Decorin Is a Biological Ligand for the Epidermal Growth Factor Receptor

Ectopic expression of decorin induces profound cytostatic effects in transformed cells with diverse histogenetic backgrounds. The mechanism of action has only recently begun to be elucidated. Exogenous decorin activates the epidermal growth factor (EGF) receptor, thereby triggering a signaling cascade that leads to phosphorylation of mitogen-activated protein (MAP) kinase, induction of p21, and growth suppression. In this study we demonstrate a direct interaction of decorin with the EGF receptor. Binding of decorin induces dimerization of the EGF receptor and rapid and sustained phosphorylation of MAP kinase in squamous carcinoma cells. In a cell-free system, decorin induces autophosphorylation of purified EGF receptor by activating the receptor tyrosine kinase and can also act as a substrate for the EGF receptor kinase itself. Using radioligand binding assays we show that both immobilized and soluble decorin bind to the EGF receptor ectodomain or to purified EGF receptor. The binding is mediated by the protein core and has relatively low affinity (K d ∼87 nm). Thus, decorin should be considered as a novel biological ligand for the EGF receptor, an interaction that could regulate cell growth during remodeling and cancer growth. Ectopic expression of decorin induces profound cytostatic effects in transformed cells with diverse histogenetic backgrounds. The mechanism of action has only recently begun to be elucidated. Exogenous decorin activates the epidermal growth factor (EGF) receptor, thereby triggering a signaling cascade that leads to phosphorylation of mitogen-activated protein (MAP) kinase, induction of p21, and growth suppression. In this study we demonstrate a direct interaction of decorin with the EGF receptor. Binding of decorin induces dimerization of the EGF receptor and rapid and sustained phosphorylation of MAP kinase in squamous carcinoma cells. In a cell-free system, decorin induces autophosphorylation of purified EGF receptor by activating the receptor tyrosine kinase and can also act as a substrate for the EGF receptor kinase itself. Using radioligand binding assays we show that both immobilized and soluble decorin bind to the EGF receptor ectodomain or to purified EGF receptor. The binding is mediated by the protein core and has relatively low affinity (K d ∼87 nm). Thus, decorin should be considered as a novel biological ligand for the EGF receptor, an interaction that could regulate cell growth during remodeling and cancer growth. Decorin plays a pivotal role in regulating the proper assembly of collagenous matrices and in the control of cell proliferation (1Iozzo R.V. Annu. Rev. Biochem. 1998; 67: 609-652Crossref PubMed Scopus (1344) Google Scholar). Most of its biological interactions occur via the central leucine-rich repeat region, which is thought to fold into an arch-shaped structure (2Weber I.T. Harrison R.W. Iozzo R.V. J. Biol. Chem. 1996; 271: 31767-31770Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar). Because of the ability of decorin to bind fibrillar collagen and to delay in vitro fibrillogenesis (3Vogel K.G. Paulsson M. Heinegård D. Biochem. J. 1984; 223: 587-597Crossref PubMed Scopus (704) Google Scholar), this proteoglycan is regarded as a key modulator of matrix assembly. Genetic studies utilizing decorin null mice have indeed proved a major role for decorin in the homeostasis of dermal collagen. Although the nullizygous animals grew to adulthood without any overt pathology, a close analysis revealed a skin fragility phenotype; the dermal collagen exhibited aberrant organization of fibrils with abnormal packing and a great variability in diameter (4Danielson K.G. Baribault H. Holmes D.F. Graham H. Kadler K.E. Iozzo R.V. J. Cell Biol. 1997; 136: 729-743Crossref PubMed Scopus (1183) Google Scholar). Another important role of decorin is linked to its ability to inhibit transforming growth factor-β, which in turns blocks cell proliferation (5Border W.A. Noble N.A. Yamamoto T. Harper J.R. Yamaguchi Y. Pierschbacher M.D. Ruoslahti E. Nature. 1992; 360: 361-364Crossref PubMed Scopus (926) Google Scholar). There is also mounting evidence that decorin is involved directly in the control of cell growth. Decorin levels are markedly elevated during growth arrest and quiescence, its expression is abrogated by viral transformation, and its transcription is suppressed in most tumorigenic cell lines (6Mauviel A. Santra M. Chen Y.Q. Uitto J. Iozzo R.V. J. Biol. Chem. 1995; 270: 11692-11700Crossref PubMed Scopus (127) Google Scholar). De novo expression of decorin induces a generalized growth suppression by activating p21, a potent inhibitor of cyclin-dependent kinase activity (7Santra M. Skorski T. Calabretta B. Lattime E.C. Iozzo R.V. