Phosphoinositide-3 Kinase–Rac1–c-Jun NH2-terminal Kinase Signaling Mediates Collagen I–induced Cell Scattering and Up-Regulation of N-Cadherin Expression in Mouse Mammary Epithelial Cells

Abstract

During epithelial-to-mesenchymal transitions (EMTs), cells must change their interactions with one another and with their extracellular matrix in a synchronized manner. To characterize signaling pathways cells use to coordinate these changes, we used NMuMG mammary epithelial cells. We showed that these cells become fibroblastic and scattered, with increased N-cadherin expression when cultured on collagen I. Rac1 and c-Jun NH₂-terminal kinase (JNK) were activated when cells were plated on collagen I, and dominant inhibitory Rac1 (RacN17) or inhibition of JNK signaling prevented collagen I–induced morphological changes and N-cadherin up-regulation. Furthermore, inhibiting phosphoinositide-3 kinase (PI3K) activity prevented Rac1 and JNK activation as well as collagen I–induced N-cadherin up-regulation. These data implicate PI3K–Rac1–JNK signaling in collagen I–induced changes in NMuMG cells. To establish a role for N-cadherin in collagen I–induced cell scattering, we generated N-cadherin overexpressing and knockdown NMuMG cells and showed that knocking down N-cadherin expression prevented collagen I–induced morphological changes. Motility assays showed that cells overexpressing N-cadherin were significantly more motile than mock-transfected cells and that N-cadherin-mediated motility was collagen I dependent. In addition, we showed that cord formation and branching in three-dimensional culture (EMT-dependent events) required N-cadherin expression and PI3K–Rac1–JNK signaling.