (1986) Proc. Natl. Acad. USA 83, 8351-8355. 36. Knudsen, K. A., Tuszynski, G. P., Huang, T.-F., and Niewiarowski, S. (1988) Exp. Cell Res. 179, 42-49.
37. Lawler, J., Weinstein, R., and Hynes, R. 0. (1988) .Z. Cell Biol. 107, 2351-2361. Received December 12, 1988 Revised version received January 16, 1989
F’rinted
in Sweden
The GPllb-llla-like Complex May Function as a Human Melanoma Cell Adhesion Receptor for Thrombospondin GEORGE P. TUSZYNSKI,’ JERZY KARCZEWSKI, LINDA ANDREW MURPHY, VICKI L. ROTHMAN, and KAREN A. KNUDSEN Lankenau
Medical
SMITH,
Research Center, Lancaster Avenue West of City Line Avenue, Philadelphia, Pennsylvania 19151
The purpose of this study was to determine whether a heterodimeric complex immunologically related to the fibrinogen receptor could function as a thrombospondin (TSP) receptor in TSP-mediated cell-substratum adhesion of human melanoma cells. We found that polyclonal antibodies to the platelet GPIIb-IIIa complex, GPIIIa, and the human vitronectin receptor inhibited TSP-mediated cell adhesion by 6348 %. Immunoprecipitation of detergent extracts of ‘251-surface-labeled melanoma cells using either anti-human platelet GPIIb-IIIa or anti-human vitronectin receptor antibody revealed the presence of a single heterodimeric complex, suggesting that both antisera recognize the same integrin receptor, GPIIb-IIIa-like antigen, Adhesion of cells to TSP is likely mediated through a region of the TSP molecule containing the arginine-glycine-aspartic (RGD) peptide sequence, since cell attachment to TSP was inhibited 5066% in the presence of peptides containing RGD. These results strongly suggest that a GPIIb-IIIa-like/vitronectin receptor can serve as a cell binding site for TSP in mediating cell-substratum adhesion. @ 19139 Academic
Press, Inc.
Thrombospondin (TSP) was first isolated and characterized from proteins secreted by activated platelets [ 13.Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed that TSP has three identical disulfide-linked polypeptide chains, each with an apparent molecular weight of 180,000 [2, 31. TSP binds specifically to a number of macromolecules, including fibrinogen [4], fibronectin [5], type V collagen [6], plasminogen [7], and heparin
HI. More recently, TSP has been found to be produced by other cells. For example, endothelial cells [8-lo], fibroblasts [ll, 121, and smooth cells [13, 141 synthesize and release TSP and incorporate it into their extracellular matrix. The presence of TSP as a component of the extracellular matrix has stimulated interest in its physiological role and has led to studies investigating the ability of TSP to promote cell-cell and cell-matrix adhesion. To date, TSP isolated from human platelets has been shown to promote platelet aggregation [ 151,adhesion of malaria-parasitized erythrocytes [ 161, adhesion of human melanoma and squamous cell carcinoma [17, 181, and the in vitro cell-substratum adhesion of many ’ To whom correspondence should be addressed at the present address: Department of Medicine, Medical College of Pennsylvania, Philadelphia, PA 19129. 473
Copyright 0 1989 by Academic Press, Inc. All rights nf~epmduction in any form resewed CO14-4827/89 603.00
