Pichard V, Honoré S, Kovacic H, Li C, Prevôt C, Briand C, Rognoni J B
UPRES CNRS 6032, Faculté de Pharmacie, Université Aix-Marseille II, 27 Bd Jean Moulin, 13385 Marseille Cedex 05, France.
Histochem Cell Biol. 2001 Oct;116(4):337-48. doi: 10.1007/s004180100324.
Both epidermal growth factor (EGF) and the extracellular matrix components have been implicated in the pathobiology of adenocarcinomas by somewhat poorly understood mechanisms. We have addressed this problem using an in vitro model comprising the colon adenocarcinoma cell line HT29-D4, wherein the role of EGF and type IV collagen on cell adhesion was examined. We demonstrated that the effect of EGF on HT29-D4 cell adhesion was regulated by type IV collagen in a time- and dose-dependent manner. The incorporation of a panel of monoclonal antibodies to integrins alpha1beta1, alpha2beta1 and alpha3beta1 in adhesion medium revealed that EGF-mediated increase in the cell adhesion was mediated essentially by alpha2beta1, and the use of flow cytometry led us to conclude that this EGF effect was mediated by an increase in alpha2beta1 activation and not by an increase in cell surface expression of integrin. An indirect immunofluorescence technique was employed to demonstrate that focal adhesion kinase (FAK) and alpha2beta1 integrin were present in focal complexes in large EGF-induced lamellipodia whereas actin cytoskeleton was organised in small tips that colocalised with FAK. This pattern was observed at early time points (15 min) with a strong FAK tyrosine phosphorylation and with an increase in mitogen-activated protein kinase activity (5-15 min) as measured by immunoprecipitation and immunoblotting. We conclude that at early time points of cell adhesion and spreading, EGF exerted an inside-out regulation of alpha2beta1 integrin in HT29-D4 cells. This regulation seemed to be mediated by EGF-dependent FAK phosphorylation entailing an increase in integrin activation and their recruitment in numerous focal complexes. Furthermore after activation, FAK induced aggregation of actin-associated proteins (paxillin, vinculin and other tyrosine phosphorylated proteins) in focal complexes, leading to organisation of actin cytoskeleton that is involved in lamellipodia formation. Finally, activated alpha2beta1 integrins intervened in all these processes clustered in small focal complexes but not in focal adhesions.
表皮生长因子（EGF）和细胞外基质成分都通过一些尚未完全清楚的机制参与了腺癌的病理生物学过程。我们使用包含结肠腺癌细胞系HT29-D4的体外模型来解决这个问题，其中研究了EGF和IV型胶原对细胞黏附的作用。我们证明，EGF对HT29-D4细胞黏附的影响受IV型胶原的时间和剂量依赖性调节。在黏附培养基中加入一组针对整合素α1β1、α2β1和α3β1的单克隆抗体表明，EGF介导的细胞黏附增加主要由α2β1介导，流式细胞术的结果使我们得出结论，这种EGF效应是由α2β1激活增加介导的，而不是由整合素细胞表面表达增加介导的。采用间接免疫荧光技术证明，在大的EGF诱导的片状伪足的粘着斑复合物中存在粘着斑激酶（FAK）和α2β1整合素，而肌动蛋白细胞骨架则组织在与FAK共定位的小尖端中。在早期时间点（15分钟）观察到这种模式，FAK酪氨酸磷酸化强烈，丝裂原活化蛋白激酶活性增加（5-15分钟），通过免疫沉淀和免疫印迹测量。我们得出结论，在细胞黏附和铺展的早期时间点，EGF对HT29-D4细胞中的α2β1整合素发挥了由内向外的调节作用。这种调节似乎是由EGF依赖性的FAK磷酸化介导的，导致整合素激活增加及其在众多粘着斑复合物中的募集。此外，激活后，FAK诱导粘着斑复合物中肌动蛋白相关蛋白（桩蛋白、纽蛋白和其他酪氨酸磷酸化蛋白）聚集，导致参与片状伪足形成的肌动蛋白细胞骨架的组织。最后，活化的α2β1整合素参与了所有这些过程，聚集在小的粘着斑复合物中，而不是粘着斑中。
