Epigenetic control of lung fluid clearance

The Na,K-ATPase regulates ion transport and fluid balance in the lung, generating the driving force for alveolar fluid clearance (AFC). TGF-β mediates acute lung injury (ALI), and impacts AFC. We hypothesized that TGF-β impacts Na,K-ATPase function and activity by modifying subunit stoichiometry. Cell surface density of the Na,K-ATPase is regulated by the ATP1B1 subunit. Quantitative PCR analysis revealed that ATP1B1 gene expression was reduced both in TGF-β-treated A549 lung epithelial cell line and in lungs from ALI patients or mice with bleomycin-induced experimental ALI. Moreover, cell surface biotinylation and 86Rb+ uptake assays revealed TGF-β depleted cell-surface Na,K-ATPase, and reduced Na,K-ATPase activity in A549 cells and primary mouse alveolar type II cells. In silico and dual-luciferase reporter analyses of ATP1B1 gene promoter detected number of putative TGF-β responding elements. RNA interference and enzymatic inhibition studies revealed that downregulation of the ATP1B1 gene relied on SMAD2, SMAD4, SNAI1 and E2F5 transcription factors. Furthermore, we have found that downregulation of the ATP1B1 gene was facilitated by epigenetic mechanisms involving class I HDAC activity and was critically dependent upon HDAC2. The HDAC pan-inhibitor, Trichostatin A, restored normal Atp1b1 gene expression as well as improved oedema resolution in bleomycin-induced experimental ALI. Collectively, our data suggest that TGF-β downregulates ATP1B1 gene expression leading to decreased Na,K-ATPase activity and consequently impaired AFC in ALI, and that these processes can be reversed by HDAC inhibition.