Cyclic stretch-induced nuclear localization of transcription factors results in increased nuclear targeting of plasmids in alveolar epithelial cells

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-kB and NF1. Specific inhibition of the nuclear import of AP1 and/or NF-kB 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.