Jiang R, Grabel L B
Department of Biology, Wesleyan University, Middletown, Connecticut 06459, USA.
Exp Cell Res. 1995 Apr;217(2):195-204. doi: 10.1006/excr.1995.1079.
F9 embryonal carcinoma cells treated with retinoic acid differentiate in monolayer into parietal endoderm (PE) or in suspension into embryoid bodies with an outer layer of visceral endoderm (VE) surrounding a core of largely undifferentiated cells. Previous reports have shown that cell-extracellular matrix interactions mediated by the beta 1 integrins play a critical role in the differentiation and migration of PE. In the present study we investigated the pattern of expression and function of the integrin alpha 6 beta 1 during the differentiation of F9 cells into VE and PE. F9 cells express integrin subunits alpha 3, alpha 5, alpha 6, and beta 1. Cell adhesion and migration assays demonstrate that alpha 6 beta 1 is the major laminin receptor in undifferentiated F9 cells as well as F9-derived PE cells. However, the amount of alpha 6 protein decreases significantly upon F9 cell differentiation into either VE or PE, as revealed by immunofluorescent staining and immunoprecipitation analysis. In contrast, the amount of steady-state alpha 6 message stays constant before and after F9 cell differentiation, suggesting that the down-regulation of alpha 6 beta 1 occurs post-transcriptionally. In view of previous reports of two alpha 6 isoforms generated by alternative RNA processing, we carried out reverse transcription-PCR analysis and show that, while alpha 6B is the major mRNA isoform before and after F9 cell differentiation, alpha 6A mRNA is weakly expressed in undifferentiated F9 cells and is substantially increased following F9 differentiation into PE. Immunoprecipitations using the isoform-specific antibodies show an increase in alpha 6A and a dramatic decrease in alpha 6B protein following PE differentiation. Pulse-chase experiments indicate that, whereas the stability of alpha 6B protein is unaltered, synthesis of alpha 6B protein is decreased at least threefold following PE differentiation. Further experiments demonstrate that alpha 6A localizes to focal contacts in PE cells. The switch from alpha 6B to alpha 6A and the localization of alpha 6A at focal contacts correlate with the acquisition of PE cell motility, which suggests distinct functions for the two alpha 6 isoforms.
用视黄酸处理的F9胚胎癌细胞在单层培养中分化为滋养层内胚层（PE），或在悬浮培养中分化为胚状体，其外层为脏内胚层（VE），围绕着一个主要由未分化细胞组成的核心。先前的报道表明，由β1整合素介导的细胞-细胞外基质相互作用在PE的分化和迁移中起关键作用。在本研究中，我们调查了整合素α6β1在F9细胞分化为VE和PE过程中的表达模式和功能。F9细胞表达整合素亚基α3、α5、α6和β1。细胞黏附和迁移试验表明，α6β1是未分化的F9细胞以及F9来源的PE细胞中的主要层粘连蛋白受体。然而，免疫荧光染色和免疫沉淀分析显示，F9细胞分化为VE或PE后，α6蛋白的量显著减少。相比之下，F9细胞分化前后稳态α6信使的量保持不变，这表明α6β1的下调发生在转录后。鉴于先前关于通过可变RNA加工产生两种α6同工型的报道，我们进行了逆转录-聚合酶链反应分析，结果表明，虽然α6B是F9细胞分化前后的主要mRNA同工型，但α6A mRNA在未分化的F9细胞中弱表达，在F9分化为PE后显著增加。使用同工型特异性抗体进行的免疫沉淀显示，PE分化后α6A增加，α6B蛋白显著减少。脉冲追踪实验表明，虽然α6B蛋白的稳定性未改变，但PE分化后α6B蛋白的合成至少减少了三倍。进一步的实验表明，α6A定位于PE细胞中的粘着斑。从α6B到α6A的转变以及α6A在粘着斑处的定位与PE细胞运动性的获得相关，这表明两种α6同工型具有不同的功能。
