Kabir-Salmani Maryam, Shiokawa Shigetatsu, Akimoto Yoshihiro, Sakai Keiji, Nagamatsu Shinya, Sakai Ken, Nakamura Yukio, Lotfi Abbas, Kawakami Hayato, Iwashita Mitsutoshi
Department of Obstetrics and Gynecology, Kyorin University School of Medicine, Mitaka, Tokyo 181-8611, Japan.
Endocrinology. 2003 Apr;144(4):1620-30. doi: 10.1210/en.2002-220886.
IGF-I and -II provide paracrine and autocrine stimuli, respectively, for extravillous trophoblast (EVT) cell migration. This study examined the role of alpha(v)beta(3) integrin and its signaling pathway in IGF-I-stimulated migration. Migration assays were conducted using cultured EVT cells treated with or without IGF-I in the presence or absence of alphaIR3, Arg-Gly-Asp (RGD) hexapeptide, and antibody against alpha(v)beta(3) integrin. Morphological changes were studied using scanning electron microscopy. Colocalization of alpha(5)beta(1) alpha(v)beta(3) integrins, vinculin, focal adhesion kinase, and paxillin were determined by immuno-cytochemistry and immunoblotting. The results showed that IGF-I could stimulate EVT cell migration in a time- and dose-dependent manner and addition of alphaIR3, Arg-Gly-Asp hexapeptide, and antibody against alpha(v)beta(3) integrin attenuated the IGF-I migratory effect. Scanning electron microscopy images revealed that IGF-I promoted lamellipodia formation. Immunostaining and immunoblotting exhibited the colocalization of alpha(v)beta(3) integrin with phosphorylated focal adhesion kinase, paxillin, and vinculin at focal adhesions after IGF-I treatment. Immunoblotting demonstrated an increase in focal adhesion kinase and paxillin tyrosine phosphorylation followed by tyrosine phosphorylation of IGF-I receptor in a time- and dose-dependent manner. These findings indicated alpha(v)beta(3) integrin localization in the core of focal adhesions of EVT cells and that alpha(v)beta(3) integrin signaling pathways are activated in IGF-I-mediated migration of these cells.
胰岛素样生长因子-I（IGF-I）和-II分别为绒毛外滋养层（EVT）细胞迁移提供旁分泌和自分泌刺激。本研究检测了α(v)β(3)整合素及其信号通路在IGF-I刺激的迁移中的作用。使用在有或无IGF-I、αIR3、精氨酸-甘氨酸-天冬氨酸（RGD）六肽以及抗α(v)β(3)整合素抗体存在的情况下处理过的培养EVT细胞进行迁移试验。使用扫描电子显微镜研究形态学变化。通过免疫细胞化学和免疫印迹法确定α(5)β(1)、α(v)β(3)整合素、纽蛋白、粘着斑激酶和桩蛋白的共定位。结果表明，IGF-I能以时间和剂量依赖性方式刺激EVT细胞迁移，添加αIR3、RGD六肽和抗α(v)β(3)整合素抗体可减弱IGF-I的迁移作用。扫描电子显微镜图像显示，IGF-I促进片状伪足形成。免疫染色和免疫印迹显示，IGF-I处理后，α(v)β(3)整合素与磷酸化粘着斑激酶、桩蛋白和纽蛋白在粘着斑处共定位。免疫印迹表明，粘着斑激酶和桩蛋白酪氨酸磷酸化增加，随后IGF-I受体酪氨酸磷酸化呈时间和剂量依赖性增加。这些发现表明α(v)β(3)整合素定位于EVT细胞粘着斑的核心，且α(v)β(3)整合素信号通路在IGF-I介导的这些细胞迁移中被激活。
