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- 10.1074/jbc.274.16.11209
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Abstract
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 274, No. 16, Issue of April 16, pp. 11209 –11219, 1999 © 1999 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Integrin-mediated Migration of Murine B82L Fibroblasts Is Dependent on the Expression of an Intact Epidermal Growth Factor Receptor* (Received for publication, October 6, 1998, and in revised form, December 28, 1998) Jing Li‡, Meei-Lih Lin, Gregory J. Wiepz, Arturo G. Guadarrama, and Paul J. Bertics§ From the Department of Biomolecular Chemistry, University of Wisconsin, Madison, Wisconsin 53706-1532 To evaluate the mechanisms by which epidermal EGF triggers many biological responses, including cell pro- liferation and differentiation (1). In addition, EGF has been growth factor (EGF) regulates actin-based cellular proc- esses such as cell migration, we first examined the shown to induce the reorganization of the actin cytoskeleton, effects of EGF on cell adhesion, which is essential for and the EGF receptor has been found to be associated with cell migration. In mouse B82L fibroblasts transfected actin filaments (2–7). In this regard, EGF has been reported to with the full-length EGF receptor, EGF promotes cell stimulate rapid cell rounding, extensive membrane ruffling, rounding and attenuates cell spreading on fibronectin, extension of filopodia, retraction of cells from the substratum laminin, and vitronectin, and thus appears to reduce the (8, 9), extensive cortical actin polymerization, and depolymer- strength of cell adhesion. Moreover, EGF synergizes ization of actin stress fibers (10, 11). Moreover, numerous stud- with multiple extracellular matrix (ECM) components in ies have shown that activation of the EGF receptor leads to the promotion of integrin-mediated cell migration of increased cell motility (12–19) and production of ECM degrad- several different cell types, including fibroblasts and ing proteases (20 –23), thereby supporting a role for the EGF various carcinoma and osteosarcoma cell lines. Interest- receptor in normal development and pathophysiological events ingly, co-presentation (co-positioning) of EGF with lami- such as tumor cell invasion and metastasis. nin or fibronectin is essential for EGF-stimulated migra- Cell migration plays a central role in a variety of biological tion. When EGF is mixed with the cells instead of the processes including embryonic development, angiogenesis, ECM components, it has little effect on cell migration. wound healing, and tumor cell metastasis (24). Although the These results suggest that co-presentation of EGF with exact mechanisms of cell migration are not well established, it ECM components can enhance the polarization events is generally believed that several coordinated events are in- required for directional cell movement. To identify the volved, including morphological polarization, membrane exten- EGF receptor elements critical for the EGF stimulation sion, formation of cell-substratum attachments, contractile of cell migration, B82L fibroblasts were transfected with force and traction, and release of attachments (24). The adhe- either mutated or wild-type EGF receptors. Surpris- ingly, we found that B82L-Parental cells that lack the sive interactions between cells and various ECM substrates are EGF receptor are not able to migrate to fibronectin, likely to be critical in determining cell migration capacity (24, even though they can adhere to fibronectin. However, 25). Many investigations have shown that an intermediate the introduction of wild-type EGF receptors into these adhesive strength generates maximal cell migration (26 –33). fibroblasts enables them to migrate toward fibronectin The adhesion between cells and substrate are largely mediated even in the absence of EGF. The requirement of the EGF by the integrins, which are a family of cell surface het- receptor for cell migration does not appear to result erodimeric receptors that bind to ECM proteins such as lami- a by the cells trans- from the secretion of EGF or TGF- nin, fibronectin, and vitronectin (34). Integrin expression, the fected with the EGF receptor. Furthermore, cells ex- affinity and specificity for their ligands, and the integrin-cy- pressing EGF receptors that are kinase-inactive, or C- toskeleton linkages are regulated by various signals including terminally truncated, exhibit little migration toward those initiated by growth factors. In fact, integrins and growth fibronectin, indicating that an intact EGF receptor ki- factor receptors share many common signaling events, such as nase is required for fibronectin-induced cell migration. increased tyrosine phosphorylation, activation of mitogen-acti- In addition, neutralizing anti-EGF receptor antibodies vated protein (MAP) kinases, protein kinase C isoforms, and attenuate cell migration in the presence of EGF, and small molecular weight GTP-binding proteins, as well as en- inhibit migration to fibronectin or laminin alone. These hanced Ca fluxes (35–38). Given their common signaling results further suggest that the EGF receptor is down- pathways, and the association of both EGF receptor and inte- stream of integrin activation in the signal transduction grin systems with the actin cytoskeleton, it is conceivable that pathways leading to fibroblast migration. the EGF receptor can be involved in the modulation of integrin- mediated cellular functions such as cell migration. Previous analyses of the EGF receptor have revealed that the receptor tyrosine kinase activity and the C-terminal autophos- * This work was funded by National Institutes of Health Grant R01 GM53271 (to P. J. B.).The costs of publication of this article were phorylation sites are critical for EGF-stimulated signal trans- defrayed in part by the payment of page charges. This article must duction (1). The C-terminal domain of the receptor exerts a therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ‡ Partially supported by National Institutes of Health Training Grant T32 HD07259. The abbreviations used are: EGF, epidermal growth factor; ECM, § To whom correspondence should be addressed: Dept. of Biomolecu- extracellular matrix; MAP, mitogen-activated protein; SH2, Src homol- lar Chemistry, University of Wisconsin, 1300 University Ave., Madison, ogy 2; PTB, phosphotyrosine binding; TGF, transforming growth factor; WI 53706-1532. Tel.: 608-262-8667; Fax: 608-262-5253; E-mail: DMEM, Dulbecco’s modified Eagle’s medium; BSA, bovine serum albu- [email protected]. min; FAK, focal adhesion kinase; PDGF, platelet-derived growth factor. This paper is available on line at http://www.jbc.org 11209 This is an Open Access article under the CC BY license. 