Purification and characterization of human laminin-8 -: Laminin-8 stimulates cell adhesion and migration through α3β1 and α6β1 integrins

Recently identified laminin isoforms containing the alpha4 chain have been shown to be expressed in the basement membrane of restricted organs such as heart, skeletal muscle, and blood vessels, especially those in embryos. We screened 38 human cell lines for the expression of the laminin alpha4 chain by reverse transcriptase-polymerase chain reaction and found that T98G glioblastoma cells express only alpha4, but not other alpha chains. Laminin-8, an isoform containing the alpha4 and beta1 chains, was purified from conditioned medium of T98G cells by gel filtration and immunoaffinity chromatography using a monoclonal antibody against laminin beta1 chain. The purified laminin isoform was composed of disulfide-linked 230-, 220-, and 200-kDa subunits, which immunoblot analysis identified as the beta1, gamma1, and alpha4 chains. Purified laminin-8 had cell adhesive activity comparable to laminin-1 but significantly weaker than laminin-5 and laminin-10/11. T98G cells adhering to laminin-8 became more elongated than those adhering to other laminin isoforms and extended multiple pseudopods. Cell adhesion to laminin-8 was abolished by an antibody against the integrin beta (1) subunit or a combination of antibodies against the integrin alpha (3) and alpha (6) subunits, but not by either anti-alpha (3) or anti-alpha (6) antibody alone, suggesting that both alpha (3)beta (1) and alpha (6)beta (1) integrins serve as adhesion receptors for laminin-8. Consistent with these observations, K562 erythroleukemic cells transfected with either integrin alpha (3) or alpha (6) cDNA were capable of adhering to laminin-8 when beta (1) integrins were stimulated by the beta (1)-activating antibody 8A2. Despite its moderate cell adhesive activity, laminin-8 was significantly potent in promoting cell migration when compared with other laminin isoforms and fibronectin. Cell migration on laminin-8 was completely inhibited by a combination of antibodies against alpha (3) and alpha (6) integrins, and substantially inhibited by anti-alpha (3) antibody alone, suggesting that laminin-8-mediated cell migration is predominantly mediated by alpha (3)beta (1) integrin. Given its potency to stimulate cell migration and preferential localization to the basement membrane of capillaries and embryonic tissues, laminin-8 may play a role in processes requiring enhanced cell migration during development, wound healing, and angiogenesis.