Article |
Address correspondence to Alan Wells, Department of Pathology, 713 Scaife, University of Pittsburgh, Pittsburgh, PA 15261. Tel.: (412) 647-7813. Fax: (412) 647-8567. E-mail: wellsa{at}msx.upmc.edu
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Abstract |
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Key Words: receptor signaling; cellsubstratum interactions; adhesion; extracellular matrix; tissue engineering
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Introduction |
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Several matrix components possess EGF-like repeats, the functions of which are largely unknown. Two of these components have been suggested to initiate signaling through the EGFR. The EGF-like repeats in laminin and tenascin-C (hexabrachion) have been shown to modulate cell adhesion and cell motility (Prieto et al., 1992; Nelson et al., 1995). Experiments have suggested that these repeats may directly trigger EGFR signaling by acting as very low affinity ligands (Engel, 1989; Panayotou et al., 1989; Nelson et al., 1995) or potentiate signaling from soluble EGF (Jones et al., 1997). However, these previous studies did not directly isolate EGFR signaling or binding and therefore the exact mechanism of signaling remains undetermined. Furthermore, low affinity ligands (dissociation constant kd in the micromolar range at best) would not be detected by standard binding assays. Low values of solution phase affinity would be predicted for matrix-embedded EGFR ligands because they effectively act from two dimensions, constrained at the cell surface. The effective concentration is increased by being constrained to the interface between the extracellular matrix and the cell surface. Further, the tethered ligand receptor complexes are physically restrained from entering the cell and thus impervious to the major long-term attenuation mechanism of ligand-dependent internalization and degradation (Herbst et al., 1994).
We decided to investigate whether the EGF-like repeats in tenascin-C activate EGFR, as tenascin-C is restricted to sites of tissue development and regeneration and is up-regulated in tumor cells, all of which are sites of EGFR functioning (Erickson, 1993; Chiquet-Ehrismann, 1995). To isolate EGFR signaling, we used NR6 mouse fibroblasts devoid of endogenous EGFR (Pruss and Herschman, 1977) that have been engineered to express various EGFR constructs (Wells, et al., 1990). We found that select EGF-like repeats of tenascin-C were capable of eliciting mitogenesis in an EGFR-dependent manner. Furthermore, although EGFR autophosphorylation was negligible at best, extracellular signalregulated kinase (ERK) mitogen-activated protein (MAP) kinase activation was comparable to subsaturating levels of known EGFR ligands. These cell responses required the extracellular ligand-binding motifs of EGFR, suggesting direct binding. EGFR-dependent adhesion was noted when the predicted avidity of the EGF-like repeat was increased by dimerization or polyvalency via tethering the ligands to inert beads, thereby simulating the physiologically relevant presentation of tenascin-C as hexabrachion. Immunofluorescent imaging further revealed EGFR-dependent binding of the EGF-like repeats to the cell surface. These bindings were abolished upon preincubation with EGF ligand. Direct proof of interaction was demonstrated by cross-linking of EGFR to the EGF-like repeats of tenascin-C.
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Results |
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This pattern of relatively small phosphorylation of EGFR but robust activation of ERK MAP kinases is not unexpected if one considers the highly nonlinear effects that result from differential rates of signal activation and attenuation of molecules activated downstream from EGFR (Bhalla and Iyengar, 1999). Such nonlinearities can result, for example, in persistent activation of MAP kinase or PKC after the EGF signal is withdrawn and maximal pathway stimulation over a wide range of concentrations of signaling pathway components (Bhalla and Iyengar, 1999). The EGFR is subject to multiple signal attentuation mechanisms, including rapid dephosphorylation and internalization (Welsh et al., 1991; Countaway et al., 1992; Hernandez-Sotomayor et al., 1993), but at the same time is associated with a prolonged activation or slower deactivation of downstream signals, such as persistence of grb2SOS interactions (Waters et al., 1996). The observed EGFR activation pattern would be expected for either a ligand at concentrations significantly below kd or a low affinity ligand with a high off-rate. This was tested with both a high affinity ligand (EGF, kd 2 nM) and a lower affinity ligand (Y13G-EGF, kd
100 nM; 41; Fig. 3 B). As observed, EGFR autophosphorylation was barely demonstrable at 0.1 kd for both these ligands. Notably, though, dually phosphorylated ERK MAP kinase could be detected at even lower concentrations: 0.01 kd for both ligands, and both ligands stimulated maximal MAP kinase phosphorylation at 0.01 kd. It was at approximately this level of fractional kd that Ten14 induced dually phosphorylated ERK MAP kinase (Fig. 3 C).
