The Functions of Exogenous and Endogenous Laminin-5 on Corneal Epithelial Cells

Abstract

Recent evidence suggests that the basement membrane not only separates basal cells from Bowman's layer, but also has a crucial role in the proliferation, differentiation and migration of corneal epithelial cells. The basement membrane is composed of a mixture of matrix components including collagens, laminins and heparan sulfate proteoglycans. In these extracellular matrixes, laminin is a major component of the basement membrane. Of 11 laminin isoformes, laminin-5 is a variant, composed of three nonidentical subunits α3, β3, γ2 and is a major component of the corneal basement membrane. However, little is known about the interactions of laminin-5 with corneal epithelial cells. In this study, we investigated the functions of laminin-5 on SV-40 transfected human corneal epithelial cells (HCE cells). We also revealed different functions between exogenous and endogenous laminin-5 on HCE cells.Laminin-5 is synthesized initially as a 490kDa molecule that undergoes specific processing to cleavaged isoforms after being secreted. The α3 subunit is processed from 200–190kDa to 160kDa/145kDa. The γ2 subunit is processed from 150kDa to 105kDa/80kDa. The β3 subunit (140kDa) is not processed. Exogenously added laminin-5 (soluble form) in this study was purified from a serum-free, conditioned medium of a human gastric carcinoma cell line STKM-I. This soluble laminin is a processed isoform containing α3 (160kDa), β3 (140kDa) and γ2 (105kDa) chains. On the other hand, immunocytochemical analysis showed that HCE cells themselves secreted laminin-5 endogenously. Western blotting analysis revealed that HCE cells initially produced unprocessed isoform containing 190kDa α3, 140kDa β3 and 150kDa γ2 chains and that after being secreted, the α3 chain was processed to 160kDa/145kDa and the γ2 chain was processed to 105kDa.Initially we investigated the functions of exogenous (processed) laminin-5 on HCE cells. Exogenously added laminin-5 strongly promoted cell adhesion via α3β1 integrin, cell spreading, assembly of hemidesmosomes and mildly inhibited cell migration. Next we estimated the effect of endogenous (unprocessed) laminin-5 on HCE cells. Using an anti laminin-5 monoclonal antibody (mAb) or anti integrin α3β1 mAbs, the blocking of the interaction between endogenously secreted laminin-5 and HCE cells caused strong inhibition of cell migration. Integrin α3β1 and α6β4 were expressed in HCE cells. These integrins are receptors of laminin-5. But, anti integrin α6β4 mAbs did not have any blocking ability against cell migration. These results indicated that endogenous (unprocessed) laminin-5 has a crucial role in cell migration on HCE cells via α3β1 integrin.In conclusion, structural differences between exogenous (processed) and endogenous (unprocessed) laminin-5 regulated their functions on HCE cells. Exogenously added laminin-5 strongly promoted cell adhesion, cell spreading and assembly of hemidesmosomes. Endogenously secreted laminin-5 had a crucial role in cell migration. In the future, processed soluble laminin-5 could be a useful drug for the prevention of recurrent corneal erosion, and unprocessed soluble laminin-5 could be applied for the treatment of prolonged corneal epithelial defects.