Tubulin acetylation and histone deacetylase 6 activity in the lung under cyclic load.

Previous studies from our lab have demonstrated that upon exposure to physiologic levels of cyclic stretch, alveolar epithelial cells demonstrate a significant decrease in the amount of polymerized tubulin (Geiger et al., Gene Therapy 2006;13:725-731). However, not all microtubules are disassembled, although the mechanisms or implications of this were unknown. Using immunofluorescence microscopy, Western blotting, and immunohistochemistry approaches, we have compared the levels of acetylated tubulin in stretched and unstretched A549 cells and in murine lungs. In cultured cells exposed to cyclic stretch (10% change in basement membrane surface area at 0.25 Hz), nearly all of the remaining microtubules were acetylated, as demonstrated using immunofluorescence microscopy. In murine lungs ventilated for 20 minutes at 12 to 20 ml/kg followed by 48 hours of spontaneous breathing or for 3 hours at 16 to 40 ml/kg, levels of acetylated tubulin were increased in the peripheral lung. In both our in vitro and in vivo studies, we have found that mild to moderate levels of cyclic stretch significantly increases tubulin acetylation in a magnitude- and duration-dependent manner. This appears to be due to a decrease in histone deacetylase 6 activity (HDAC6), the major tubulin deacetylase. Since it has been previously shown that acetylated microtubules are positively correlated to a more stable population of microtubules, this result suggests that microtubule stability may be increased by cyclic stretch, and that tubulin acetylation is one way in which cells respond to changes in exogenous mechanical forces.

Microtubule acetylation through HDAC6 inhibition results in increased transfection efficiency.

The success of viral and nonviral gene delivery relies on the ability of DNA-based vectors to traverse the cytoplasm and reach the nucleus. We, as well as other researchers, have shown that plasmids utilize the microtubule network and its associated motor proteins to traffic toward the nucleus. While disruption of microtubules with nocodazole was shown to greatly inhibit cytoplasmic plasmid trafficking, it did not abolish it. It has been demonstrated that a pool of stabilized post-translationally acetylated microtubules exists in cells, and that this acetylation may play a role in protein trafficking. In order to determine whether this modification could account for the residual DNA trafficking in nocodazole-treated cells, we inhibited or knocked down the levels of the tubulin deacetylase, histone deacetylase 6 (HDAC6), thereby generating higher levels of acetylated microtubules. Electroporation of plasmids into cells with inhibited or silenced HDAC6 resulted in increased gene transfer. This increased transfection efficiency was not because of increased transcriptional activity, but rather, because of increased cytoplasmic trafficking. When plasmids were cytoplasmically microinjected into HDAC6-deficient cells, they entered the nucleus within 5 minutes of injection, almost 10 times faster than in wild-type cells. Taken together, these results suggest that modulation of HDAC6 and the microtubule network can increase the efficiency of gene transfer.

KGF prevents oxygen-mediated damage in ARPE-19 cells.

PURPOSE: Oxidative stress has been implicated in a variety of diseases of the eye. In several other tissues, keratinocyte growth factor (KGF) has been shown to prevent negative cellular changes associated with oxidative insult, such as permeability increases and nuclear DNA damage. In this study, we looked at whether KGF provided these same protective effects to cultured human retinal pigmented epithelial (RPE) cells (ARPE-19). METHODS: Reverse transcriptase-polymerase chain reaction (RT-PCR) using a published primer pair sequence followed by restriction endonuclease digestion with AvaI and HincII was used to look for the KGF receptor message in ARPE-19 cells. Cellular response to KGF was verified through proliferation assays and Western blot analysis for mitogen-activated protein kinase (MAPK). Single-cell gel electrophoresis was used to assess DNA damage, Western blot analysis was used to assay actin cytoskeletal changes, and electrical resistance and tracer experiments with Transwell tissue plates were used to assess permeability changes. Immunostaining was used to verify the existence of the tight junction protein occludin. RESULTS: It was verified through RT-PCR that the ARPE-19 cell line exhibited the message for FGFR2-IIIb, otherwise known as KGFR. KGF was also shown to increase cellular proliferation and activated the MAPK p44/p42 cascade. KGF ameliorated nuclear DNA damage and cytoskeletal rearrangement caused by oxidative stress through the addition of exogenous hydrogen peroxide but was unable to prevent permeability changes. CONCLUSIONS: KGF was shown to significantly reduce DNA damage and cytoskeletal rearrangement caused by oxidative stress in cultured ARPE-19 cells. This result may be useful in targeting future therapies to combat a multitude of diseases of the eye that result from increases in reactive oxygen species.

Intracellular trafficking of nucleic acids.

Until recently, the attention of most researchers has focused on the first and last steps of gene transfer, namely delivery to the cell and transcription, in order to optimise transfection and gene therapy. However, over the past few years, researchers have realised that the intracellular trafficking of plasmids is more than just a "black box" and is actually one of the major barriers to effective gene delivery. After entering the cytoplasm, following direct delivery or endocytosis, plasmids or other vectors must travel relatively long distances through the mesh of cytoskeletal networks before reaching the nuclear envelope. Once at the nuclear envelope, the DNA must either wait until cell division, or be specifically transported through the nuclear pore complex, in order to reach the nucleoplasm where it can be transcribed. This review focuses on recent developments in the understanding of these intracellular trafficking events as they relate to gene delivery. Hopefully, by continuing to unravel the mechanisms by which plasmids and other gene delivery vectors move throughout the cell, and by understanding the cell biology of gene transfer, superior methods of transfection and gene therapy can be developed.