Receptor Tyrosine Kinase Signaling Required for Integrin c v / 35-directed Cell Motility but Not Adhesion on Vitronectin

FG human pancreatic carcinoma cells adhere to vitronectin using integrin o~vB5 yet are unable to migrate on this ligand whereas they readily migrate on collagen in an ot2/31-dependent manner. We report here that epidermal growth factor receptor (EGFR) activation leads to de novo av/35-dependent FG cell migration on vitronectin. The EGFR specific tyrosine kinase inhibitor tyrphostin 25 selectively prevents EGFR autophosphorylation thereby preventing the EGF-induced FG cell migration response on vitronectin without affecting constitutive migration on collagen. Protein kinase C (PKC) activation also leads to otv/55-directed motility on vitronectin; however, this is not blocked by tyrosine kinase inhibitors. In this case, PKC activation appears to be associated with and downstream of EGFR signaling since calphostin C, an inhibitor of PKC, blocks FG cell migration on vitronectin induced by either PKC or EGE These findings represent the first report implicating a receptor tyrosine kinase in a specific integrin mediated cell motility event independent of adhesion. C ELL migration plays a critical role in a variety of biological processes including embryonic development, angiogenesis, wound repair, and tumor cell metastasis. Though the detailed mechanisms of cell migration are poorly understood, it is clear that adhesion events are crucial to this process. The adhesive interactions between cells and the extracellular matrix are mediated by integrins, a family of cell surface receptors that bind to extracellular matrix proteins including vitronectin, collagen, and fibronectin (Hynes, 1992). While many cells express integrin receptors and are capable of adhesion their capacity to promote cell motility is likely to be regulated independently by growth factors or cytokines. In fact, cytokines such as epidermal growth factor (EGF) and insulin-like growth factor one (IGF-1)' induce cell motility of a wide variety of cells (Stracke et al., 1989; Stoker and Gherardi, 1990; Chen et al., 1993; Matthay et al., 1993). However, the mechanism regulating cytokine-induced cell motility has not been clearly defined. FG human carcinoma cells provide a useful model for assessing the role of physiological factors that regulate cell migration. Specifically, FG cells attach to vitronectin using integrin t~v~5 yet are unable to migrate on this ligand whereas they are competent to migrate on collagen in an ot2~l-dependent manner (Leavesley et al., 1992). These findings imply that FG cell motility on collagen is regulated Address all correspondence to D. A. Cheresh, The Scripps Research Institute, Dept. of Immunology, 10666 N. Torrey Pines Rd., La Jolla, CA 92037. Tel. : (619) 554-8281. Fax: (619) 554-6705. 1. Abbreviations used in this paper: FBM, fibroblast basal medium; IGF-1, insulin-like growth factor one; PKC, protein kinase C. by intrinsic intracellular events whereas ore/~5-dependent migration on vitronectin requires an exogenous stimulus. In this report, we provide direct evidence that the EGF receptor (EGFR) stimulates a protein kinase C-dependent signaling pathway leading to the induction of c~v/~5-dependent FG cell migration on a vitronectin substrate. These findings provide the first link between the activation of a tyrosine kinase receptor, protein kinase C, and a specific integrin-mediated motility event. Moreover, evidence is presented that this signaling pathway leads to otv/~5-directed motility but not adhesion to vitronectin. Materials and Methods Cells and Cell Culture FG human pancreatic carcinoma cells were provided by Dr. Shama Kajiji and Vito Quaranta (The Scripps Research Institute, La Jolla, CA). FG-B is a subline stably transfected with a full-length eDNA encoding human/~3 gene and, thus, expresses functional c~v/33 (Leavesley et al., 1992). All cells were grown in RPMI 1640 (GIBCO BRL, Gaithersburg, MD) supplemented with 10% FBS and 50 izg/ml geatamycin and were free from mycoplasma during these studies. Before testing all cells were starved for 24 h by replacing serum containing culture media with FBS-free RPMI. Monoclonal Antibodies and Reagents Integrin-spexific mAbs P3G2 (anti-c~v~/5; Wayner et al., 1991), 6F1 (anti~2/~1; Coller et al., 1990), LM609 (anti-otv~3; Cheresh and Spiro, 1987), P4CI0 (anti-~l; Carter et al., 1990), and AP3 (anti-/~3; Dr. P. Newman, Blood Center of Southwestern Wisconsin, Milwaukee, WI) 661 (antivitronectin, Nip et al., 1992) were purified from ascites on protein A-Sepharos¢. MAbs to the epidermal growth factor and insulin-like growth factor receptors were obtained from Oncogene Science (Uniondale, NY) and PY20 Coiotinylated anti-phosphotyrosine) was from ICN Biomedicals, © The Rockefeller University Press, 0021-9525/94/11/859/8 $2.00 The Journal of Cell Biology, Volume 127, Number 3, November 1994 859-866 859 on A uust 8, 2017 jcb.rress. org D ow nladed fom Inc. (Costa Mesa, CA). Horseradish peroxidase-conjugated strepavidin/biotin complex was from Amersham Corp. (Arlington Heights, IL). Tyrphostin 25 ([3,4,5-trihydroxyphenyl]-methylene propanedinitfile) was purchased from Calbiochem (San Diego, CA). Tyrphostin 63 ([4-hydroxybenzyl] malonitxile) an analogue that does not inhibit EGFR kinase activity was obtained from LC Laboratories (Woburn, MA). Phorbol, 12-myristate, 13-acetate (PMA), genistein, and calphostin C were acquired from Calbiochem. Human recombinant EGF and IGF-1 were obtained from Genzyme (Cambridge, MA).