11210 EGF Receptor Regulation of Cell Migration with purified matrix or control proteins (laminin, 20 mg/ml; fibronectin, competitive autoinhibitory restraint on the receptor tyrosine 10 mg/ml; vitronectin, 10 mg/ml; polylysine, 10 mg/ml) diluted in phos- kinase activity that can be removed by autophosphorylation of phate-buffered saline using either an overnight incubation at 4 °C or a the tyrosine residues located within this domain (39, 40). Fur- 2-h incubation at 37 °C. Nonspecific binding sites were blocked using a thermore, the autophosphorylated tyrosine residues at the 2% (w/v) bovine serum albumin (BSA) solution (in phosphate-buffered EGF receptor C terminus provide anchoring sites for down- saline) and a 1-h incubation at 37 °C. The Clone B3 cells were detached stream Src homology 2 (SH2) or phosphotyrosine binding (PTB) by digestion with 0.1% trypsin, and the cells were then resuspended in DMEM containing 0.1% BSA. The cells were mixed with EGF (at the domain-containing effectors that are involved in the transduc- indicated final concentrations), seeded at a density of 2 3 10 cells/ml, tion of EGF-stimulated intracellular signals (1). Chen et al. (17, and incubated at 37 °C in a humidified incubator containing 5% CO . 18) have reported that the receptor kinase activity and at least Samples were viewed using a light microscope equipped with phase one C-terminal tyrosine autophosphorylation site are required contrast (Nikon Inc., Instrument Group, Melville, NY), and photo- for cell movement. In addition, EGF stimulation of phospho- graphs were taken at different times (magnification, 340). lipase C-g and protein kinase C has been linked to the EGF Cell Motility Assays—The experiments assessing chemotactic motil- ity were performed using a 48-well migration chamber as described induction of cell motility (14, 18). previously (42). In these studies, cells were grown as described under In the present study, we evaluated the effects of EGF on cell “Cell Culture Conditions” for 3– 4 days. After being detached from the migration and assessed the capacity of various EGF receptor plastic dishes using a 0.1% trypsin solution, the cells were stabilized for constructs to modulate the motility of mouse B82L-Parental 1 h at 37 °C in DMEM containing 0.1% BSA. Cells were then counted fibroblasts that possess no detectable endogenous EGF recep- and resuspended in DMEM plus 0.1% BSA at a final concentration of tors. We found that EGF synergistically stimulates integrin- 1 3 10 cells/50 ml. The lower compartment of the migration chamber was filled with the indicated proteins dissolved in DMEM 1 0.1% BSA mediated chemotaxis, and that the co-positioning of EGF and (29 ml/well), and the cells were added to the upper compartment of the the chemoattractant is critical for EGF-stimulated migration. migration chamber (50 ml/well). To ascertain the effects of EGF, the In particular, it is noteworthy that, although B82L-Parental growth factor was added either to the upper or to the lower compart- cells do not exhibit fibronectin-induced chemotaxis, they do ments at indicated concentrations. To evaluate the effects of anti-EGF adhere to fibronectin, but it is the introduction of an intact EGF receptor, anti-EGF, or anti-TGF-a antibodies, the antibodies were in- receptor into these cells that allows for fibronectin-induced cubated with the cells for 30 min before the cells were placed into the motility. In addition, neutralizing anti-EGF receptor antibod- upper wells. In all experiments, the two compartments of the migration chamber were separated by a polycarbonate filter (5-mm pore size, ies inhibit cell migration toward fibronectin or laminin alone. Nucleopore Corp.). The cells were allowed to migrate for4hat37°Cin These findings demonstrate that the EGF receptor is important a humidified atmosphere containing 5% CO . The cells that did not for B82L fibroblast motility, suggesting that the EGF receptor migrate through the membrane remained on the upper surface of the may act downstream of integrin activation and may directly filter and were removed mechanically by scraping; the migrant cells on engage in the signal transduction events leading to cell the lower surface were fixed in methanol/acetone (1:1) for 2 min, and migration. then stained with 1% crystal violet. The filters were densitometrically analyzed using the OFOTO program (Light Source Computer Images, MATERIALS AND METHODS Inc.) and quantified by Scanner Analysis (Biosoft, Ferguson, MO). Reagents and Antibodies—Mouse laminin was obtained from Collab- orative Research (Bedford, MA). Human fibronectin and vitronectin RESULTS were generous gifts from Dr. Deane F. Mosher (University of Wisconsin, To explore the effects of EGF on cell migration, mouse B82L Madison, WI). Recombinant human EGF was purchased from Upstate fibroblasts that lack measurable endogenous EGF receptors Biotechnology Inc. (UBI, Lake Placid, NY). Nucleopore membranes and the 48-well chemotaxis chamber were purchased from Nucleopore Corp. (B82L-Parental) were transfected with wild-type EGF recep- (Pleasanton, CA). Anti-EGF receptor monoclonal antibodies were ob- tors (these include two separate transfectants designated as tained from the following sources: antibody 528 was affinity-purified B82L-wt and B82L-wt2) or mutant human EGF receptors. A from hybridoma cell line HB 8509 (ATCC, Rockville, MD), antibody cell clone possessing laminin binding activity was also isolated C225 was obtained from Imclone Systems, Inc. (New York, NY), and from B82L-wt cells (labeled as Clone B3) (42). The two EGF antibody LA22 was purchased from UBI. Neutralizing anti-mouse EGF receptor mutants used in these studies include a construct and anti-human TGF-a antibodies were purchased from UBI and R&D Systems (Minneapolis, MN), respectively. encoding a kinase-inactive receptor that contains a lysine to Cell Culture Conditions—Murine B82L-Parental fibroblasts and methionine substitution at residue 721 (B82L-K721M), which those expressing the wild-type or the K721M or c’1022 EGF receptor is involved in ATP binding, and a construct encoding a kinase- constructs were provided by Dr. Gordon Gill (University of California, active receptor that has been truncated at residue 1022 (B82L- San Diego, CA). Mouse B82L-Parental fibroblasts were maintained in c’1022), which lacks four major receptor autophosphorylation Dulbecco’s modified Eagle’s medium (DMEM) containing 10% cosmic sites. These cell lines were used to explore the involvement of calf serum (HyClone, Logan, UT). Cells transfected with wild-type or mutated EGF receptors were cultured in the same medium that con- EGF receptor activation in cell migration toward various ECM tained 10 mM methotrexate because a mutant dihydrofolate reductase components. gene