May 10-14, 2000.
Annual Meeting of The Society of Investigative Dermatology. Sheraton Chicago Hotel and Towers, Chicago, Illinois. The abstract was selected for an oral presentation.

BMP-1 Processing of Laminin-5 Controls Migration of Human Epithelial Cells. K. McGowan, D. Veitch, P. Findell, A. Russell, W-B. Ho and M.P. Marinkovich, Stanford University, Stanford, CA; and FibroGen Inc., South San Francisco, CA.

This study sought to characterize the enzyme(s) which process laminin 5 and their role in cell migration. In a survey of proteases, only bone morphogenetic protein 1 (BMP-1) efficiently cleaved both the gamma2 and alpha3 chains of human laminin-5 to sizes identical to endogenously processed laminin 5, shown by immunoblot analysis. The putative BMP-1 gamma2 and alpha3 cleavage sites were identified and gamma2 site was confirmed by sequencing. In contrast, MMP-2 readily cleaved collagen XVII and collagen IV, but failed to cleave human laminin-5 even at six times the enzyme to substrate ratio used for BMP-1 cleavage studies. Two hydroxamic acid inhibitors of BMP-1, FG-1731 and FG-1226, were developed which were highly selective, being 500-1000 times more potent in inhibiting BMP-1 compared to MMPs 1, 2 and 9. and nontoxic to cultured cells even at 100 times inhibitory levels. Keratinocyte (KC) cultures containing 1uM of either inhibitor showed a complete absence of laminin-5 processing by immunoblot. Both FG-1731 and FG-1226 each inhibited KC migration in a dose dependant manner, shown in Boyden and scratch assays and removal of the inhibitors restored normal migration. Both inhibitors completely halted migration of KC and SCC cells but not EB KC lacking laminin-5, suggesting the inhibitors' effect on migration is specific to laminin-5 processing. Antibody blocking experiments showed increased binding of unprocessed laminin-5 with alpha3beta1 integrin and processed laminin-5 with alpha6beta4 integrin. These results demonstrate that BMP-1 processes both the laminin-5 gamma2 and alpha3 chains and that this processing is crucial in controlling cell migration. This study also introduces two BMP-1 inhibitors that hold promise as nontoxic and highly specific anti-cancer agents.