These data suggest that EGFR phosphorylation would be enhanced by limiting attenuation or increasing ligand accessibility. We used sodium vanadate to block receptor dephosphorylation, as this is the most rapid attenuation event (Hernandez-Sotomayor et al., 1993; Fig. 3 D). Treatment with this generalized tyrosine phosphatase inhibitor increased EGFR phosphotyrosine content after exposure to Ten14; similar vanadate-increased EGFR phosphorylation was noted in response to low levels of EGF (0.01 nM), demonstrating fidelity of the assay. To increase signaling persistence and/or ligand accessibility, we tethered EGFR ligands via the NH2-termini to 1-um diameter latex beads using 20 nm polyethylene oxide (PEO) flexible spacer chains to ensure ligand accessibility. This represents an initial attempt to present low affinity ligands in a context that mirrors ligands constrained within the extracellular matrix (Kuhl and Griffith-Cima, 1996). When the tenascin 14 repeat was covalently tethered to these beads, a significantly higher level of tyrosyl-phosphorylation was observed over that obtained with soluble, monomeric ten14 (Fig. 3 E). It was noted that EGFR phosphorylation increased with time exposed to the tenascin-tethered beads; however, it remains to be determined whether this is due to slow diffusion and settling of beads or reflects a situation akin to eph receptor activation (Davis et al., 1994). These data support the finding that select tenascin EGFlike repeats directly activate the EGF receptor from an insoluble presentation mode. Furthermore, the initial findings with these insoluble ligand complexes strongly suggest that manner of ligand presentation alters the balance between signaling and attenuation.
The MAP kinase signaling pathway is activated by the EGF-like repeats of tenascin-C through their direct activation of EGFR
That EGFR kinase activity is required for downstream cell responses was corroborated directly by inhibiting EGFR using the pharmacological agent PD153035. This selective inhibitor of EGFR kinase blocked ERK MAP kinase activation by the tenascin EGFlike repeats (Fig. 4
A).
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Tenascin EGFlike repeats directly bind to EGFR
Final proof that these EGF-like repeats act as novel direct EGFR ligands, however, requires a visualization of EGFR-dependent binding and/or a demonstration of an interaction with EGFR. In direct binding assays, we were unable to detect specific binding to the EGFR by the EGF-like repeat proteins at micromolar concentration (data not shown). This indicates that the ligand is quite low affinity (10 uM) when compared with prototypical growth factor ligands for EGFR (in the low nanomolar range). This proposed low solution affinity of the tenascin 14 repeats appears to be commensurate with the solution affinities for integrin ligands, which are in the micromolar range for fibronectin (Akiyama and Yamada, 1985) and the millimolar range for linear arginineglycineaspartic acid peptides (Pierschbacher and Ruoslahti, 1987). Since specific binding to these integrin ligands can be readily detected by presenting the ligands from the solid phase (i.e., bound to beads or the substrate; Pierschbacher and Ruoslahti, 1984) in a relatively normal physiological manner, we reasoned that specific binding to the tenascin 14 repeats might also be detected by presenting the repeats in a method that resembles their presentation in ECM. We generated
1-um diameter beads that presented a high surface density of ligand (either tenascin 14 or EGF). Beads presenting EGF or the tenascin 14 fragment exhibited specific adhesion to WT NR6 cells compared with control beads (Fig. 5)
. That this occurred via EGFR is demonstrated by blocking of binding by anti-EGFR antibodies (number of bound beads were reduced by >90% in each of three independent experiments). There was negligible binding to M721 NR6 cells, which are devoid of EGFR.