was used as a selectable marker (41). The Clone B3 cells were Effect of EGF on B82L Fibroblast Spreading on ECM Sub- derived from B82L-wt cells based on their ability to bind laminin (42), strates—Cell migration is a process that requires temporally and were cultured in the same medium as the B82L-wt cells. The GD25 and spatially coordinated cell attachment and detachment (24, cells are differentiated and immortalized cells derived from the embry- onic stem cell clone G201, which are deficient in the integrin subunit b 25). Given the importance of adhesive strength between the cell as the result of the introduction of a null mutation in the b integrin and the substrate in determining the migration speed and gene via homologous recombination (43). The stably transfected cell line persistence, we tested whether EGF could influence the adhe- GD25-b was obtained by electroporating wild-type integrin b cDNA 1A 1A siveness of Clone B3 cells on several ECM substrates. As shown into GD25 cells. In the present study, the GD25 cells were cultured in in Fig. 1A, when Clone B3 cells were plated onto fibronectin- nonselection medium consisting of DMEM plus either 10% fetal calf coated wells in the presence of different levels of EGF, the serum (HyClone) or 10% cosmic calf serum, whereas the GD25-b cells 1A were continuously cultured in the same medium plus puromycin (10 ability of these cells to spread on the fibronectin surface was mg/ml) (43). Both the GD25 and the GD25-b cells were provided by Dr. attenuated in a time- and EGF dose-dependent manner. In the 1A Deane F. Mosher. Other cell lines used in this study, including human absence of EGF, nearly all the clone B3 cells were attached to mammary carcinoma cell line MDA468, human osteosarcoma cell line the fibronectin surface in the first 30 min. Within 1 h, the cells MG63 and human epidermal carcinoma cell line A431, were cultured in became flattened and exhibited the characteristic protrusive DMEM plus 10% fetal calf serum. 3T3-F442A fibroblasts were cultured structures of spreading cells, a process that was completed by in DMEM plus 10% bovine calf serum (HyClone). Cell Spreading Analysis—Cell culture plates (24-well) were coated 2.5 h after plating. However, upon the addition of EGF, cell EGF Receptor Regulation of Cell Migration 11211 initiation of cell spreading to approximately 2.5 h after plating. Because Clone B3 cells express receptors for other ECM proteins such as laminin and vitronectin, we examined the effects of EGF on Clone B3 spreading onto laminin or vitronec- tin-coated wells. Similar to the results shown in Fig. 1A, the addition of EGF was found to dose-dependently reduce the capacity of the cells to spread on all three substrates tested (Fig. 1B). It was also noted that the spreading of these mouse fibroblasts onto laminin- or vitronectin-coated wells was less extensive than that observed on fibronectin-coated surfaces. Polylysine was used as a negative control for cell spreading onto ECM components because cells attach to polylysine with- out the activation of integrin-associated processes (45). Inter- estingly, EGF treatment had no effect on the attachment of Clone B3 cells onto fibronectin and laminin, as measured by the number of cells adhered to these ECM substrates (data not shown). These results suggest that EGF does not substantially regulate the initial interaction between the cell surface inte- grins, but that the later events (e.g. focal contacts maturation and stress fiber formation) are more likely to be EGF-sensitive. Effect of EGF on Laminin-induced Chemotaxis of Clone B3 Fibroblasts—Based on the ability of EGF to modulate the ad- hesiveness of Clone B3 cells on various substrates, we investi- gated whether EGF could regulate the chemotactic capacity of Clone B3 cells using a 48-well chemotaxis chamber. As shown in Fig. 2A, EGF synergized with laminin in enhancing the migratory activity of these cells. This synergistic effect of EGF on cell chemotaxis was about 2–3-fold above that observed with laminin alone, whereas EGF alone had little or no effect on cell migration. Interestingly, the effect of EGF on laminin-induced chemotaxis could only be achieved when EGF was co-present with laminin in the lower wells. When EGF was mixed with the cells located in the upper wells, EGF was unable to augment laminin-induced chemotaxis. Because the development of cell polarization, such as the formation of distinct cellular leading and trailing edges, is essential for directional cell movement (24), our findings suggest that EGF, when co-present with laminin, may help to enhance the cell polarization required for directional cell movement by forming an EGF concentration gradient parallel to the laminin gradient. Conversely, when EGF is present with the cells in the upper wells, the cells were exposed to EGF from all directions, and thus may not be able to enhance a polarization process. Although several studies have shown that EGF can stimulate cell movement (14, 16 –18, 46), this appears to be the first report concerning the importance of EGF positioning in promoting directional cell movement. FIG.1. EGF attenuates the spreading of Clone B3 fibroblasts, To further test the hypothesis that EGF-induced cell polar- which contain the full-length active EGF receptor, on various ization is important in facilitating cell migration toward lami- ECM substrates. A, the time- and EGF dose-dependent spreading of nin, EGF was added to both the lower and upper wells, based Clone B3 fibroblasts on fibronectin. Cells were collected by trypsiniza- tion and allowed to attach and spread on wells coated with fibronectin on the rationale that the uniform exposure of cells to EGF (10 mg/ml) in the absence or presence of the specified concentrations of would disrupt the cell polarization induced by EGF present EGF as described under “Materials and Methods.” The cells were pho- with laminin in the lower wells. As shown in Fig. 2B, the tographed at the indicated times using 40X magnification. B, EGF maximal cell migration induced by the co-presentation of lami- dose-dependent spreading of Clone B3 fibroblasts on various ECM components. Cells were collected by trypsinization and allowed to at- nin and EGF (1 nM) in the lower wells was reduced in a tach and spread on wells coated with either fibronectin (10 mg/ml), dose-dependent fashion (33– 85%) when EGF (1–10 nM) was laminin (20 mg/ml), vitronectin (10 mg/ml), or the non-integrin-activat- also mixed with cells in the upper wells. These studies further ing protein polylysine (10 mg/ml) in the absence or presence of the support the concept that the stimulation of cells with a co- specified concentrations of EGF. At the end of a 2-h incubation, the cells were photographed (original magnification, 