TGF-β1 stimulates HDAC4 nucleus-to-cytoplasm translocation and NADPH oxidase 4-derived reactive oxygen species in normal human lung fibroblasts

Myofibroblasts are important mediators of fibrogenesis; thus blocking fibroblast-to-myofibroblast differentiation (FMD) may be an effective strategy to treat pulmonary fibrosis (PF). Previously, we reported that histone deacetylase 4 (HDAC4) activity is necessary for transforming growth factor-beta 1 (TGF-beta 1)-induced human lung FMD. Here, we show that TGF-beta 1 increases NADPH oxidase 4 (NOX4) mRNA and protein expression in normal human lung fibroblasts (NHLFs) and causes nuclear export of HDAC4. Application of the NOX family inhibitor diphenyleneiodonium chloride reduces TGF-beta 1-induced HDAC4 nuclear export, expression of the myofibroblast marker alpha-smooth muscle actin (alpha-SMA), and alpha-SMA fiber formation. Inhibition of HDAC4 nucleus-to-cytoplasm translocation using leptomycin B (LMB) had little effect on alpha-SMA expression but blocked alpha-SMA fiber formation. A coimmunoprecipitation assay showed that HDAC4 associates with alpha-SMA. Moreover, LMB abolishes TGF-beta 1-induced alpha-SMA fiber formation and cell contraction. Relevant to human pulmonary fibrosis, idiopathic PF specimens showed significantly higher NOX4 RNA expression and scant HDAC4 staining within nuclei of fibroblast foci myofibroblasts. Taken together, these results indicate that reactive oxygen species promote TGF-beta 1-mediated myofibroblast differentiation and HDAC4 nuclear export. The physical association of HDAC4 with alpha-SMA suggests that HDAC4 has a role in regulating the alpha-SMA cytoskeleton arrangement.