Laminin synthesis and the adhesion characteristics of immortalized human corneal epithelial cells to laminin isoforms

We have studied the synthesis of laminins (Ln) and determined the specific integrins mediating the adhesion of immortalized human corneal epithelial c ells to mouse Ln-1, and human Lns-5 and -10. Immunofluorescence microscopy of the cells demonstrated integrin alpha (2), alpha (3), alpha (6), beta (1 ) and beta (4) subunits, integrins alpha (6) and beta (4) being found in a typical 'leopard-skin' like manner. Immunoprecipitation studies showed that the cells produced alpha3, beta3 and gamma2 chains of Ln-5, but not Lns-1 or -10. In culture Ln-5 was found as small plaques beneath the adhering cel ls within 1 hr, while in 4 hr widely spread Ln-5 plaques were observed in c olocalization with beta (4) integrin subunit. By using a quantitative cell adhesion assay and function-blocking monoclonal antibodies we showed that i ntegrin beta (1) subunit plays a role in mediating corneal epithelial cell adhesion to mouse Ln-1. However, none of the available function-blocking an tibodies to integrin alpha -subunits inhibited the adhesion. Integrin alpha (3)beta (1) complex mediated the adhesion of corneal epithelial cells to h uman Lns-5 and -10. Integrin complex alpha (3)beta (1), as well as laminin alpha (3) chain, was also shown to mediate cell adhesion to newly produced endogenous Ln-5. The present results show that integrin alpha (3)beta (1) c omplex mediates the adhesion of corneal epithelial cells to Lns-5 and -10, while a yet unknown integrin a subunit appears to play a role in the adhesi on to Ln-1. The results also show that among corneal basement membrane lami nins, Ln-5 is synthetized by epithelial cells while Ln-10 may be a product of keratocytes. (C) 2000 Academic Press.