474 Tuszynski et al.
cell types regardless of species [19]. These studies suggest that TSP, like tibronectin, is an extracellular protein involved in cell-matrix attachment. The ability of cells to adhere to a matrix is a basic property of most cell types. A number of extracellular proteins, including fibronectin, vitronectin, fibrinogen, and thrombospondin, have been shown to promote cellular adhesion. TSP, like fibronectin, vitronectin, and fibrinogen, contains in its primary amino acid structure the amino acid sequence, RGD [20], a sequence that has been shown to be critical to a cell surface recognition domain of fibronectin, vitronectin, and fibrinogen [21]. The RGD domains of these proteins have recently been shown to interact with members of a family of cell surface, heterodimeric glycoprotein receptors, including the platelet fibrinogen receptor GPIIb-IIIa [22], the vitronectin receptor (VnR) [23], the mammalian fibronectin receptor (FnR) [24], and the avian fibronectin receptor, CSAT antigen [25]. The identification of the cell binding site(s) for TSP is of great interest. Recent observations in our laboratories suggested that human melanoma cells possess GPIIb-IIIa-like adhesion receptors (26). In this paper we explore the role of the GPIIb-IIIa-like complex in TSP-promoted cell-substratum adhesion. MATERIALS AND METHODS Materials. The peptides, GRGDS, GRGDSP, and GRGESP were from Penninsula. TSP was purified from the secreted proteins of ionophore A23187-activated human platelets as previously described [4]. Purified TSP preparations contained undetectable levels of libronectin, vitronectin, PF,, von Willebrand factor, and less than 0.1% fibrinogen as previously described [4, 191. Cell culture. The human melanoma cell line, C32 (ATCC CRL 1585)was purchased from American Type Culture Collection and grown in Dulbecco’s modified essential medium (DMEM) (Flow) supplemented with glutamine and 10% fetal calf serum (Hyclone). Antibodies. The monoclonal antibody, A2A9 [27], to human platelet GPIIb-IIIa was a gift from Dr. J. Bennett, University of Pennsylvania School of Medicine. The specific polyclonal antiserum to GPIIb-IIIa was generated to the complex purified from human platelets by Lens culinaris lectin affinity chromatography, anion exchange, and monoclonal antibody aftinity chromatography using A& coupled to CnBr-Sepharose 4B (Pharmacia) [26]. This antisemm recognizes both GPIIb and GPIIIa by immunoblot analysis [26]. Anti-GPIIIa was generated by injecting a rabbit with GPIIIa, purified as above and eluted from nonreduced SDS-polyacrylamide gels as previously described 1261. This antiserum immunoblots only GPIIIa but immunoprecipitates the complex [26]. Immunoglobulin fractions (IgG) were prepared by protein A-Sepharose 4B affinity chromatography (Pharmacia). Antisera to the vitronectin receptor (anti-VnR) isolated from human placenta [23] was a gift from Dr. E. Ruoslahti, La Jolla Cancer Research Foundation. Anti-VnR cross-reacts with GPIIb-IIIa because it recognizes the /I subunit of the highly conserved integrin family of adhesion receptors. SDS-polyacrylamide gel electrophoresis. SDS-PAGE was performed according to the procedure of Laemmli [28], except that reducing agents were sometimes omitted. Molecular weight markers included myosin (200 kDa), ,!I-galactosidase (116 kDa), phosphorylase b (94 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), and carbonic anhydrase (30 kDa). Cell adhesion assays. Cell binding to TSP was assayed essentially as previously described 1191, except that 0.5% BSA was included in the assay. Briefly, cells were harvested with trypsin/EDTA and washed to remove any serum. Wells (1 cm’) of eight-well glass tissue culture slides were treated overnight (refrigerated in a humidified atmosphere) with a S-pl drop of TSP (50-200 &ml). Cells were added and incubated for 60 min at 37°C. Nonadherent cells were washed away; adherent cells were fmed with 2% glutaraldehyde for 10 min, stained with Giemsa for 20 min, viewed microscopically at 100x, and counted using a l-mm’ reticle in the 10x eyepiece. For each well of the eight-chamber slide, cells in one representative area circumscribed by the grid were counted and recorded. Antibody and peptide concentrations yielding maximal effects were used in these studies. The absolute number of cells in a l-mm’ field under control conditions varied by as much as 60% from day to day. Therefore, experiments done on different days cannot be compared directly.
GPIIb-IIIa-like
complex as a thrombospondin receptor
475
Fig. I. Effect of anti-GPIIb-IIIa IgG on C32 melanoma cell attachment to TSP. The cell adlheziion assay \vas performed as described under Materials and Methods. (A) Preimmune IgG, 400 1d ml. @I Anti-G IIb-IIIa IgG, 400 &ml. Cells were tied and photographed at a final magnification of lo(OX. Back@pund debris is due to dye adhering to the adsorbed TSP.
416 Tuszynski et al.
O-
control Sor”m
antiIlb-llla
antIllla
antlVnR
Fig. 2. Effect of anti-GPIIb-IIIa, anti-GPIIIa, and anti-VnR on C32 melanoma cell attachment to TSP. The adhesion assays were performed as described under Materials and Methods. The bars represents the mean number of cells attached f the standard deviation. The assays were done in the presence of a 1: 5 dilution of serum. The number of replicates are as follows: control=7, anti-GPIIbIIIa=7, anti-GPIIIa=4, anti-VnR=2.
Zmmunoprecipitarion.Immunoprecipitation studies were performed as previously described [291. Briefly, cells were surface labeled with ‘*‘I using Iodogen (Pierce) and extracted with 1.O% NP-40 in 20 mM ‘B-is acetate, pH 8.0, containing 2 mkf PMSF. The extract was incubated with antisera and then exposed to fured Staphylococcusaureus. The immune complex was eluted with 2% SDS and analyzed by SDS-PAGE followed by autoradiography.
