688. The Effects of Cyclic Stretch on Microtubule Stability and Exogenous Gene Expression in the Alveolar Epithelium

Abstract

Top of pageAbstract Cells in the lung undergo cyclic deformation under both normal breathing processes as well as mechanical ventilation. While large deformations due to disease or mechanical ventilation can cause cellular damage such as that seen in ventilator induced lung injury, our lab has found that smaller, physiologically relevant deformations have a positive effect on gene transfer in an in vitro pulmonary epithelium model, increasing reporter gene products up to 12-fold in A549 cells, a lung adenocarcinoma cell line frequently used in cellular models of the pulmonary epithelium. This result is not that surprising considering exogenous mechanical forces have been shown by a number of groups to alter the same barriers efficient gene transfer faces as a vector moves from the outside of the cell to the nucleus; namely alterations in the cytoskeleton and extracellular matrix, activation of cell signaling pathways and changes in transcription factor activation. We have previously shown that this increase in exogenous gene expression is due to increased trafficking of the plasmid DNA through the cytosol toward the nucleus, and that cytoskeletal rearrangement, including depolymerization of the microtubule network, is required for stretch-enhanced gene transfer and expression to occur. These results are somewhat puzzling as our lab and others have also shown that the cytoskeleton, specifically the microtubule network, is required for cytoplasmic trafficking of plasmid DNA. One type of post-polymerization modification that microtubules undergo is acetylation, an activity that is not well understood, but has been shown through the use of a variety of drugs to potentially increase microtubule stability against depolymerization from cold or drug-treatment. We wanted to see if this stabilization also applied to exogenous mechanical forces, and if this stabilization allowed for stretch-enhanced gene transfer and expression to occur. To do this, A549 cells were exposed to equibiaxial stretch (10% change in membrane surface area, 15 cycles per minute). Significant increases in gene expression were seen within 15 minutes of the applied stretch. Overexpression of acetylated microtubules through the addition of either tricostatin A, a drug known to inhibit all histone deacetylases (HDACs), or tubacin, a small-molecule inhibitor of only HDAC6, the specific deacetylase for microtubules, caused a significant increase in gene expression over untreated controls. Changes in acetylated microtubule levels were examined via western blot analysis and immunofluoresent microscopy in both cells treated with drugs that affected HDAC activity as well as cells that were stretched. These data demonstrated that a majority of the microtubules in cells that were stretched were also acetylated. Taken together, these results suggest that acetylated microtubules may be resistant to depolymerization due to mechanical forces, a result not previously demonstrated in the literature, and that acetylation may also enhance gene expression through enhanced trafficking of the plasmid DNA through the cytosol.