Laminin Synthesis and the Adhesion Characteristics of Immortalized Human Corneal Epithelial Cells to Laminin Isoforms

Abstract

We have studied the synthesis of laminins (Ln) and determined the specific integrins mediating the adhesion of immortalized human corneal epithelial cells to mouse Ln-1, and human Lns-5 and -10. Immunofluorescence microscopy of the cells demonstrated integrin α2, α3, α6, β1and β4subunits, integrins α6and β4being found in a typical ‘leopard-skin’ like manner. Immunoprecipitation studies showed that the cells produced α3, β3 and γ2 chains of Ln-5, but not Lns-1 or -10. In culture Ln-5 was found as small plaques beneath the adhering cells within 1hr, while in 4hr widely spread Ln-5 plaques were observed in colocalization with β4integrin subunit. By using a quantitative cell adhesion assay and function-blocking monoclonal antibodies we showed that integrin β1subunit plays a role in mediating corneal epithelial cell adhesion to mouse Ln-1. However, none of the available function-blocking antibodies to integrin α-subunits inhibited the adhesion. Integrin α3β1complex mediated the adhesion of corneal epithelial cells to human Lns-5 and -10. Integrin complex α3β1, as well as laminin α3chain, was also shown to mediate cell adhesion to newly produced endogenous Ln-5. The present results show that integrin α3β1complex mediates the adhesion of corneal epithelial cells to Lns-5 and -10, while a yet unknown integrin α subunit appears to play a role in the adhesion to Ln-1. The results also show that among corneal basement membrane laminins, Ln-5 is synthetized by epithelial cells while Ln-10 may be a product of keratocytes.