RESULTS The efSect of anti-GPIIb-IIIa, anti-GPIIIa, and anti-vitronectin receptor on attachment of cells to TSP. The adhesion of C32 melanoma cells to TSP was perturbed by the addition of polyclonal antisera to GPIIb-IIIa, GPIIIa, or the vitronectin receptor. The antisera to GPIIb-IIIa and GPIIIa are monospecific and have been previously characterized by us [26], while the antiserum to the vitronectin receptor was a gift of E. Ruoslahti and has also been previously characterized (23). In the adhesion assay, 63-86% fewer cells attached to TSP in the presence of anti-GPIIb-IIIa IgG (Fig. 1) or antisera to GPIIb-IIIa, GPIIIa, or VnR (Fig. 2) as compared to cells in the presence of control IgG (Fig. 1) or preimmune serum (Fig. 2). In addition to decreasing the number of cells attached to TSP, each immune antiserum or purified immune IgG visibly inhibited cell spreading (Fig. 1). Thus, it appeared that TSP-promoted cell-substratum adhesion of C32 melanoma cells proceeded by a mechanism involving GPIIb-IIIa-like receptors and/or the melanoma receptor. Immunoprecipitation of ‘251-labeled cells. We previously showed that our antisera to purified platelet GPIIb-IIIa and GPIIIa both immunoprecipitated a heterodimeric complex from ‘251-surface-labeled C32 melanoma cells (26), indicating that the antisera recognized a GPIIb-IIIa-like complex on the surface of the melanoma cells. Previous immunoblotting studies revealed that the band with lower apparent molecular weight (~5subunit) was recognized by both monoclonal and polyclonal antibodies to GPIIIa, while the band with the higher apparent molecular weight (a subunit) was not recognized by either polyclonal or mono-
GPZZb-ZZZa-likecomplex as a thrombospondin receptor
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Fig. 3. SDS-PAGE analysis of ‘%labeled material immunoprecipitated by anti-GPIIb-IIIa and antiVnR. Cells were surface labeled with radioactive iodine and extracted, and the extract was immunoprecipitated with the antisera as described under Materials and Methods. Lane 1, preimmune serum. Lane 2, anti-IIb-IIIa serum. Lane 3, anti-VnR serum. Lane 4, whole extract.
clonal antibodies to GPIIb [26]. These results indicated that the heterodimeric complex on the surface of melanoma cells shares immunological similarity to platelet GPIIb-IIIa by virtue of having a similar, and perhaps identical, fi subunit, but is not GPIIb-IIIa. Here we show that a complex apparently identical to the GPIIb-IIIa-like complex was immunoprecipitated from extracts of surface-labeled melanoma cells by an antiserum to the vitronectin receptor isolated from human placenta (Fig. 3), suggesting that anti-GPIIb-IIIa and anti-VnR antisera recognize the same glycoprotein complex expressed on the surface of C32 cells. The effect of RGD-containing peptides on cell attachment to TSP. If a GPIIbIIIa-like integrin receptor was serving as a cell binding site for TSP, one would expect that the adhesion of C32 melanoma cells to TSP would be perturbed by addition of RGD-containing peptides. We found 50-66% fewer cells attached to TSP in the presence of 200 p,M GRGDS or GRGDSP, two RGD-containing peptides, than in the presence of 200 @4 GRGESP, an inactive peptide having glutamic acid (E) substituted for aspartic acid (D) (Figs. 4 and 5). In addition to decreasing the number of cells attached to TSP, the RGD-containing peptides visibly inhibited cell spreading (Fig. 4). Thus, it appeared that TSP-promoted cellsubstratum adhesion proceeds at least in part through a mechanism involving the RGD sequence. DISCUSSION Recent studies using a variety of cell types including melanoma cells 1171, squamouscell carcinoma cells [18], and endothelial cells [193have indicated that TSP can function to support cell-substratum adhesion. The identity of the cellular
