Purification and characterization of human laminin-8 -: Laminin-8 stimulates cell adhesion and migration through α3β1 and α6β1 integrins
Purification and characterization of human laminin-8 -: Laminin-8 stimulates cell adhesion and migration through α3β1 and α6β1 integrins
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DOI：
10.1074/jbc.m010155200
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发表时间：
2001-05-18
影响因子：
4.8
通讯作者：
Sekiguchi, K
中科院分区：
文献类型：
--
作者：
Fujiwara, H;Kikkawa, Y;Sekiguchi, K
最近发现的含有 α4 链的层粘连蛋白亚型已显示在心脏、骨骼肌和血管等受限器官的基底膜中表达，特别是在胚胎中。我们通过逆转录酶-聚合酶链反应筛选了38个人类细胞系的层粘连蛋白α4链的表达，发现T98G胶质母细胞瘤细胞仅表达α4，而不表达其他α链。 Laminin-8 是一种含有 α4 和 β1 链的异构体，使用抗层粘连蛋白 β1 链的单克隆抗体，通过凝胶过滤和免疫亲和层析从 T98G 细胞的条件培养基中纯化。纯化的层粘连蛋白亚型由二硫键连接的 230、220 和 200 kDa 亚基组成，免疫印迹分析将其鉴定为 β1、γ1 和 α4 链。纯化的层粘连蛋白-8的细胞粘附活性与层粘连蛋白-1相当，但明显弱于层粘连蛋白-5和层粘连蛋白-10/11。粘附层粘连蛋白 8 的 T98G 细胞比粘附其他层粘连蛋白亚型的 T98G 细胞变得更长，并延长了多个伪足。细胞对层粘连蛋白 8 的粘附可被整合素 β (1) 亚基的抗体或整合素 α (3) 和 α (6) 亚基的抗体组合所消除，但单独的抗 α (3) 或抗 α (6) 抗体不会消除细胞粘附，这表明 α (3) β (1) 和 α (6) β (1) 整合素均可作为层粘连蛋白 8 的粘附受体。与这些观察结果一致，当β(1)整合素被β(1)激活抗体8A2刺激时，用整合素α(3)或α(6)cDNA转染的K562红白血病细胞能够粘附到层粘连蛋白-8。尽管其细胞粘附活性中等，但与其他层粘连蛋白亚型和纤连蛋白相比，层粘连蛋白-8 在促进细胞迁移方面具有显着的功效。抗 α (3) 和 α (6) 整联蛋白的抗体组合可完全抑制层粘连蛋白 8 上的细胞迁移，并且单独抗 α (3) 抗体可显着抑制细胞迁移，表明层粘连蛋白 8 介导的细胞迁移主要由 α (3) β (1) 整联蛋白介导。鉴于其刺激细胞迁移的能力以及优先定位于毛细血管和胚胎组织的基底膜的能力，层粘连蛋白-8可能在发育、伤口愈合和血管生成过程中需要增强细胞迁移的过程中发挥作用。
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.