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Discussion |
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It has been suggested previously that EGF-like repeats potentiate signaling from growth factor receptors through the activation of integrins and other signaling transducers (Jones et al., 1997). The entire matrix protein tenascin-C presents numerous interactive and signaling elements. However, we do not feel that this indirect action explains the signaling from the EGF-like repeats. First, these repeats activated biochemical and biological responses in the absence of soluble factors (i.e., serum-free conditions) dependent on binding to a signaling competent EGFR (Figs. 14). These data do not exclude the possibility of signaling through release of membrane-associated EGFR ligands (Daub et al., 1996; Prenzel et al., 1999). This scenario is countered by the second set of data, those demonstrating direct interactions between Ten14 and EGFR (Figs. 57). We could demonstrate EGFR-specific bead binding and Ten14 binding and cross-linking. These interactions were prevented by anti-EGFR antibodies and competed by unlabeled EGF, demonstrating that they occurred through the ligand binding site of EGFR. In short, we have provided definitive evidence for direct binding of EGFR by EGF-like repeats.
The physiological role of such a low affinity ligand remains an open question. One of the main attenuation mechanisms necessary to prevent excessive signaling is EGFR internalization and subsequent degradation of receptor and/or ligand (Welsh et al., 1991; Reddy et al., 1996b). For a ligand that is embedded in the extracellular matrix, such internalization would be physically limited and constantly represent itself even after dissociation due to its physical proximity to the cell surface. A matrix-embedded ligand with high affinity would lead to continuous, strong signaling through EGFR; this has been shown to lead to cellular transformation (DiFiore et al., 1987; Wells et al., 1990). In contrast, a ligand with low affinity (fast off rate; Ebner and Derynck, 1991) would result in ligand decoupling from receptor, allowing dephosphorylation and other mechanisms (Welsh et al., 1991; Countaway et al., 1992; Hernandez-Sotomayor et al., 1993) to function more efficiently at attenuating signaling.
Tenascin-C is an excellent candidate to present such activators of a receptor that can stimulate both cell proliferation and migration. Tenascin-C is expressed, by and large, only during periods of organogenesis and remodeling, such as the fetal/neonatal growth period and during wound repair, and is expressed as a hexamer allowing for physiological presentation of multiple potential matricrine ligands (Schalkwijk et al., 1991; Whitby et al., 1991; Erickson, 1993; Chiquet-Ehrismann, 1995). During these events EGFR signaling is required both for proliferation and migration of the cells (Ashcroft et al., 1995; Miettinen et al., 1995; Sibilia and Wagner, 1995; Threadgill et al., 1995; Xie et al., 1998; Kim et al., 1999). In support of such a model, we have found that NR6 fibroblasts expressing motility competent WT EGFR (8) present greater transmigration of the human extracellular matrix Amgel than NR6 cells expressing the motility-deficient c'973 EGFR (26.8 ± 0.2% vs. 9.6 ± 0.3% of the invasiveness of highly invasive HT1080 fibrosarcoma cells; P < 0.01). This result is similar to transmigration actuated by EGFR triggered by ligand derived either from autocrine signaling (Xie et al., 1995) or the extracellular milieu (Chakrabarty et al., 1995; Kassis et al., 1999). This is intriguing as NR6 cells do not produce known EGFR ligands, and Amgel, derived from human amniotic membranes (Siegal et al., 1993), does not contain detectable levels of EGF, TGF-, or other soluble EGFR ligands, but does contain appreciable levels of tenascin (75 mg/ml out of
1,300 mg/ml proteinaceous material). Thus, low affinity ligands encrypted within matrix components might represent a new mode of modulation of cellular responses by matrix acting directly through growth factor receptors.
