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Idiopathic pulmonary fibrosis (IPF) is a fibroproliferative lung disease, and fibroblast-myofibroblast differentiation (FMD) is thought to be a key event in the pathogenesis of IPF. Histone deacetylase-8 (HDAC8) has been shown to associate with α-smooth muscle actin (α-SMA; a marker of FMD) and regulates cell contractility in vascular smooth muscle cells. However, the role of HDAC8 in FMD or pulmonary fibrosis has never been reported. This study investigated the role of HDAC8 in pulmonary fibrosis with a focus on FMD. We observed that HDAC8 expression was increased in IPF lung tissue as well as transforming growth factor (TGF)β1-treated normal human lung fibroblasts (NHLFs). Immunoprecipitation experiments revealed that HDAC8 was associated with α-SMA in TGFβ1-treated NHLFs. HDAC8 inhibition with NCC170 (HDAC8-selective inhibitor) repressed TGFβ1-induced fibroblast contraction and α-SMA protein expression in NHLFs cultured in collagen gels. HDAC8 inhibition with HDAC8 siRNA also repressed TGFβ1-induced expression of profibrotic molecules such as fibronectin and increased expression of antifibrotic molecules such as peroxisome proliferator-activated receptor- (PPAR). Chromatin immunoprecipitation quantitative PCR using an antibody against H3K27ac (histone H3 acetylated at lysine 27; a known HDAC8 substrate and a marker for active enhancers) suggested that HDAC8 inhibition with NCC170 ameliorated TGFβ1- induced loss of H3K27ac at the PPAR gene enhancer. Furthermore, NCC170 treatment significantly decreased fibrosis measured by Ashcroft score as well as expression of type 1 collagen and fibronectin in bleomycin-treated mouse lungs. These data suggest that HDAC8 contributes to pulmonary fibrosis and that there is a therapeutic potential for HDAC8 inhibitors to treat IPF as well as other fibrotic lung diseases. [ABSTRACT FROM AUTHOR]

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-β1 (TGF-β1)-induced human lung FMD. Here, we show that TGF-β1 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-β1-induced HDAC4 nuclear export, expression of the myofibroblast marker α-smooth muscle actin (α-SMA), and α-SMA fiber formation. Inhibition of HDAC4 nucleus-to-cytoplasm translocation using leptomycin B (LMB) had little effect on α-SMA expression but blocked α-SMA fiber formation. A coimmunoprecipitation assay showed that HDAC4 associates with α-SMA. Moreover, LMB abolishes TGF-β1-induced α-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-β1-mediated myofibroblast differentiation and HDAC4 nuclear export. The physical association of HDAC4 with α-SMA suggests that HDAC4 has a role in regulating the α-SMA cytoskeleton arrangement. [ABSTRACT FROM AUTHOR]