Purification and characterization of human laminin-8 - Laminin-8 stimulates cell adhesion and migration through alpha(3)beta(1) and alpha(6)beta(1) 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 chai n by reverse transcriptase-polymerase chain reaction and found that T98G gl ioblastoma cells express only alpha4, but not other alpha chains. Laminin-8 , an isoform containing the alpha4 and beta1 chains, was purified from cond itioned medium of T98G cells by gel filtration and immunoaffinity chromatog raphy using a monoclonal antibody against laminin beta1 chain. The purified laminin isoform was composed of disulfide-linked 230-, 220-, and 200-kDa s ubunits, which immunoblot analysis identified as the beta1, gamma1, and alp ha4 chains. Purified laminin-8 had cell adhesive activity comparable to lam inin-1 but significantly weaker than laminin-5 and laminin-10/11. T98G cell s 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) subu nits, but not by either anti-alpha (3) or anti-alpha (6) antibody alone, su ggesting that both alpha (3)beta (1) and alpha (6)beta (1) integrins serve as adhesion receptors for laminin-8. Consistent with these observations, K5 62 erythroleukemic cells transfected with either integrin alpha (3) or alph a (6) cDNA were capable of adhering to laminin-8 when beta (1) integrins we re stimulated by the beta (1)-activating antibody 8A2. Despite its moderate cell adhesive activity, laminin-8 was significantly potent in promoting ce ll migration when compared with other laminin isoforms and fibronectin. Cel l migration on laminin-8 was completely inhibited by a combination of antib odies against alpha (3) and alpha (6) integrins, and substantially inhibite d by anti-alpha (3) antibody alone, suggesting that laminin-8-mediated cell migration is predominantly mediated by alpha (3)beta (1) integrin. Given i ts 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.