Migratory phenotypes of HSC-3 squamous carcinoma cell line induced by EGF and PMA: relevance to migration of loosening of adhesion and vinculin-associated focal contacts with prominent filopodia.

Cell migration is involved in carcinoma cell invasion and wound healing. We examined motogenic cytokines that potentiated migration of human HSC-3 carcinoma cells. To assess migratory activity, modified Boyden chambers were used. Among a variety of potential motogenic cytokines, epidermal growth factor (EGF) enhanced migration of HSC-3 cells both on collagen and fibronectin. Phorbol myristate acetate (PMA) also enhanced migration. Inhibitors of protein kinase C completely inhibited PMA-induced migration, but only partly inhibited EGF-induced migration. Protein kinase A was also involved in the EGF-induced signaling pathway for migration. Although the signaling pathways were independent, and the cell shape on collagen was different from that on fibronectin, migratory cells stimulated by EGF or PMA showed common morphology on different ligands. The cells were polygonal or round in shape and the loss of long cytoplasmic extensions was noted. Migratory HSC-3 cells stimulated by EGF or PMA became less adhesive to collagen and fibronectin. Since both EGF- and PMA-stimulated migration did not require de novo protein synthesis, the signaling pathways possibly lead to assembly and disassembly of an actin cytoskeleton. Immunofluorescence for vinculin was concentrated into focal contacts in EGF- and PMA-stimulated HSC-3 cells, whereas the fluorescence signal was hardly detected in non-stimulated cells. Talin and beta1 integrin were immunolocalized at focal contacts in non-stimulated cells, and it remained unchanged in stimulated cells. Numerous filopodia visualized with actin immunofluorescence were formed around stimulated HSC-3 cells, whereas filopodia were short and sparse around elongated cytoplasms in non-stimulated cells. Thus, shortening of cytoplasmic extensions with numerous filopodia, loosening of adhesion, and vinculin-associated focal contacts were regarded as migratory phenotypes.