340). For both A and B, gradient of laminin and EGF facilitates cell polarization and analogous results were observed in three other experiments. efficient directional cell locomotion. EGF Effects on Cell Migration with Multiple Cell Lines and spreading was impaired, i.e. the cells remained in a rounded ECM Substrates—Clone B3 cells were isolated by their ability to bind laminin (42). To assess whether the effects of EGF on shape for a longer period of time and appeared to form fewer focal contacts as assessed by immunofluorescent staining of cell migration were restricted to laminin, we performed similar vinculin (data not shown). As also illustrated in Fig. 1A, the migration assays using fibronectin and vitronectin as the che- addition of 10 nM EGF delayed cell spreading until 1.5 h after moattractants. We found that EGF addition to the upper wells plating, whereas the presence of 50 nM EGF suppressed the had no measurable effects on fibronectin or vitronectin-induced 11212 EGF Receptor Regulation of Cell Migration FIG.3. EGF stimulates the migration of Clone B3 and B82L-wt cells toward multiple ECM components. Cell migration was deter- mined using a 48-well microchemotaxis chamber as discussed under “Materials and Methods.” Clone B3 fibroblast migration in response to laminin (200 mg/ml), fibronectin (100 mg/ml) (A), or vitronectin (100 mg/ml) (B) was measured in the absence or presence of EGF in the lower wells (co-positioned with the chemoattractant). C, the migration of B82L-wt fibroblasts toward fibronectin (100 mg/ml) or vitronectin (100 mg/ml) was assessed in the absence or presence of EGF in the lower wells (co-positioned with the chemoattractant). For each experiment, the bars represent the mean (6 S.D.) of triplicate determinations. FIG.2. The synergistic action of EGF in promoting laminin- Similar results were obtained in at least three different experiments. induced migration of Clone B3 fibroblasts. Cell migration toward laminin was determined using a 48-well microchemotaxis chamber as discussed under “Materials and Methods.” In the experiments using cells toward fibronectin or vitronectin in the absence or pres- laminin as the chemoattractant, 200 mg/ml laminin was placed in the lower wells. A, EGF is required to be co-positioned with laminin to ence of EGF were almost completely blocked by RGDS peptides stimulate chemotaxis. EGF (0 –10 nM) was mixed either with the lami- but not by the control peptide SDGRG, whereas laminin-in- nin in the lower wells or with B82L-Clone B3 fibroblasts in the upper duced migration was not affected by RGDS peptides at the wells. Cell motility induced by EGF alone (placed in lower wells) was same concentrations (data not shown). This observation is con- also measured. B, the direct addition of EGF to the cells reduces the maximal migration conferred by the EGF co-positioned with laminin. In sistent with the previous finding that, although the RGD se- the indicated cases (filled circles),1nM EGF was added to the lower quence is present in the A chain of murine laminin, it is cryptic, wells, whereas the EGF concentration in the upper wells was varied and therefore not accessible in native laminin (47, 48). In from0to10nM. The cell migration to laminin alone (square)orinthe addition, the laminin receptor on Clone B3 cells mediating absence of both EGF and laminin (triangle) was also measured. In all motility has been identified to be a -containing integrins (42), cases, the data points represent the mean 6 S.D. of triplicate determi- nations. Similar results were obtained in at least five different whose recognition sites on laminin are different from the RGD- experiments. containing region (49). These results support the concept that fibronectin- or vitronectin-induced chemotaxis of Clone B3 cells chemotaxis (data not shown); however, when EGF was mixed is mediated by integrins. with fibronectin or vitronectin in the lower wells, EGF substan- To verify that the EGF stimulation of chemotaxis is not tially elevated Clone B3 cell migration (Fig. 3, A and B). More- limited to B82L cells, we examined the chemotactic behavior of over, because Clone B3 cells were isolated from B82L fibro- another mouse fibroblastic cell line in response to EGF treat- blasts that were transfected with the human EGF receptor ment, namely GD25 cells. These cells were derived from the (B82L-wt) (42), we compared the chemotactic capacity of Clone mouse embryonic stem cell clone G201, which are deficient in B3 and B82L-wt cells in order to ascertain whether EGF stim- the integrin subunit b due to the introduction of a null muta- ulation of cell migration reflected clonal variation. We observed tion in the b integrin gene. In addition, we evaluated a related that both cell types exhibited comparable responses to EGF, cell line, i.e. GD25-b cells, which were derived from GD25 1A fibronectin, and vitronectin (Fig. 3C). cells that were transfected with the integrin b gene and have 1A The arginine-glycine-aspartate (RGD) sequence on many laminin binding ability. Both cell lines attach to fibronectin matrix proteins, including fibronectin and vitronectin, has and form focal contacts that contain a b integrins (43). As v 3 been shown to interact directly with cell surface receptor inte- shown in Fig. 5, we observed that the migration of both GD25 grins (34). Synthetic RGDS peptides can block such matrix- and GD25-b cells toward fibronectin (Fig. 5A) and vitronectin 1A integrin interactions. To identify whether ECM-induced che- (Fig. 5B), and of GD25-b cells toward laminin (Fig. 5A), was 1A motaxis was mediated by integrins, Clone B3 cells were mixed stimulated by the addition of EGF (1 nM) to the lower wells. with RGDS peptides before they were placed into the chemo- There was no detectable difference between the two cell lines in taxis chamber. As shown in Fig. 4, the migration of Clone B3 terms of their migration toward ECM components and their EGF Receptor Regulation of Cell Migration 11213 FIG.4. The fibronectin- and vitronectin-induced chemotaxis of Clone B3 fibroblasts is dependent on the broadly selective integrin-binding sequence RGDS. The cells were mixed with RGDS peptides at the indicated doses before they were loaded into the microchemotaxis chamber. Cell migration toward fibronectin (100 mg/ml) (A) or vitronectin (100 mg/ml) (B) in the absence or presence of EGF (1 nM) in the lower wells (co-positioned with the chemoattractants) was determined as described under “Materials and Methods.” Each point represents the mean 6 S.D. of triplicate determinations. Comparable results were obtained in three different experiments. EGF responses. Moreover, in all other cell lines tested, as long EGF receptor overexpression may lead to ligand-independent as the cells were able to migrate toward