The functions of exogenous and endogenous laminin-5 on corneal epithelial cells

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 alpha 3, beta 3, gamma 2 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 490 kDa molecule that undergoes specific processing to cleavaged isoforms after being secreted. The alpha 3 subunit is processed from 200-190 kDa to 160 kDa/ 145 kDa. The gamma 2 subunit is processed from 150 kDa to 105 kDa/80 kDa. The beta 3 subunit (140 kDa) 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 alpha 3 (160 kDa), beta 3 (140 kDa) and gamma 2 (105 kDa) 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 190 kDa alpha 3, 140 kDa beta 3 and 150 kDa gamma 2 chains and that after being secreted, the alpha 3 chain was processed to 160 kDa/145 kDa and the gamma 2 chain was processed to 105 kDa. Initially we investigated the functions of exogenous (processed) laminin-5 on HCE cells. Exogenously added laminin-5 strongly promoted cell adhesion via alpha 3 beta 1 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 alpha 3 beta 1 mAbs, the blocking of the interaction between endogenously secreted laminin-5 and HCE cells caused strong inhibition of cell migration, Integrin alpha 3 beta 1 and alpha 6 beta 4 were expressed in HCE cells. These integrins are receptors of laminin-5. But, anti integrin alpha 6 beta 4 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 alpha 3 beta 1 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. (C) 2000 Academic Press.