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7016-7020Crossref PubMed Scopus (206) Google Scholar, 8De Luca A. Santra M. Baldi A. Giordano A. Iozzo R.V. J. Biol. Chem. 1996; 271: 18961-18965Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar, 9Santra M. Mann D.M. Mercer E.W. Skorski T. Calabretta B. Iozzo R.V. J. Clin. Invest. 1997; 100: 149-157Crossref PubMed Scopus (184) Google Scholar). Decorin activates the EGF 1The abbreviations EGFepidermal growth factorEGFREGF receptorp21the cyclin-dependent kinase inhibitor p21WAF1/CIP1BSAbovine serum albuminMAPmitogen-activated proteinPAGEpolyacrylamide gel electrophoresisNTAnitrilotriacetic acidBS3bissulfosuccinimidylsuberate receptor (EGFR) (10Moscatello D.K. Santra M. Mann D.M. McQuillan D.J. Wong A.J. Iozzo R.V. J. Clin. Invest. 1998; 101: 406-412Crossref PubMed Scopus (245) Google Scholar) that in turn triggers a signal cascade leading to activation of MAP kinases, mobilization of intracellular calcium (11Patel S. Santra M. McQuillan D.J. Iozzo R.V. Thomas A.P. J. Biol. Chem. 1998; 273: 3121-3124Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar), up-regulation of p21, and growth suppression. epidermal growth factor EGF receptor the cyclin-dependent kinase inhibitor p21WAF1/CIP1 bovine serum albumin mitogen-activated protein polyacrylamide gel electrophoresis nitrilotriacetic acid bissulfosuccinimidylsuberate In this report we demonstrate a direct interaction between the EGFR and decorin. Exogenous decorin causes dimerization of the EGFR in A431 squamous carcinoma cells. Decorin induces autophosphorylation of the EGFR by activating the EGFR tyrosine kinase in a cell-free system and can also act as a substrate for the EGFR kinase itself. Both immobilized and soluble decorin bind to the soluble EGFR ectodomain or to immunopurified EGFR. The binding is solely mediated by the protein core and has relatively low affinity (K d ∼87 nm). Thus, decorin should be considered as a novel biological ligand for the EGFR. Media and fetal bovine serum were obtained from Hyclone Laboratories, Inc. (Logan, UT). γ-32PATP (6000 Ci/mmol) and Hybond ECL membranes were from Amersham Pharmacia Biotech. The EGFR preparation purified from A431 cells by immunoaffinity chromatography (Sigma) contained ∼500 units of active EGFR; 1 unit catalyzes the incorporation of 1 pmol of phosphate/min from γ-32PATP into poly(Glu,Tyr) at 30 °C. Confluent A431 cells, which were rendered quiescent by serum deprivation for 36 h, were incubated with decorin or decorin protein core (1 μm) or EGF (16 nm) or various combinations at 4 °C for 1 h. The cells were washed extensively and incubated for 10 min with the membrane-impermeable cross-linker bissulfosuccinimidylsuberate (BS3, (Pierce). The cell lysates were separated in 3–15% SDS-PAGE and subjected to immunoblotting (10Moscatello D.K. Santra M. Mann D.M. McQuillan D.J. Wong A.J. Iozzo R.V. J. Clin. Invest. 1998; 101: 406-412Crossref PubMed Scopus (245) Google Scholar) with anti-EGFR antibody. The anti-phospho-MAP kinase antibody (New England BioLabs) detects p42 and p44 MAP kinases (Erk1/Erk2) only when they are catalytically activated by phosphorylation at Thr202 and Tyr204. Constant amounts (∼300 ng) of immunopurified EGFR were preincubated with buffer alone or containing increasing concentrations (5–20 μg) of decorin, its decorin protein core, or collagen type I. After 15 min of incubation in kinase buffer (20 mm HEPES, pH 7.4, 2 mm MnCl2, 10 mm p-nitrophenyl phosphate, 40 μmNa3VO4, 0.01% BSA, 15 μm ATP) 1 μCi of γ-32PATP and 0.2% Nonidet P-40 was added to reach a final volume of 60 μl. The mixture was incubated for an additional 10 min, stopped by boiling in SDS buffer, and analyzed by SDS-PAGE. Phosphorylated proteins were visualized by autoradiography. Control samples omitting either γ-32PATP or EGFR showed no activity. Because A431 cells synthesize a soluble form of EGFR of ∼105 kDa lacking the transmembrane and intracytoplasmic domains (12Weber W. Gill G.N. Spiess J. Science. 