a given ECM compo- activation of the EGF receptor. However, in our system, we nent, EGF could further enhance their migration when it was found that the EGF receptor is expressed at ;100,000 mole- co-positioned with the ECM component, and had no effect when cules/cell (42), which is within the physiological range, suggest- mixed with the cells. Specifically, these cell lines include the ing that the EGF receptor in B82L cells is not likely to be 3T3-F442A murine fibroblasts, the human osteosarcoma cell activated due to the overexpression of the receptor. A second line MG63, the human mammary carcinoma cell line MDA468, possibility, which may be of physiological significance, is that and the human epidermal carcinoma cell line A431 (data not the cells may secrete an EGF receptor ligand such as EGF or shown). The broad spectrum of cell lines that are able to re- TGF-a, which in turn stimulates fibronectin-induced migration spond to EGF in terms of their migration toward ECM compo- in an autocrine manner. Although this feature would still nents suggests that the synergistic effect of EGF on chemotaxis strongly support the notion that the EGF receptor is critical for is a general phenomenon. cell migration, it was important to assess if this mechanism EGF Receptor Expression Is Critical for Fibronectin-induced was occurring in our system. To examine this possibility, anti- Cell Migration—To identify EGF receptor elements that are bodies that can neutralize the most common EGF receptor necessary for mediating the stimulatory effects of EGF on ligands, EGF and TGF-a, were used to assess whether they integrin-mediated chemotaxis, B82L-Parental cells, which con- could reduce the migration of Clone B3 cells toward fibronectin. tain no detectable endogenous EGF receptors, were used as a As shown in Fig. 6C, neither anti-EGF nor anti-TGF-a anti- negative control for cells transfected with either wild-type or bodies, alone or together, at the concentrations tested, exhib- mutated EGF receptors. B82L-Parental cells have been shown ited any effects on the fibronectin-induced migration of Clone to adhere and spread on fibronectin-coated surfaces (42), indi- B3 cells. These results suggest that the capacity of B82L cells cating that they express functional fibronectin receptors. Al- expressing the EGF receptor to migrate toward fibronectin though it was predicted that the B82L-Parental cells would alone does not appear to result from an EGF or TGF-a auto- migrate toward fibronectin but not show EGF responses due to crine activation of the EGF receptor. the lack of EGF receptor expression, we instead unexpectedly The EGF Receptor Kinase Activity and C Terminus Are Im- observed that these cells exhibited little detectable migration portant for Conferring Fibronectin-induced Chemotaxis—Upon toward fibronectin (6 EGF) (Fig. 6, A and B). In addition, cell EGF binding, EGF receptor autophosphorylation is linked to a migration was not observed, even when the concentrations of cascade of signaling events leading to cellular responses such fibronectin and EGF were varied from 50 to 200 mg/ml and from as cell proliferation and cell motility (50). Both the receptor 0.1 nM to 10 nM, respectively (data not shown). However, the kinase activity and the C terminus, which provides docking introduction of functional EGF receptors into these cells clearly sites for SH2 and PTB domain-containing cellular proteins, are enabled them to migrate toward fibronectin alone (B82L-wt or critical for EGF-induced signal transduction (1). Chen et al. Clone B3) (Fig. 6A; also see Figs. 2 and 3). To evaluate whether (17, 18) have shown that EGF-elicited random cell movement these responses were unique to the B82L-wt transfectant and requires receptor tyrosine kinase activity and autophosphoryl- Clone B3 cells, we generated a separate population of trans- ation. In order to map EGF receptor regions that are involved fected B82L cells expressing wild-type EGF receptors, which in the synergistic stimulation of EGF on integrin-mediated we termed B82L-wt2. As shown in Fig. 6B, B82L-wt2 cells were motility, B82L-Parental cells were transfected with EGF recep- also capable of migrating toward fibronectin, albeit at a lower tors that are kinase-inactive (B82L-K721M) or C-terminally level than that observed with B82L-wt cells. These observa- truncated (B82L-c’1022). As shown in Fig. 7A, B82L-K721M tions indicate that the introduction of the wild-type EGF re- cells did not migrate toward fibronectin alone, nor did they ceptor into B82L fibroblasts enable them to switch from a respond to EGF stimulation. Similar results were obtained non-migratory cell type into a migratory one. with B82L cells expressing EGF receptors that retain only one In these studies, it is conceivable that the necessity of the of the five C-terminal autophosphorylation sites (c’1022) (Fig. EGF receptor for B82L cells to migrate toward fibronectin 7B), although these cells exhibit increased kinase activity due alone may reflect an artifact of EGF receptor transfection, e.g. to the removal of the C-terminal autoinhibitory restraint (51, 11214 EGF Receptor Regulation of Cell Migration FIG.5. EGF synergistically stimulates the migration of GD25 and GD25-b murine fibroblasts toward multiple ECM substrates. 1A Cell migration was determined using a 48-well microchemotaxis chamber as discussed under “Materials and Methods.” The migration of GD25 and GD25-b fibroblasts toward medium alone, EGF (1 nM) alone, or laminin (100 mg/ml) (A), fibronectin (100 mg/ml) (B), and vitronectin (100 mg/ml) 1A (C) in the absence or presence of EGF (1 nM), in the lower wells (co-positioned with the chemoattractant) was measured. Each bar represents the mean (6 S.D.) of triplicate determinations. Similar results were obtained in three different experiments. 