Whey acidic protein (WAP) is a major whey protein in milk that has structural similarity to the family of serine protease inhibitors with WAP motif domains characterized by a four-disulfide core. We previously reported that enforced expression of the mouse WAP transgene in mammary epithelial cells inhibits their proliferation in vitro and in vivo by means of suppressing cyclin D1 expression (Nukumi et al., 2004, Dev Biol 274: 31-44). This study was conducted in order to clarify the molecular mechanism of the inhibitory function of WAP in HC11 cells, a mammary epithelial cell line. The assembly of laminin, a component in the extracellular matrix, was much more prominent around WAP-clonal HC11 cells that stably expressed the WAP transgene than around mock-clonal HC11 cells, and the proliferation of WAP-clonal HC11 cells was particularly inhibited in the presence of laminin. A laminin degradation assay demonstrated that WAP inhibited the activity of the pancreatic elastase-mediated cleavage of laminin B1 and the phosphorylation of ERK1/2. ERK1/2 phosphorylation was blocked by an inhibitor of the epidermal growth factor (EGF) receptor AG1478. Treatment with pancreatic elastase was found to enhance the proliferation of mock-clonal HC11 cells, but had no effect on that of WAP-clonal HC11 cells. The proliferation of WAP-clonal HC11 cells was recovered by the addition of exogenous EGF. We concluded that WAP plays some role in regulating the proliferation of mammary epithelial cells by preventing elastase-type serine protease from carrying out laminin degradation and thereby suppressing the MAP kinase signal pathway.
2007 Wiley-Liss, Inc.