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Materials and methods |
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Expression and purification of EGF-like repeat proteins
Midlog phase cultures of Escherichia coli strain BL21/DE3/pLys-S (Stratagene) transformed with the individual expression plasmids were induced for recombinant protein expression with 1 mM isopropyl-b-D-thiogalactopyranoside for 4 h at 37°C. Bacteria were harvested by centrifugation for 10 min at 5,000 g at 4°C, and bacterial lysates were prepared by extraction with 0.02 culture volumes of B-PER detergent (Pierce Chemical Co.). Recombinant proteins were purified from bacterial lysates by nickel-agarose chromatography with imidazole elution. Purified protein was dialyzed against PBS, 0.25 mM 2-mercaptoethanol for 24 h at room temperature.
Mitogenesis assay
Cells were quiesced for 24 h under normal growth conditions in starvation medium (serum-free growth medium supplemented with 1% dialyzed fetal calf serum). The ligand-induced 3H-thymidine incorporation assay has been described previously (Chen et al., 1996). In brief, cells were exposed to EGF (1 nM), serum (1%), or various concentrations of EGF-like repeat proteins for 24 h. 3H-thymidine was added to the cells for the last 8 h to determine stimulation of proliferation.
Phosphorylation assays
For assaying EGFR activation, quiesced cells were treated with ligand for 5 min in quiescence medium (for the experiments described in Fig. 3 D only, the medium was supplemented with 0.1 mM sodium vanadate during this time period and treated for 30 min). When indicated in the figure legends, after cells were quiesced the experiment was performed under serum-free conditions; this further reduces background phosphorylation of ERK. Detergent lysates were immunoprecipitated at 4°C with anti-EGFR antibody (Ab-1; Oncogene Research Products) bound to protein Aconjugated agarose (GIBCO BRL). Immunoprecipitated EGFR was analyzed for tyrosine phosphorylation by immunoblotting using antiphopshotyrosine antibody (PY20; Transduction Laboratories). For assessing MAP kinase activation, quiesced cells were treated with ligand for 5 min in the presence or absence of anti-EGFR Ab-1 (4 ug/ml; Calbiochem) or PD153035 (1 uM). Whole cell lysates were analyzed for dually-phosphorylated ERK MAP kinase by immunoblotting using antiphospho-MAP kinase antibody (New England Biolabs, Inc.). Equal loading was assured using the pan-erk antibody. Relative densitometric values were derived with the NIH Image shareware and Adobe Photoshop® software.
Tethered ligands
Tenascin 14 fragments and EGF were covalently tethered to surfaces to present the ligands in a manner analogous to physiological presentation of matrix-associated tenascin. Poly(methyl methacrylate) latex beads were synthesized by dispersion polymerization using an amphiphilic comb copolymer stabilizer, following a procedure adapted from (Banerjee et al., 2000). A comb stabilizer comprised of methylmethacrylate, polyethylene glycol methacrylate (Mn = 526 g/mol), and methoxypolyethylene glycol methacrylate (Mn = 425 g/mol) in a weight ratio of 30:10:10 was synthesized by free radical polymerization using AIBN as initiator. The hydroxy-terminated polyethylene glycol side chains were subsequently carboxylated by refluxing 16 g of comb with 10 g succinic anhydride, and 0.15 ml N-methyl imidazole in 300 ml of dichloroethane overnight at 80°C. The carboxylated product was precipitated and washed with acidified water. Polyethylene methylmethacrylate latexes were synthesized by the addition of 9 ml methylmethacrylate, 1.25 g of carboxylated comb stabilizer, 1.2 ml vinyl methacrylate cross-linking agent, and 0.50 g of ammonium persulfate initiator to 45 ml of 70:30 (vol/vol) methanol/water. The reaction proceeded at 50°C for 3 h, resulting in a highly stable dispersion of micron-sized polyethylene methylmethacrylate latex beads, each coated by comb polymers that situate and become grafted at the water/bead interface. The carboxylated latex suspension was purified by repeated centrifugation and redispersion, before peptide coupling.
