Abstract
Epidermal growth factor stimulates migration of a number of cell types, yet the signaling pathways that regulate epidermal growth factor-stimulated migration are poorly defined. In this report, we employ a transient transfection migration assay to assess the role of components of the Ras-mitogen-activated protein (MAP) kinase signaling pathway in epidermal growth factor-stimulated chemotaxis of rat embryo fibroblasts. Expression of dominant negative Ras blocks epidermal growth factor-mediated chemotaxis, while constitutively active Ras has no effect on chemokinesis or chemotaxis. PD98059 and U0126, inhibitors of MAP kinase kinase (MEK) activity, decreased epidermal growth factor-stimulated migration, while kinase-defective MEK1, an inhibitor of MAP kinase activation, enhanced migration. To understand the paradoxical effects of these molecules on epidermal growth factor-induced migration, we examined the role of c-Raf on migration. Expression of either wild type c-Raf or the catalytic domain of c-Raf effectively inhibited epidermal growth factor-stimulated cell migration. We suggest that, whereas Ras activity is necessary to promote epidermal growth factor-stimulated migration, sustained activation of c-Raf may be important in down-regulating migratory signaling pathways triggered by epidermal growth factor receptor activation. Further, activation of c-Raf upon inhibition of the MEK-MAP kinase pathway may contribute to the inhibition of cell migration observed with pharmacological MEK inhibitors.
Highlights
Regulated cell migration is physiologically important for embryonic development, wound healing, and immunological responses associated with inflammation
Cells treated with PI3K inhibitors wortmannin or LY294002, failed to inhibit EGFstimulated migration (Table I) at concentrations that blocked PDGF-stimulated migration
We provide evidence that epidermal growth factor (EGF)-stimulated migration of REF52 cells is differentially regulated by Ras and its downstream effector, c-Raf
Summary
Reagents—REF52 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (Life Technologies, Inc.), 10 g/ml penicillin (Life Technologies, Inc.), and 0.25 g/ml streptomycin (Life Technologies, Inc.). Between 24 and 48 h after the addition of DNA, the transfection efficiency was determined by calculating the ratio of GFP-positive cells to total cells (counted in four random (magnification, ϫ320) fields using a Ziess Axiovert 135TV inverted fluorescence microscope). Migration of transfected cells was assessed using the Boyden chamber assay described below. Cells transfected with individual test constructs and GFP were stained with monoclonal antibodies to the peptide tag expressed on each ectopically expressed protein (12CA5 for HA-tagged Ras and MEK, M2 (Sigma) for FLAG-tagged c-Raf) at a final concentration of 1.5–5 g/ml. The number of cells expressing both green (GFP) and red (test protein) fluorescence was determined by counting four random 400ϫ magnification fields using a Leitz DMR fluorescence microscope.
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