1984; 224: 294-297Crossref PubMed Scopus (140) Google Scholar), serum-free medium conditioned by confluent A431 cells was concentrated by Centricon-50 and incubated with decorin, or its protein core immobilized on nitrocellulose filters, washed, and subjected to immunodetection with anti-EGFR antiserum. Briefly, serial dilutions of BSA, decorin, or its protein core were slot-blotted onto ECL nitrocellulose, blocked overnight at 4 °C with 5% fetal bovine serum and 5% nonfat milk, and washed several times in TBS-T (Tris-buffered saline, 25 mm Tris-HCl, 150 mm NaCl, 0.1% Tween 20, pH 7.4). The membranes were incubated with the serum-free medium conditioned by the A431 cells, washed 3 more times, incubated with an antibody raised against the N-terminal sequence LEEKK of the human EGFR, and affinity-purified on a peptide linked to Sepharose. This antibody would not interfere with the binding because it recognizes the N-terminal end of the EGFR. As additional control, we used a mouse monoclonal antibody 225 raised against the EGF-binding domain of the EGFR (13Fan Z. Lu Y. Wu X. Mendelsohn J. J. Biol. Chem. 1994; 269: 27595-27602Abstract Full Text PDF PubMed Google Scholar). We utilized columns containing nickel-nitrilotriacetic acid (Ni-NTA) as ligand (Qiagen) since the recombinant human decorin contains an N-terminal tag with 6 histidine residues (His6) that allows a rapid and efficient purification via Ni-NTA affinity chromatography (14Ramamurthy P. Hocking A.M. McQuillan D.J. J. Biol. Chem. 1996; 271: 19578-19584Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). First we phosphorylated the immunopurified EGFR to reach a specific activity of ∼1.7 × 1017 cpm/mol using thein vitro phosphorylation assay described above. Following purification by Sephadex G-50 chromatography, constant amounts of32P-labeled EGFR were incubated with increasing concentrations of decorin or its core protein for 30 min at 4 °C under gentle agitation. The spin columns were equilibrated with 3 column volumes of binding buffer (300 mmNaH2PO4, pH 8.0, 300 mm NaCl), and then the samples were applied and spun at 750 × g for 5 min. Following two consecutive washes, the bound decorin-EGFR complexes were eluted with buffer containing 250 mmimidazole. Parts of the fractions were counted in a scintillation counter, and parts were analyzed by SDS-PAGE and autoradiography. Radioligand binding assays were performed as described before (15Milev P. Chiba A. Margolis R.K. Schachner M. Ranscht B. Margolis R.U. J. Biol. Chem. 1998; 273: 6998-7005Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). Decorin was coated in removable Immulon 4HXB wells (Dynex Technologies, Chantilly, VA), and binding of 32P-labeled EGFR was measured in TBS supplemented with 2 mm CaCl2, 2 mm MgCl2, 0.02% NaN3, and 1 mg/ml heat-inactivated BSA following incubation under gentle shaking (60 rpm) for 4–14 h. Reversible binding was demonstrated by incubation with 100-fold molar excess EGFR. After incubation the wells were washed with ice-cold TBS-T and then counted in toto. Scatchard plots were generated using the Ligand program as described before (16Milev P. Monnerie H. Popp S. Margolis R.K. Margolis R.U. J. Biol. Chem. 1998; 273: 21439-21442Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). It is well established that the general mechanism transmitting extracellular signals starts with binding of a growth factor to a cell surface receptor that in many cases carries an intrinsic tyrosine kinase activity (17van der Geer P. Hunter T. Lindberg R.A. Annu. Rev. Cell Biol. 1994; 10: 251-337Crossref PubMed Scopus (1245) Google Scholar). For example, EGF stimulates the formation of non-covalent homo- and heterodimers with other members of the family of receptor tyrosine kinase (17van der Geer P. Hunter T. Lindberg R.A. Annu. Rev. Cell Biol. 1994; 10: 251-337Crossref PubMed Scopus (1245) Google Scholar). The growth of some tumor cells bearing high levels of EGF receptors is paradoxically suppressed. For instance, A431 vulvar squamous carcinoma cells are stimulated to grow with picomolar amounts of EGF but are markedly growth inhibited with nanomolar amounts of the growth factor (18Barnes D.W. J. Cell Biol. 1982; 93: 1-4Crossref PubMed Scopus (235) Google Scholar). EGF treatment of A431 cells causes a protracted induction of p21(19, 20), and similarly, antibodies that recognize the EGF-binding region of the EGFR inhibit the proliferation of normal and transformed cells (13Fan Z. Lu Y. Wu X. Mendelsohn J. J. Biol. Chem. 1994; 269: 27595-27602Abstract Full Text PDF PubMed Google Scholar, 21Fan Z. Baselga J. Masui H. Mendelson J. Cancer Res. 1993; 53: 4637-4642PubMed Google Scholar, 