478 Tuszynski et al.
Fit :. 4. Effect of RGD-containing peptide on C32 melanoma cell adhesion 1to, and spraading on, TSP. The cell-TSP ’ adhesion assay was performed as described under Materi als andI Mc:thods. (4 Cells on TSP in the absence of added peptides. (B) Cells on TSP in the presence:of200 w GRGE SP. d at (c)C :ells on TSP in the presence of 200 @%fGRGDSP. Cells were fixed, stained , and pl1oto 100x
GPIIb-Ma-like
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Fig. 5. Effect of RGD-containing peptides on C32 melanoma cell attachment to TSP. Adhesion assays were performed as described under Materials and Methods. Peptides were added at a concentration of 200 t&f. The bar denotes the average of six replicates + the standard deviation. The number of cells binding to TSP in the presence of GRGDS and GRGDSP differed significantly (P
TSP receptor mediating TSP-promoted cell adhesion has yet to be determined. Recent work by Asch et al. [30] indicated that an 88,000-Da membrane glycoprotein present in platelets, endothelial cells, and various tumor cells is a TSP membrane binding site. However, a functional role for this glycoprotein in cellsubstratum adhesion has not been described. GPIIb-IIIa is a surface heterodimeric glycoprotein complex that mediates platelet aggregation and other platelet adhesive functions by its interaction with fibrinogen, fibronectin, and von Willebrand factor [21]. Our recent studies suggest that GPIIb-IIIa mediates TSP-promoted thrombin-induced platelet aggregation [ 151.We have also shown that TSP binds GPIIb-IIIa in vitro [3 11.Briefly, we found that polystyrene tubes coated with TSP bound radiolabeled GPIIb-IIIa in a dose-dependent and saturable manner. The binding was divalent-cation dependent and specific as assessed by anti-GPIIb-IIIa antibodies. The association of TSP with GPIIb-IIIa has also been suggested from electron microscopic studies showing that TSP colocalizes with GPIIb-IIIa on the surface of thrombin-activated platelets [32]. In recent years numerous adhesion receptors have been identified in eukaryotic cells that show biochemical properties similar to the platelet GPIIb-IIIa complex. These receptors belong to a superfamily of heterodimeric complexes composed of noncovalently-linked protein subunits termed a subunits (140-200 kDa) and /3 subunits (95-120 kDa) [21]. The receptors recognize the RGD-containing sequences in such extracellular adhesion proteins as fibronectin and vitronectin 1231.Platelet GPIIb-IIIa and the vitronectin receptor belong to a subfamily of adhesion receptors with apparently identical B subunits and somewhat similar, but not identical, a subunits 1331.Recently, a large variety of different cell types, such as melanoma cells 1261,endothelial cells [34], fibroblasts [35], and smooth muscle cells [35], have been shown to contain adhesion receptors with immunological cross-reactivity to GPIIb-IIIa. Based on studies in our laboratory indicating an in vitro interaction between TSP and platelet GPIIb-IIIa [31], it seemed reasonable to speculate that GPIIb-
480 Tuszynski et al. IIIa-like proteins might function as TSP receptors in TSP-mediated cellular adhesion. Furthermore, previous studies indicated that melanoma cells expressed a heterodimeric complex with a b subunit immunologically and biochemically similar to GPIIIa. This complex was shown to be involved in melanoma cell attachment to fibronectin, vitronectin, and fibrinogen [26]. To ascertain whether melanoma cell GPIIb-IIIa-like proteins might function as TSP adhesion receptors, human melanoma cells were evaluated for their ability to adhere to TSP in the presence of antisera (or IgG) to platelet GPIIb-IIIa, GPIIIa, or the human vitronectin receptor. In addition, the effect of RGD-containing peptides was evaluated on TSP-promoted cell adhesion. Our results suggest that TSP-promoted C32 melanoma cell adhesion is mediated by a GPIIb-IIIa-like receptor that appears similar to the vitronectin receptor. In general, TSP-promoted melanoma cell adhesion was qualitatively and quantitatively similar to tibronectin-promoted melanoma cell adhesion as previously described [26, 361. The conclusion that GPIIb-IIIa-like receptors mediate C32 melanoma cell adhesion to TSP is based on several lines of evidence. First, antibodies to GPIIb-IIIa, IIIa, or the vitronectin receptor block adhesion. Second, anti-GPIIb-IIIa and anti-VnR immunoprecipitate the same heterodimeric complex from 1251-surface-labeledmelanoma cells. Third, RGD-containing peptides block TSP-promoted adhesion, an observation consistent with the RGD recognition site being present on GPIIb-IIIa-like adhesion receptors. Our data do not exclude the possibility that other molecules may function as TSP receptors since a significant number of cells remained attached even at high concentrations of either antibody or peptide. While our work was being considered for publication, Lawler et al. [37] showed that human endothelial, smooth muscle, U937 monocyte, and normal rat kidney cell attachment to thrombospondin was RGD-dependent and mediated by an integrin complex related to GPIIb-IIIa. This research was supported by Grants HL28149 and AR37945 from the Department of Health Services, Grant PDT-287 from the American Cancer Society, and a grant from W. W. Smith Charitble -Rust.
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