Cyclic stretch-induced nuclear localization of transcription factors results in increased nuclear targeting of plasmids in alveolar epithelial cells
Cyclic stretch-induced nuclear localization of transcription factors results in increased nuclear targeting of plasmids in alveolar epithelial cells
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DOI：
10.1002/jgm.1187
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发表时间：
2008-06-01
影响因子：
3.5
通讯作者：
Dean, David A.
中科院分区：
文献类型：
--
作者：
Lam, Anna P.;Dean, David A.
我们之前已经表明，与正常呼吸时肺上皮所经历的循环拉伸相对应，通过增加质粒细胞内运输，增强了肺泡上皮细胞中的非病毒基因转移和表达。虽然微管和肌动蛋白细胞骨架的循环拉伸重组对于增加质粒运输是必要的，但核进入在这种增强运输中的作用尚未阐明。方法对肺泡上皮细胞进行双轴循环拉伸（0.5 Hz下表面积变化10%），检测其RNA表达、核定位和关键转录因子的激活情况。拉伸的上皮细胞通过电穿孔转染质粒，并暴露于转录因子激活抑制剂中。结果原位杂交检测发现，与未拉伸的细胞相比，电穿孔后拉伸的细胞有更多的质粒定位在细胞核上。循环拉伸还增加了被认为参与质粒核进入的多种转录因子的核定位，包括AP1、AP2、NF-kappa B和NF1。特异性抑制AP1和/或NF-kappa B的核输入可消除拉伸所见的质粒核定位增强。结论质粒的核进入被认为是通过核孔与DNA的蛋白结合介导的。拉伸增强的转录因子核定位增加了质粒的核靶向性，而抑制特定转录因子的核输入则取消了拉伸增强的质粒核定位。综上所述，这些结果表明，循环拉伸增加了细胞质和核膜的基因运输。版权所有(C) 2008约翰威利父子有限公司
Background We have shown previously that cyclic stretch corresponding to that experienced by the pulmonary epithelium during normal breathing enhances nonviral gene transfer and expression in alveolar epithelial cells by increasing plasmid intracellular trafficking. Although reorganization of the microtubule and actin cytoskeletons by cyclic stretch is necessary for increased plasmid trafficking, the role of nuclear entry in this enhanced trafficking has not been elucidated.Methods Alveolar epithelial cells were subjected to biaxial cyclic stretch (10% change in surface area at 0.5 Hz) and assayed for RNA expression, nuclear localization and activation of key transcription factors. Stretched epithelial cells were transfected with plasmids via electroporation and exposed to inhibitors of transcription factor activation.Results When assayed by in situ hybridization, more plasmids were localized to the nuclei of cells that were stretched following electroporation compared to unstretched cells. Cyclic stretch also increases the nuclear localization of multiple transcription factors thought to be involved in plasmid nuclear entry, including AP1, AP2, NF-kappa B and NF1. Specific inhibition of the nuclear import of AP1 and/or NF-kappa B abolishes the enhanced plasmid nuclear localization seen with stretch.Conclusions Nuclear entry of plasmids is thought to be mediated by the binding of proteins that chaperone the DNA through the nuclear pore. Stretch- enhanced nuclear localization of transcription factors increases nuclear targeting of plasmids, whereas inhibition of the nuclear import of specific transcription factors abrogated stretch-enhanced plasmid nuclear localization. Taken together, these results suggest that cyclic stretch increases gene trafficking in the cytoplasm and at the nuclear envelope. Copyright (C) 2008 John Wiley & Sons, Ltd.