22Fan Z. Mendelsohn J. Masui H. Kumar R. J. Biol. Chem. 1993; 268: 21073-21079Abstract Full Text PDF PubMed Google Scholar, 23Van de Vijver M. Kumar R. Mendelsohn J. J. Biol. Chem. 1991; 266: 7503-7508Abstract Full Text PDF PubMed Google Scholar, 24Fan Z. Masui H. Altas I. Mendelsohn J. Cancer Res. 1993; 53: 4322-4328PubMed Google Scholar). Thus, we reasoned that decorin might induce growth suppression by activating a similar signal-transducing pathway. To test whether decorin could cause dimerization of the EGFR, we added purified decorin proteoglycan or its protein core to quiescent A431 cells at 4 °C for 1 h, washed extensively, and incubated for 10 min with BS3, a non-cleavable, membrane-impermeable cross-linker. Decorin induced significant dimerization of the EGFR (Fig.1 A, lanes 4 and 8), estimated to be ∼40% of that induced by EGF (Fig. 1 A, lane 2). There were no additive effects when decorin or its protein core were added concurrently with EGF (Fig. 1, A, lanes 6 and 10, and B, lane 6). The protein core of decorin was capable of mediating the full effect of the decorin proteoglycan (Fig. 1 B). To investigate this further, we iodinated decorin and performed similar cross-linking experiments followed by immunoprecipitation. We found a very high M r complex that could not penetrate the gel (not shown), further suggesting the decorin might be directly involved in binding to the EGFR. To investigate whether decorin induces rapid and sustained activation of the MAP kinase signal-transducing pathway, we exposed quiescent A431 cells to 1 μm decorin or its protein core for either 18 h or 10 min. As positive and negative controls we used EGF (16 nm) and collagen (1 μm), respectively. The results showed both a sustained (Fig.2 A) and rapid (Fig. 2,B and C) activation of the MAP kinase signal pathway, which leads to prolonged activation of the endogenous p21 and block of the cells in G1 (10Moscatello D.K. Santra M. Mann D.M. McQuillan D.J. Wong A.J. Iozzo R.V. J. Clin. Invest. 1998; 101: 406-412Crossref PubMed Scopus (245) Google Scholar). To more directly investigate the interaction between the EGFR and decorin, we used EGFR purified by immunoaffinity chromatography from plasma membranes of A431 cells (25Yarden Y. Harari I. Schlessinger J. J. Biol. Chem. 1985; 260: 315-319Abstract Full Text PDF PubMed Google Scholar). The EGFR was not exposed to EGF during the purification procedure, and thus it contains optimal kinase activity and is suitable for both binding and in vitro kinase assays. First, we established by Western immunoblotting the purity of the EGFR and the lack of degradation products. A single ∼170-kDa band was seen using an antibody against the N terminus of the EGFR (Fig. 2 D). When the EGFR was subjected to in vitro kinase assay there was a dose-dependent induction of EGFR autophosphorylation only in the presence of decorin (Fig. 2 E, lanes 2–4) or its protein core (Fig. 2 but in the presence of collagen (Fig. 2 E, lanes the EGFR kinase phosphorylated decorin protein core as seen by the of the two and proteins seen only when the protein core was added (Fig. 2 E, lanes significant phosphorylation of the decorin proteoglycan would have as a was that the phosphorylation of the protein core be inhibited by the The binding of decorin to the EGFR was by °C for 15 of both decorin and its protein core (not The treatment of the EGFR the binding to decorin. Thus, proper protein is for this The for studies is on the that A431 cells synthesize and into the medium a soluble form of EGFR of ∼105 kDa lacking the transmembrane and intracytoplasmic domains (12Weber W. Gill G.N. Spiess J. Science. 1984; 224: 294-297Crossref PubMed Scopus (140) Google Scholar, W. Gill G.N. J. Biol. Chem. 1984; Full Text PDF PubMed Google Scholar). This soluble protein is from the from a EGFR on Annu. Rev. Biochem. PubMed Scopus Google Scholar). As the extracellular domain has both a high and of the ectodomain is and to It with high affinity EGF (12Weber W. Gill G.N. Spiess J. Science. 1984; 224: 294-297Crossref PubMed Scopus (140) Google Scholar, W. Gill G.N. J. Biol. Chem. 1984; Full Text PDF PubMed Google Scholar), and show that 1 of EGFR 1 of First, we the purity of the medium conditioned for h by A431 cells by Western A single band of ∼105 kDa was using the anti-EGFR (Fig. 3 This was used in assays in which both proteins immobilized and the soluble are under medium conditioned for h by A431 cells was concentrated by Centricon-50 and incubated with decorin, its protein core, or BSA immobilized on nitrocellulose filters, washed, and subjected to immunodetection with anti-EGFR A. E. M. S. D.J. