Laminin-10/11 and fibronectin differentially regulate integrin-dependent Rho and Rac activation via p130(Cas)-CrkII-DOCK180 pathway.

The alpha(5) chain-containing laminin isoforms, laminins-10 and -11 (laminin-10/11), are the major components of the basement membrane, having potent cell-adhesive activity. We examined the cell-adhesive and integrin-mediated signaling activities of laminin-10/11 in comparison to fibronectin, the best characterized extracellular adhesive ligand. We found that laminin-10/11 are more active than fibronectin in promoting cell migration and preferentially activate Rac, not Rho, via the p130(Cas)-CrkII-DOCK180 pathway. Cells adhering to fibronectin develop stress fibers and focal contacts, whereas cells adhering to laminin-10/11 do not, consistent with the high cell migration-promoting activity of laminin-10/11. Pull-down assays of GTP-loaded Rac and Rho demonstrated the preferential activation of Rac on laminin-10/11, in contrast to the activation of Rho on fibronectin. Activation of Rac by laminin-10/11 was associated with the phosphorylation of p130(Cas) and an increased formation of a p130(Cas)-CrkII-DOCK180 complex. Cell migration on laminin-10/11 was suppressed by the expression of either a dominant-negative Rac or CrkII mutants defective in p130(Cas) or DOCK180 binding. This is the first report demonstrating a distinct activation of Rho family GTPases resulting from adhesion to different extracellular ligands.

Purification and characterization of human laminin-8. Laminin-8 stimulates cell adhesion and migration through alpha3beta1 and alpha6beta1 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 beta1 subunit or a combination of antibodies against the integrin alpha3 and alpha6 subunits, but not by either anti-alpha3 or anti-alpha6 antibody alone, suggesting that both alpha3beta1 and alpha6beta1 integrins serve as adhesion receptors for laminin-8. Consistent with these observations, K562 erythroleukemic cells transfected with either integrin alpha3 or alpha6 cDNA were capable of adhering to laminin-8 when beta1 integrins were stimulated by the beta1-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 alpha3 and alpha6 integrins, and substantially inhibited by anti-alpha3 antibody alone, suggesting that laminin-8-mediated cell migration is predominantly mediated by alpha3beta1 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.

Differential expression of laminin alpha chains during proliferative and differentiation stages in a model for skin morphogenesis.

The purpose of this study was to determine the mRNA and protein expression of laminin alpha chains at various stages of in vitro skin morphogenesis. Fibroblasts in mono-cultures express low levels of the mRNA of laminin alpha1,alpha2, alpha3 and alpha4 chains. When co-cultured with keratinocytes for 28 days, they expressed the mRNA for all these chains. Keratinocytes in monolayer expressed the laminin alpha3 chain mRNA and very low levels of the mRNA of the alpha1 and alpha2 chains, although, when recombined with fibroblasts they also expressed laminin alpha1and alpha2 mRNA, but not the laminin alpha4 mRNA. Immunocytochemistry of cells in co-culture showed that laminin alpha1, alpha3 and alpha5 chains were expressed in the epidermis, while the laminin alpha2, beta1, and gamma1 chains were noted in the dermis and at the epidermo-dermal interface. The laminin alpha1chain was first expressed during the proliferative stage (14-21 days) and the laminin alpha2 and alpha5 chains appeared later, during the differentiation stage (28-42 days). The above results suggest that epithelial-mesenchymal interactions are involved in the expression of laminin alpha chain mRNA during in vitro skin morphogenesis. In addition, there is distinct temporal and spatial expression of these chains during proliferative and differentiation stages, possibly reflecting different functions.

Integrin binding specificity of laminin-10/11: laminin-10/11 are recognized by alpha 3 beta 1, alpha 6 beta 1 and alpha 6 beta 4 integrins.