52). The observation that the migratory behavior of K721M or of neutralizing anti-EGF receptor antibodies on Clone B3 cell c’1022 cells are indistinguishable from the Parental cells sug- migration. Monoclonal anti-EGF receptor antibodies (LA22, gest that both the receptor tyrosine kinase activity and the 528, and C225) were used that recognize the EGF binding site, C-terminal region are important for the EGF-induced enhance- compete for EGF binding, and block EGF-induced receptor ment of cell motility and for the chemotaxis induced by fi- autophosphorylation. As shown in Fig. 8 (A and B), neutraliz- bronectin alone. In addition, Tyr-992, which has been shown to ing anti-EGF receptor antibodies inhibited not only EGF stim- be sufficient to confer EGF-elicited random cell movement (17), ulation of fibronectin-induced cell migration, but also cell mi- does not appear to be sufficient to support EGF-stimulated gration induced by fibronectin alone. This effect appeared to be chemotaxis, suggesting that EGF regulation of chemokinesis overcome as the EGF concentration was increased. These re- and chemotaxis may involve different signaling mechanisms. sults support the hypothesis that the EGF receptor may par- Effects of Neutralizing Anti-EGF Receptor Antibodies on Cell ticipate, at least in part, as a downstream mediator in the Migration—Our studies have shown that the expression of integrin-stimulated signaling pathways leading to cell motility. functional EGF receptors is critical for the induction of motility This concept is consistent with the studies of Klemke et al. (14), in B82L fibroblasts. These results suggest that the EGF recep- where the EGF-selective inhibitor tyrphostin 25 blocks human tor may be used as a downstream mediator by integrins in carcinoma cell migration on vitronectin. transducing signals leading to cell migration. Such ligand- When the effects of anti-EGF receptor antibodies on laminin- independent activation of the EGF receptor by other receptor induced cell migration were examined, we observed that the systems has been reported for G-protein-coupled-receptor in addition of monoclonal anti-EGF receptor antibodies LA22 and Rat-1 cells (53). Conversely, the EGF receptor could act up- 528 to the cells not only reversed EGF stimulation of laminin- stream of integrins, for example, by regulating integrin expres- induced chemotaxis, but also blocked cell migration toward sion, activation or coupling to key components involved in cell laminin alone (Fig. 8, C and D). As also shown in Fig. 8D,a motility. To further explore this system, we examined the effect relatively weaker inhibition of cell migration was observed EGF Receptor Regulation of Cell Migration 11215 FIG.6. Expression of wild-type EGF receptors is required for B82L fibroblasts to migrate toward fibronectin. In panels A and B, B82L-Parental cells expressing no endogenous EGF receptor were transfected on two separate occasions with the wild-type (full-length) EGF receptor designated wt (A) and wt2 (B). Clone B3 cells were used as positive control for fibronectin-induced chemotaxis in response to EGF. The migration of B82L cells toward medium alone, EGF alone (1 nM), fibronectin (100 mg/ml) alone, fibronectin (100 mg/ml) and EGF (1 nM) co-positioned in the lower wells, or fibronectin in the lower well and EGF in the upper well (co-positioned with the cells), was measured. In panel C, the effects of neutralizing anti-EGF or anti-TGF-a antibodies on the fibronectin-induced migration of B82L-Clone B3 cells was determined. Clone B3 cells were incubated with anti-EGF and/or anti-TGF-a monoclonal antibodies at the indicated concentrations (legend at right) (for 0.5 h before they were loaded to the migration chamber. Each bar represents the mean 6 S.D. of triplicate determinations. Similar results were obtained in two different experiments. when the anti-EGF receptor antibodies were put into the lower These results appear to be specific because only anti-EGF wells with laminin. This may result from the lower effective receptor antibodies could block cell motility induced by laminin antibody concentration available to the cells due to diffusion alone whereas nonspecific IgG had no effect (Fig. 8D). Trypan and dilution of the antibodies upon entry into the upper wells. blue exclusion assays showed that all the cells treated with or 11216 EGF Receptor Regulation of Cell Migration without the antibodies exhibited similar viability (data not shown), indicating that the inhibition of laminin-induced cell motility by anti-EGF receptor antibodies does not appear to be due to cell death. DISCUSSION In the present study, we demonstrate that EGF can potently stimulate the motility of mouse B82L fibroblasts that express a full-length EGF receptor. Furthermore, these investigations reveal that the co-positioning of EGF and the chemoattractant is critical for EGF stimulation of cell migration, i.e. it is essen- tial that the cell is exposed to an EGF and substrate gradient oriented in the same direction. This EGF-dependent stimula- tion of cell motility may in part be due to the cell rounding and the reduction of cell spreading and focal adhesion formation caused by EGF treatment. Another major observation made in these studies is that a fully functional EGF receptor appears critical for integrin-mediated migration, in particular, the EGF receptor C-terminal domain and an intact kinase domain are critical for fibronectin and fibronectin plus EGF-stimulated cell migration. In this regard, it is noteworthy that B82L-Parental cells that do not express the EGF receptor exhibit little fi- bronectin-induced chemotaxis, whereas the introduction of the wild-type EGF receptor enables these cells to now undergo fibronectin-induced migration. Moreover, the inhibition of fi- bronectin or laminin-induced chemotaxis by neutralizing anti- EGF receptor antibodies supports the concept that the EGF receptor may act downstream of integrin activation. Many studies have shown that cell migration is a process that requires dynamic interactions between the cell, the sub- strate, and the cytoskeleton-associated motile apparatus (24, 25). Reduced cell motility can result if cell-substratum interac- tions conferred by growth factor/cytokine (32, 33) or anti-inte- grin antibody stimulation (28, 29), as well as by modulation of the levels of substrates (26, 27), integrins (30, 31), or cytosketal proteins present at focal contacts are too strong or too weak. These observations are consistent with earlier findings that EGF can induce rapid cell rounding (9), membrane ruffling and retraction (8), and the promotion of extensive cortical actin polymerization and depolymerization of actin stress fibers (10, 11). The present studies suggest that an EGF-induced reduc- tion of adhesiveness may augment the motility of B82L fibro- blasts expressing functional EGF receptors as well as the mo- bility of other EGF-responsive cell types. The synergistic action of EGF and matrix proteins to en- hance fibroblast motility suggests that an interaction exists between the signaling pathways triggered by the activation of these two receptor systems. One possible site of convergence could be at the level of the tyrosine kinase Src, which has been reported to modulate EGF-induced mitogenesis (54), actin-cy- toskeleton reorganization (via the small molecular weight G- protein Rho) (55), and integrin-initiated signal transduction. Of note, the transformation of chicken embryo fibroblasts by the Rous sarcoma virus is associated with a general loss of sub- stratum adhesion, changes in cell shape, and the disorganiza- tion of the cytoskeleton (56), all of which are similar to those observed in EGF-treated cells. Moreover, the integrin b sub- unit is tyrosine-phosphorylated in v-Src-transformed cells, and the highly homologous integrin b subunit has been reported to be tyrosine-phosphorylated (57) and to bind the Shc PTB do- FIG.7. The EGF receptor tyrosine kinase activity and the C- terminal region are important for conferring fibronectin-in- duced chemotaxis in B82L fibroblasts. B82L-Parental cells ex- pressing no endogenous EGF receptor were transfected with an EGF of B82L cells toward medium alone, EGF alone (1 nM), fibronectin (100 receptor that carries a lysine to methionine substitution at position 721, mg/ml) alone, fibronectin (100 mg/ml) and EGF (1 nM) co-positioned in which abolishes the receptor kinase activity (K721M) (A), or a kinase the lower wells, or toward fibronectin placed in the lower well and EGF active EGF receptor that has been C-terminally truncated from residue added to the upper well (co-positioned with the cells), was measured. 