Abstract
Activation of the Met receptor tyrosine kinase through its ligand, hepatocyte growth factor, stimulates cell spreading, cell dispersal, and the inherent morphogenic program of various epithelial cell lines. Although both hepatocyte growth factor and epidermal growth factor (EGF) can activate downstream signaling pathways in Madin-Darby canine kidney epithelial cells, EGF fails to promote the breakdown of cell-cell junctional complexes and initiate an invasive morphogenic program. We have undertaken a strategy to identify signals that synergize with EGF in this process. We provide evidence that the overexpression of the CrkII adapter protein complements EGF-stimulated pathways to induce cell dispersal in two-dimensional cultures and cell invasion and branching morphogenesis in three-dimensional collagen gels. This finding correlates with the ability of CrkII to promote the breakdown of adherens junctions in stable cell lines and the ability of EGF to stimulate enhanced Rac activity in cells overexpressing CrkII. We have previously shown that the Gab1-docking protein is required for branching morphogenesis downstream of the Met receptor. Consistent with a role for CrkII in promoting EGF-dependent branching morphogenesis, the binding of Gab1 to CrkII is required for the branching morphogenic program downstream of Met. Together, our data support a role for the CrkII adapter protein in epithelial invasion and morphogenesis and underscores the importance of considering the synergistic actions of signaling pathways in cancer progression.
Highlights
Epithelial morphogenesis is essential for normal embryonic development and involves proliferation, migration, cellular invasion, turnover of surrounding extracellular matrix, and the deposition of newly synthesized extracellular matrix [1]
To examine why epidermal growth factor (EGF) fails to induce the dispersal of colonies of MadinDarby canine kidney (MDCK) cells, we examined the response of MDCK cells to EGF
hepatocyte growth factor (HGF) but not EGF promotes cell dispersal and branching morphogenesis in MDCK cells. This provides an experimental system to identify HGF-dependent signals and to dissect signals that synergize with EGF in mediating the dispersal of epithelial sheets, epithelial remodeling, invasion, and morphogenesis
Summary
The generation of stable cell lines overexpressing CrkII was described previously [52]. Collagen Assays—The ability of MDCK cells to form branching tubules was assayed as described previously [22]. The cells were maintained in Liebowitz medium containing 5% FBS and allowed to form cysts for 5–7 days. HGF (15 units/ml), CSF-1 (5 units/ml), or EGF (20 or 100 ng/ml) was added to Liebowitz medium containing 3% FBS. The invasion assays were carried out in the same fashion as described above with the exception that 104 cells were seeded and allowed to form small colonies for 2 days prior to stimulation with growth factor. Growth Factor Stimulations—MDCK and MDCK cells overexpressing CrkII were plated at 6 ϫ 105/100-mm dish and were serum-starved the day for 20 h in Dulbecco’s modified Eagle’s medium containing 0.02% FBS. Cells were stimulated with 70 units/ml HGF or 70 ng/ml EGF for the indicated times and lysed in Rac lysis buffer [53]. 700 g of cell lysate was used for pull down assays with the CRIB domain of PAK1 fused to glutathione S-transferase as described previously [53]
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