Laminin-10/11, the laminin isoforms containing the alpha 5 chain, are major components of basement membranes of many fetal and adult tissues. Laminin-10/11 purified from the conditioned medium of human lung carcinoma cells were potent in mediating adhesion of the carcinoma cells in an integrin alpha 3 beta 1-dependent manner. To further define the type(s) of integrins involved in cell adhesion to laminin-10/11, we examined the effects of a panel of function-blocking anti-integrin antibodies on the adhesion of different cell types to laminin-10/11. Although anti-integrin beta 1 antibody inhibited the adhesion of all cell types tested, anti-alpha 3 antibody inhibited the adhesion of carcinoma and glioma cells but not fibroblastic cells. Adhesion of fibroblastic cells was inhibited, however, by a combination of anti-alpha 3 and anti-alpha 6 antibodies, suggesting that both alpha 3 beta 1 and alpha 6 beta 1 integrins function as laminin-10/11 receptors in these cells. To explore this possibility, we examined the adhesion of K562 leukemic cells transfected with integrin alpha 3 or alpha 6 subunit to laminin-10/11 or other laminin isoforms. Laminin-10/11 were potent adhesive ligands for both the alpha 3 beta 11 and alpha 6 beta 1 transfectants, whereas laminin-5 was the preferred ligand for the alpha 3 beta 1 transfectants. Upon stimulation with the activating anti-integrin beta 1 antibody, both transfectants became more adherent to the substratum regardless of the type of laminins coated, although their preference for laminin isoforms remained unaltered. K562 cells transfected with alpha 6 and beta 4 subunits were also capable of adhering to laminin-10/11, indicating that integrin alpha 6 beta 4 is another receptor for laminin-10/11. Even with lung carcinoma cells, the alpha 6-containing integrins partly contributed to adhesion to laminin-10/11 at higher coating concentrations, although non-integrin receptor(s) might also be involved under such conditions. These results indicated that laminin-10/11 are potent and versatile adhesive ligands in basement membranes capable of binding to both alpha 3 beta 1 and alpha 6 beta 1 integrins with high avidity and also to alpha 6 beta 4 integrin.

Isolation and characterization of laminin-10/11 secreted by human lung carcinoma cells. laminin-10/11 mediates cell adhesion through integrin alpha3 beta1.

A panel of human tumor cell lines was screened for selective expression of laminin alpha5 chain, a newly identified laminin subunit comprising laminin-10 (alpha5 beta1 gamma1) and -11 (alpha5 beta2 gamma1). The lung adenocarcinoma cell line A549 was found to express the alpha5 chain at relatively high levels but no detectable amounts of other alpha chains. The laminin variants containing alpha5 chain were purified from the conditioned medium of A549 cells by immunoaffinity chromatography using the anti-laminin monoclonal antibody 4C7 which was shown recently to recognize the laminin alpha5 chain (Tiger, C.-F., Champliaud, M.-F., Pedrosa-Domellof, F., Thornell, L.-E., Ekblom, P., and Gullberg, D. (1997) J. Biol. Chem. 272, 28590-28595). The purified laminin variants consisted of three chains with molecular masses of 350, 220, and 210 kDa. The 350-kDa chain was specifically recognized by another anti-alpha5 chain monoclonal antibody capable of recognizing denatured alpha5 chain on immunoblots, whereas the 210-kDa chain was recognized by an anti-gamma1 chain antibody. The purified alpha5 chain-containing laminin variants (hereafter referred to as laminin-10/11) were highly active in mediating adhesion of A549 cells to the substratum with potency as high as that of laminin-5 and significantly higher than those of laminin-1, laminin-2/4, or fibronectin. Adhesion to substrata coated with laminin-10/11 was specifically inhibited by anti-integrin antibodies directed against the integrin alpha3 or beta1 subunit but not by those against alpha2 or alpha6 subunit, indicating that laminin-10/11 is specifically recognized by integrin alpha3 beta1. Given the wide distribution of laminin-10/11 in the basement membrane of various tissue types and dominant expression of integrin alpha3 beta1 in most epithelial cells, specific interaction of laminin-10/11 with integrin alpha3 beta1 may play an important role in in vivo regulation of proliferation and differentiation of epithelial cells through the basement membrane.