1022 through residue 1186 (c’1022) (B). This mutant lacks four of the Each bar represents the mean (6 S.D.) of triplicate determinations. major EGF receptor tyrosine autophosphorylation sites. The migration Similar results were obtained in three different experiments. EGF Receptor Regulation of Cell Migration 11217 FIG.8. Neutralizing anti-EGF receptor antibodies inhibit migration of B82L-Clone B3 cells toward fibronectin or laminin. A, cell migration toward fibronectin (50 mg/ml) alone, or fibronectin (50 mg/ml) together with different doses of EGF (0 –1 nM) was measured. Cells were incubated with anti-EGF receptor monoclonal antibodies LA22 (10 or 100 nM) for 0.5 h before they were loaded to the migration chamber. B, cell migration toward fibronectin alone (0 –200 mg/ml) was measured. Cells were incubated with anti-EGF receptor monoclonal antibodies LA22 (30 nM), 528 (30 nM), or C225 (30 nM) for 0.5 h before they were loaded to the migration chamber. C, cell migration toward laminin (200 mg/ml) alone, or laminin (200 mg/ml) together with different doses of EGF (0 –1 nM) was measured. Cells were incubated with anti-EGF receptor monoclonal antibodies LA22 (10 or 100 nM) for 0.5 h before they were loaded to the migration chamber. D, cell migration toward laminin (200 mg/ml) alone, or laminin (200 mg/ml) and EGF (1 nM) together was measured. Anti-EGF receptor monoclonal antibodies 528, LA22, or nonspecific IgG at 10 nM were added either to the cells in the upper wells or to the laminin in the lower wells. LA22, 528, and C225 can compete with EGF for the EGF receptor binding and block EGF-induced autophosphorylation of the EGF receptor. Each point represents the mean 6 S.D. of triplicate determinations. Similar results were obtained in two different experiments. main upon platelet aggregation (58). Tyrosine-phosphorylated rylated integrins from focal contacts, thereby reducing cell b integrin subunit exhibits a decreased ability to bind to both adhesiveness and enhancing cell motility. fibronectin and talin (59), implying that the phosphorylated b The observation that a non-polarized addition of EGF to the subunit may be able to escape from focal contacts. The release cells does not affect cell migration (Fig. 2) suggests that a of b integrins from focal adhesions may result in the disrup- generalized EGF-induced attenuation of cell adhesion strength tion of focal contacts, and a reduction of adhesion strength that is not sufficient to fully explain EGF action. EGF increases cell allows the cell to enter a motile state. In fact, certain Tyr to Phe migration toward matrix proteins only when EGF and the mutations in the b cytoplasmic domain results in the loss of chemoattractant are co-presented to the cells at the same time cell motility (60). Although the b subunit could be a direct and in the same direction. The exposure of the cell to EGF and substrate of Src, it is also likely that the phosphorylation of b chemoattractant gradients originating from the same direction is mediated by other kinases such as focal adhesion kinase is likely to augment the cell polarization known to be necessary (FAK), which has been shown to bind a peptide mimicking the for directional cell movement (24). When EGF is added directly b cytoplasmic domain (61). Additionally, Src and the Src fam- to the cells, cell polarization can be generated only by the ily kinase Fyn are known to form stable complexes with FAK chemoattractant and not by an EGF gradient, and thus EGF through phosphotyrosine-SH2 domain interactions, thereby does not increase cell migration. bringing these kinases into focal contacts (62– 64). Src can also EGF-enhanced cell polarization may occur in several non- phosphorylate FAK at additional sites to fully activate its ty- exclusive ways. For example, a high local EGF concentration at rosine kinase activity (65, 66). Therefore, as a downstream the leading edge of a migrating cell may facilitate the induction mediator of both EGF receptor and integrin activation, Src or of various active membrane processes. EGF is known to pro- Src family kinases may integrate signals from both receptor mote F-actin redistribution (10, 11) and the formation of lamel- systems, thus leading to events such as the release of phospho- lipodia and filopodia (8, 9), which are processes occurring at the 11218 EGF Receptor Regulation of Cell Migration leading edge of migrating cells (24). In addition, EGF may growth factor receptors such as the PDGF receptor and the induce integrin association/interaction with the EGF receptor insulin receptor substrate (IRS-1) upon PDGF or insulin treat- at the cell leading edge, and thus generate an asymmetric ment (58, 67, 73) has been reported. The present study provides distribution of integrins when the cells are co-exposed to an evidence for an EGF receptor-integrin system interaction, EGF gradient. Growth factors such as PDGF (58) and insulin which is consistent with earlier observations that the EGF (67) have been reported to induce the association of integrins receptor interacts directly with b-catenin, a vinculin homol- (such as a b ) with these growth factor receptors. Furthermore, ogous protein involved in cell-cell adhesion (74), and that v 3 as integrins engaged in cell migration become clustered at the EGF receptors are accumulated and potentially activated by leading edge in the form of macroaggregates (68, 69), EGF integrins (45, 70). receptors may co-cluster with the integrins at the cell leading Inactivation of the EGF receptor kinase activity by replacing edge when the cells are exposed to a chemoattractant gradient. Lys-721 with Met reduces EGF-induced tyrosine phosphoryla- The idea that the clustering of EGF receptors by activated tion of cellular proteins, including phospholipase C-g, and in- integrins could be involved is supported by the observation that