William G. Carter

We seek to understand how changes in cell adhesion and cell signaling induced by wounding of epidermis activates repair but prevents invasion of normal tissue. We suggest that normal wound repair mechanisms are re-directed in oncogenically transformed epithelial cells to promote invasion of normal tissue. Cultures of normal keratinocytes in organ culture on a collagen dermis assemble a morphologically normal epidermis. In contrast, keratinocytes transformed with E6 and E7 oncogenes from human papilloma virus invade the dermis under the same culture conditions. To understand the invasion, we have examined how normal keratinocytes migrate, survive and repair wounds and how these mechanisms prevent invasion. Wounding of quiescent epidermis changes cell signals and adhesion followed by transcriptional responses as follows: (i) Quiescent HFKs adhere to endogenous laminin 5 in the basement membrane (BM) via integrins a6b4 and a3b1. In contrast, quiescent keratinocytes cannot adhere to exposed dermal ligands (fibronectin or collagen) due to insufficient levels of integrin receptors (a5b1) or appropriate cell signals (RhoGTP). However, wounding induces initial cell spreading on endogenous laminin 5 via integrin a3b1at the wound edge. This initial adhesion change is necessary for the subsequent increase in expression of integrins, cell signals and laminin 5 necessary for subsequent adhesion to exposed dermal ligands in the wound. (ii) Wounding of quiescent epidermis generates two sub-populations of activated keratinocytes; leading cells at the wound edge and more quiescent following cells. Leading cells are distinct from following cells but together they closure the wound. Leading cells express laminin 5 for deposition and repair of the BM, activation transcription factor 3 (ATF3), a stress response protein and L6 protein, a tumor antigen thought to participate in cell migration in both tumors and wounds. Leading cells are autonomous and do not communicate via gap junctions. In contrast, following cells communicate as an integrated cell sheet that migrates over the path of laminin 5. Thus, deposits of laminin 5 by leading cells promote the communication in following cells necessary for re-establishing the epidermis as a barrier. (iii) Deposition of laminin 5 is required for migration of the leading and following cell sheet over collagen in response to soluble factors (growth factors, cytokines, etc). Laminin 5 promotes sustained directional polarized migration in response to growth factors when deposited over dermal ligands. Defects in laminin 5 in man and mice generate lethal blistering of epidermis, impaired migration in wounds and chronic up-regulation of activation events in wounds (eg. ATF3 upregulation). Chronic wound activation is a tumor promoter resulting in high incidence of invasive tumors at sites of blistering. Thus, deposition of laminin 5 is required for migration of the epidermal outgrowth but also limits the duration of the wound activation by returning the cells to a quiescent state. (iv) Laminin 5 is also required for survival of keratinocytes. Wounding exposes the migratory epidermis to stresses (shear, oxygen deprivation and bacterial toxins) that are fatal to most cells. Keratinocytes from the laminin 5 null mice fail to adhere or survive in culture because they arrest in G1 of the cell cycle. Adhesion to laminin 5, but not dermal ligands, protects keratinocytes from the toxic effects of stress activators (toxin B, a bacterial inhibitor of RhoGTP, and nocodazole or taxol as inhibitors of microtubules). Thus, deposits of laminin 5 contribute to survival of the outgrowth in the stressed environment of the wound. (v) In attempts to identify adhesive, signaling and survival domains in laminin 5, we prepared recombinant laminin 5 isoforms. Expression of the isoforms rescues adhesion and survival of laminin 5 null keratinocytes. These domain studies have identified sequences within the a3 chain of laminin 5 that regulate assembly of the laminin 5 heterotrimer, necessary for adhesion and survival. Together, these studies provide a basic understanding of migration, repair and survival mechanisms in normal epidermal wound repair. They also provide a basis for understanding how normal repair mechanisms may be redirected by transforming oncogenes in epithelial cells that promote invasion of normal tissue.