Purification and Characterization of Human Laminin-8: LAMININ-8 STIMULATES CELL ADHESION AND MIGRATION THROUGH α3β1 AND α6β1INTEGRINS

Hironobu Fujiwara,Yamato Kikkawa,Noriko Sanzen,Kiyotoshi Sekiguchi

doi:10.1074/jbc.m010155200

Hironobu Fujiwara, Yamato Kikkawa + Show 2 more

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https://doi.org/10.1074/jbc.m010155200

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Abstract

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.

Highlights

Identified laminin isoforms containing the ␣4 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
Cell adhesion to laminin-8 was abolished by an antibody against the integrin ␤1 subunit or a combination of antibodies against the integrin ␣3 and ␣6 subunits, but not by either anti-␣3 or anti-␣6 antibody alone, suggesting that both ␣3␤1 and ␣6␤1 integrins serve as adhesion receptors for laminin-8
We found that one of the glioma cell lines, T98G, expresses only one of the five known laminin ␣ chains, the ␣4 chain (Fig. 1A)

Summary

Introduction

Identified laminin isoforms containing the ␣4 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. Laminin-8, an isoform containing the ␣4 and ␤1 chains, was purified from conditioned medium of T98G cells by gel filtration and immunoaffinity chromatography using a monoclonal antibody against laminin ␤1 chain. We purified laminin-8 from conditioned medium of human glioma cells and characterized its biological activities, including cell adhesive and cell migration promoting properties and integrin binding specificity in comparison with other laminin isoforms and fibronectin.

Results

Conclusion