Histone Deacetylase 6 Regulates TGF-β1–mediated Epithelial–Mesenchymal Transition

Emerging evidence strongly suggests the importance of epithelial–mesenchymal transition (EMT) in pulmonary fibrosis. TGF-b1 can induce EMT in vitro and in vivo. Histone deacetylase 6 (HDAC6), unlike other HDACs, deacetylates a-tubulin, a major structural component of the microtubule. Overexpression of HDAC6 increases cell motility, which is one aspect of the myriad of TGF-b1–mediated cellular responses. These prior studies prompted us to investigate a potential regulation of HDAC6 by TGF-b1 in EMT. The A549 human alveolar epithelial cell line was employed as our experimental system because TGF-b1 can induce EMT in these cells. Serum-starved A549 cells were exposed to TGF-b1 for up to 72 hours. The expression of HDAC6, acetylated a-tubulin, and EMT markers were assessed using immunoblot and immunofluorescence analyses. HDAC6 activity was compromised using Tubacin, a small molecule inhibitor, or by using an HDAC6-targeting siRNA (HDAC6si). We confirmed TGF-b1–induced EMT in A549 cells by demonstrating: (1) a decrease in E-cadherin, an epithelial cell marker; (2) an increase in vimentin, a mesenchymal cell marker; and (3) the formation of stress fibers. We uncovered two novel isoforms of HDAC6 with apparent molecular masses of 190 kD (HDAC6p190) and 130 kD (HDAC6p130), respectively. HDAC6p190 is expressed predominantly in the cytoplasm, whereas HDAC6p130 is expressed mainly in the nucleus. TGF-b1 up-regulated HDAC6 activity, as demonstrated by HDAC6-dependent deacetylation of a-tubulin. Intriguingly, the expression of only HDAC6 p130, but not HDAC6p190 was up-regulated by TGF-b1. Similar TGF-b1–mediated regulation of HDAC6 isoforms was observed in human pulmonary arterial endothelial cells, but not in human lung fibroblasts. Inhibition of HDAC6 by HDAC6si or Tubacin attenuated TGF-b1–induced EMT, as demonstrated by preserved expression of E-cadherin, and by reduced expression of vimentin and stress fibers. These findings strongly suggest a pivotal role for HDAC6 in TGF-b1–mediated EMT. Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.