Laminin fragment E4 inhibition studies: basement membrane assembly and embryonic lung epithelial cell polarization requires laminin polymerization.

Laminins (LMs), the main constituents of basement membranes (BMs), are heterotrimeric glycoproteins composed of alpha, beta, and gamma chains held together by disulfide bonds. In the presence of Ca2+, some laminins, such as laminin-1 self-assemble into a polymer through the interaction of their three NH2 termini. Here we exposed lung organotypic cultures to a proteolytic fragment of laminin-1 that blocks laminin polymerization. This fragment, referred as E4, comprises the outer globular region of laminin beta1 chain. Inhibition of laminin polymerization in lung organotypic cultures resulted in impaired basement membrane assembly and failure of epithelial cells to polarize. In addition, we found that in control organotypic cultures, the bronchial smooth muscle cells were arranged in concentric layers around the newly formed epithelium. However, in E4-treated cultures, the smooth muscle cells were in disarray. Exposure of organotypic cultures to laminin-1 proteolytic fragment P1', that comprises part of alpha1, beta1, and gamma1 chains, but does not overlap with fragment E4, had no effect in basement membrane assembly. Exposure to fragment E4 also caused an increased release of laminin-1 into the culture medium, suggesting a failure to retain laminin at the epithelial-mesenchymal interface. These studies provide the first direct evidence linking epithelial cell polarization to laminin polymerization at the epithelial-mesenchymal interface and assign a key role to the outer globular region of laminin beta1 chain.