The NH2-terminal amine groups of EGF and tenascin 14 fragments were used to covalently link the peptides to ends of the PEO chains emanating from the surface of the latex beads. Beads were resuspended in dry ethanol with 20 mg/mL sulfo-NHS (Pierce Chemical Co.) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (Pierce Chemical Co.) and activated at room temperature for 3 h, then centrifuged to remove supernatant and resuspended in ethanol for a total of three washes. Beads were washed a final time in 100 mM phosphate buffer, pH 7, and resuspended in 5 ug/mL mouse EGF (Collaborative Biomedical), tenascin 14 fragment, or buffer alone in 100 mM phosphate buffer, pH 7. The coupling reaction was allowed to proceed for 24 h at 4°C. Unreacted peptide was removed and residual NHS reactivity blocked by washing three times in 100 mM tris buffer, pH 7. The beads were then washed once with sterile PBS before addition to cells.
Immunofluorescence
WT NR6 cells were quiesced at 50% confluency. Mouse anti-HisG antibody (Invitrogen) was incubated 24 h at 4°C with Ten14 or mEGF-His6 for dimerization. After quiescence, ligand, either without antibody or antibody-ligand mix, was added to cells in serum-free media and incubated for 10 min at room temperature. 100 nM EGF served as a competitor and was added 5 min before the Ten14 or mEGF-His6. Cells were fixed with 3% formaldehyde for 15 min at room temperature, washed three times with PBS and incubated in 1% BSA for 30 min. Cells that were exposed to ligand alone were washed twice with PBS and incubated with mouse anti-HisG antibody (Invitrogen; 1:1000) for 30 min at 37°C. Cells were washed five times with PBS and secondary goat antimouse conjugated to Oregon green (Molecular Probes; 1:1,000) was added at 37°C for 30 min. Cells were once again washed three times, mounted, and viewed.
Immunoprecipitation
WT NR6 cells were quiesced at 80% confluency. Cells were washed once with PBS. 100 nM EGF served as a competitor and was added 5 min before the Ten14 or mEGF-His6. Cells were than washed with PBS and incubated with ligands Ten14 (2 uM) or mEGF-His6 (10 nM) in PBS for 5 min at room temperature. In parallel, cells were incubated with just PBS (no tx) or with monoclonal anti-HisG (0.01 uM) in PBS. Dithiobis(succinimidyl propionate) (Pierce Chemical Co.) was added to the solution and the cells were placed at 4°C for 30 min. Cells were than washed with 0.2% glycine solution in PBS twice and incubated with 0.2% glycine in PBS for 5 min at 4°C followed by a final wash with 0.2% glycine in PBS once again. Cells were lysed with RIPA lysis buffer with PMSF, aprotinin, and leupeptin as protease inhibitors. 30 ul of protein G agarose beads (GIBCO BRL) and mouse anti-HisG (Invitrogen; final concentration, 0.01 uM) was added to the lysate and incubated overnight. Beads were washed for a total of five times. Lysates were separated by SDS-PAGE with 2-mercaptoethanol (to cleave the cross-linker), transferred, and immunoblotted. The upper half of the membrane was probed with a monoclonal anti-EGFR (Zymed Laboratories; 1:500) and the bottom for polyclonal antiinsulin receptor ß-subunit (Transduction Laboratories; 1:1,000).
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Footnotes |
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* Abbreviations used in this paper: EGFR, EGF receptor; ERK, extracellular signalregulated kinase; M, mutant; MAP, mitogen-activated protein; mEGF, murine EGF; PEO, polyethylene oxide; WT, wild-type.
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Acknowledgments |
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These studies were supported by grants from the National Institutes of Health/National Institute of General Medical Sciences and the National Science Foundation.
Submitted: 22 March 2001
Revised: 24 May 2001
Accepted: 29 May 2001
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References |
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