Kovala A T, Harvey K A, McGlynn P, Boguslawski G, Garcia J G, English D
Experimental Cell Research Program, Methodist Research Institute, Clarian Health Partners, Inc., Indianapolis, Indiana 46202, USA.
FASEB J. 2000 Dec;14(15):2486-94. doi: 10.1096/fj.00-0147com.
The definition of signaling pathways in endothelial cells has been hampered by the difficulty of transiently transfecting these cells with high efficiency. This investigation was undertaken to develop an efficient technique for the transfection of endothelial cells for functional analyses. Cells cotransfected with plasmid expressing green fluorescent protein (GFP) and the plasmid of interest were isolated by fluorescence-activated cell sorting (FACS) based on GFP expression. In the sorted cell population, a 2.5-fold enhancement in the number of cells expressing the gene of interest was observed, as confirmed by FACS analysis and Western blotting. Sorted cells retained functional properties, as demonstrated by chemotaxis to the agonist sphingosine 1-phosphate (SPP). To demonstrate the usefulness of this method for defining cellular signaling pathways, cells were cotransfected with plasmids encoding GFP and the carboxyl-terminal domain of the beta-adrenergic receptor kinase (beta ARKct), which inhibits signaling through the beta gamma dimer of heterotrimeric G-proteins. SPP-induced chemotaxis in sorted cells coexpressing beta ARKct was inhibited by 80%, demonstrating that chemotaxis was driven by a beta gamma-dependent pathway. However, no significant inhibition was observed in cells transfected with betaARKct but not enriched by sorting. Thus, we have developed a method for enriching transfected cells that allows the elucidation of crucial mechanisms of endothelial cell activation and function. This method should find wide applicability in studies designed to define pathways responsible for regulation of motility and other functions in these dynamic cells.
内皮细胞信号通路的定义一直受到高效瞬时转染这些细胞的困难的阻碍。本研究旨在开发一种用于内皮细胞转染以进行功能分析的有效技术。基于绿色荧光蛋白（GFP）表达，通过荧光激活细胞分选（FACS）分离共转染表达GFP的质粒和感兴趣质粒的细胞。通过FACS分析和蛋白质印迹证实，在分选的细胞群体中，观察到表达感兴趣基因的细胞数量增加了2.5倍。分选的细胞保留了功能特性，如对激动剂鞘氨醇1-磷酸（SPP）的趋化作用所证明。为了证明该方法在定义细胞信号通路方面的有用性，将编码GFP的质粒与β-肾上腺素能受体激酶（βARKct）的羧基末端结构域共转染细胞，该结构域抑制通过异源三聚体G蛋白的βγ二聚体的信号传导。在共表达βARKct的分选细胞中，SPP诱导的趋化作用被抑制了80%，表明趋化作用是由βγ依赖性途径驱动的。然而，在用βARKct转染但未通过分选富集的细胞中未观察到明显的抑制作用。因此，我们开发了一种富集转染细胞的方法，该方法能够阐明内皮细胞激活和功能的关键机制。这种方法在旨在定义负责调节这些动态细胞运动性和其他功能的途径的研究中应具有广泛的适用性。