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). Both protein core (Fig. 3 and decorin (Fig. 3 C) bound to soluble EGFR in to BSA, which was a protein containing the N terminus of and the decorin results (not Thus, the binding to EGFR is not mediated by the N terminus since not cause activation of EGFR (10Moscatello D.K. Santra M. Mann D.M. McQuillan D.J. Wong A.J. Iozzo R.V. J. Clin. Invest. 1998; 101: 406-412Crossref PubMed Scopus (245) Google Scholar). When similar assays were with monoclonal antibody 225 that with the EGF binding region of the EGFR (13Fan Z. Lu Y. Wu X. Mendelsohn J. J. Biol. Chem. 1994; 269: 27595-27602Abstract Full Text PDF PubMed Google Scholar), there was no (not shown), suggesting that decorin might bind or in the EGF To further the we immobilized on nitrocellulose amounts of conditioned for various of by A431 cells and performed assays with soluble decorin followed by Western immunoblotting 1995; Scopus Google Scholar). The results showed specific binding of decorin to serum-free medium conditioned by A431 cells, which contains the EGFR ectodomain (Fig. 3 D). As negative and positive controls we used either medium (Fig. 3 or dilutions ng) of purified decorin (Fig. 3 respectively. results show that decorin with the ectodomain of the EGFR either in or immobilized on nitrocellulose we to investigate whether soluble decorin could with the EGFR under concentrations and in The for this is that decorin contains an N-terminal tag that allows a rapid and efficient purification via Ni-NTA affinity chromatography (14Ramamurthy P. Hocking A.M. McQuillan D.J. J. Biol. Chem. 1996; 271: 19578-19584Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Constant amounts of32P-labeled EGFR were incubated with increasing concentrations of decorin or its core protein for 30 min at 4 °C under gentle agitation. The Ni-NTA columns were equilibrated with 3 column volumes of binding buffer, and then the samples were applied and spun at 750 × g for 5 min. Following two consecutive washes, the bound decorin-EGFR complexes were eluted with buffer containing 250 mm In two experiments there was specific binding of the EGFR to decorin since the of EGFR to the of decorin (Fig. of both experiments a with r 2 (Fig. 4 D). to more the binding affinity we utilized a radioligand binding assay increasing concentrations of32P-labeled EGFR were on Immulon wells coated with decorin protein core Binding of EGFR to decorin protein core was (Fig. 4 and a Scatchard a single with The binding of decorin to the EGFR was by 100-fold molar excess of EGFR (not In experiments more decorin protein core was used to the the binding of EGFR was and the also Scatchard plots (not the affinity constant obtained in experiments is that for the low affinity binding for EGF nm) and the high affinity receptor for EGF nm) Annu. Rev. Biochem. PubMed Scopus Google Scholar). We have demonstrated that decorin protein core causes a rapid phosphorylation of the EGFR in A431 cells, which leads to a specific activation of the MAP kinase signal and to induction of endogenous lines of evidence a specific interaction between decorin and the EGFR. We demonstrate for the that decorin is capable of dimerization of the EGFR in cells, a to be a to receptor The specific binding when decorin is either immobilized on nitrocellulose membranes or in In a cell-free system, decorin induces autophosphorylation of purified EGFR by activating the receptor tyrosine kinase and can also act as a substrate for the EGFR kinase it is to be a substrate in since decorin is an extracellular Decorin is capable of EGFR tyrosine kinase and that both the binding and activation protein and two receptor tyrosine kinases, have to be the receptors for fibrillar collagen A. E. M. S. D.J. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, W. T. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). to the of the receptor tyrosine kinase activity the structure of collagen and an of W. T. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). The that extracellular matrix can directly as for receptor tyrosine kinases the by which cells and to the extracellular matrix Because decorin is with fibrillar a complex interactions in an should be in decorin in the tumor could a interaction with the EGFR, to be in most tumor cells, which would in turn a signaling that directly the cell