hibits EGF-induced receptor internalization. Although cells ex- an accumulation of EGF receptors occurs on fibronectin-coated pressing this tyrosine kinase negative EGF receptor still bind beads where integrin interaction with fibronectin takes place EGF with the same affinity, and exhibit EGF-induced MAP (45). Moreover, a recent report by Moro et al. (70) indicates that kinase activation (75), EGF-stimulated cell proliferation is b integrin stimulation can enhance EGF receptor tyrosine blocked (76). Our study indicates that an intact kinase domain phosphorylation. The consequences of this EGF receptor distri- appears essential for cell migration as well. The inability of this bution/activation on the leading edge of the cell surface may kinase-inactive mutant to mediate cell motility may reside in then be further amplified by the higher concentration of EGF at the transient nature of EGF-induced MAP kinase activation the cell front when cells are exposed to an EGF gradient in (75), which has been shown to be important for focal adhesion addition to a chemoattractant gradient. The enriched distribu- disassembly (77), and/or in the blockage of activation of phos- tion of activated EGF receptors at the leading edge may trigger pholipase C-g, which has been shown to be important for me- asymmetric phosphorylation of cellular proteins resulting in an diating random cell movement (17, 18) and haptotaxis (14). augmentation of the cell polarization induced by chemoattrac- Unlike the kinase-negative mutant, the c’1022 mutant EGF tant alone. This hypothesis is consistent with the observation receptor mediates enhanced tyrosine phosphorylation of mul- that tyrosine-phosphorylated proteins are found at the tips of tiple cellular substrates (39), it undergoes normal receptor mobilization (78), and it can be ty- growth cone filopodia and that this process is correlated with internalization and Ca the length of the filopodia (71). rosine-phosphorylated (39) upon EGF treatment. The observa- Another critical observation of this study is that B82L fibro- tion that c’1022-expressing cells cannot migrate suggest that blasts appear to require an intact EGF receptor to efficiently an SH2- or PTB domain-containing protein that binds to the migrate toward fibronectin alone. B82L-Parental cells contain C-terminal autophosphorylation sites is involved in mediating functional fibronectin receptors in that they are able to adhere signals in the motility pathway, or that the altered substrate to a fibronectin-coated surface (42) yet they are essentially specificity of this mutant may modify the activity of key effec- non-migratory. Interestingly, the introduction of the wild-type tors that regulate the motility pathway. EGF receptor into these cells converts them from a non-migra- One additional issue that is related to the present work is tory cell type into a migratory one. Although we cannot rule out that the inhibition of fibronectin- and laminin-induced chemo- the possibility that the transfection of intact EGF receptor may taxis by function-blocking anti-EGF receptor antibodies could alter the expression of certain proteins that are critical for cell arise from EGF contamination of the ECM preparations, i.e. motility, the present investigations support the concept that EGF accounts for part of the ECM-induced chemotaxis. Al- the EGF receptor can act downstream of integrin activation though this scenario would still strongly support a key role for and serves as a key component of the motility machinery of the EGF receptor system in B82L cell migration, the argument B82L fibroblasts. In this regard, if the EGF receptor is not of EGF contamination is not supported by the observations that directly involved in cell migration, the blockade of EGF recep- EGF alone, at all the concentrations tested, induced little che- tor function should not abolish cell migration ability, yet anti- motaxis, that EGF was not detected in the laminin prepara- EGF receptor antibodies can block cell migration toward fi- tions as assessed by immunoblotting using anti-EGF and bronectin alone. TGF-a antibodies, and that anti-EGF or TGF-a antibodies do Altogether, these observations suggest that the EGF recep- not alter fibronectin-induced chemotaxis. A related issue is tor may be activated in an EGF-independent manner upon that because laminin contains EGF-like motifs and it may bind fibronectin engagement and that the EGF receptor plays a EGF receptor to induce cell migration, and that the anti-EGF direct role in the migration of these cells. This situation may be receptor antibodies may block laminin-induced chemotaxis by analogous to that seen in other systems, i.e. the EGF receptor abolishing laminin binding to the EGF receptor. However, this has been reported to be activated by G-protein-coupled recep- is not likely because intact laminin (79) and a peptide encom- tors (53) and by UV light (72). Moreover, Miyamoto et al. (45) passing the EGF-like fragment of laminin (80) cannot compete and Moro et al. (70) have shown an interaction between inte- with EGF receptor binding. Thus, the data are most indicative grins and growth factors for triggering the tyrosine phospho- of a system wherein anti-EGF receptor antibodies eliminate rylation of the EGF receptor under various conditions. Al- laminin-induced chemotaxis by binding to the EGF receptor though this kind of cross-talk between the EGF receptor and and antagonizing the receptor dimerization/activation, thereby integrins may be accomplished by intervening proteins such as suggesting that EGF receptor function is directly important for Src or FAK (discussed above), it may also occur directly be- ECM-induced chemotaxis. tween the two receptors. The two NPXY motifs in the cytoplas- In summary, EGF can exert a synergistic effect on integrin- b subunit are homologous to the mediated chemotaxis, and this process requires the co-expo- mic domain of the integrin tyrosine autophosphorylation sites on the EGF receptor (1, 34), sure of the cell to both an EGF and an ECM substrate gradient. indicating that the b integrin and the EGF receptor may We also provide evidence to support the concept that the EGF interact directly or may be modulated by a common system. receptor can act downstream of integrin activation and may Similarly, a physical association between integrin a b and play a direct role in cell migration toward matrix proteins. v 3 EGF Receptor Regulation of Cell Migration 11219 34. Hynes, R. O. (1992) Cell 69, 11–25 These events are not only likely to be critical for both normal 35. Sastry, S. K., and Horwitz, A. F. (1993) Curr. Opin. Cell Biol. 5, 819 – 831 growth and development (e.g. embryogenesis, organ develop- 36. 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Published: Apr 1, 1999