Abrogation of TGF-beta1-induced fibroblast-myofibroblast differentiation by histone deacetylase (HDAC) inhibition.

摘要

Idiopathic pulmonary fibrosis (IPF) is a devastating disease with no known effective pharmacologic therapy. The fibroblastic foci of IPF contain activated myofibroblasts which are the major producers of type I collagen. TGF-beta1 promotes differentiation of fibroblasts into myofibroblasts in vitro and in vivo. In the current study, we investigated the molecular link between TGF-beta1-mediated myofibroblast differentiation and HDAC activity. Treatment of normal human lung fibroblasts (NHLFs) with the pan-HDAC inhibitor trichostatin A (TSA) inhibited TGF-beta1-mediated alpha-smooth muscle actin (alpha-SMA) and alpha--1 Type I collagen mRNA induction. TSA also blocked the TGF-beta1 driven contractile response in NHLFs. The inhibition of alpha-SMA expression by TSA was associated with reduced phosphorylation of Akt, and a pharmacological inhibitor of Akt blocked TGF-beta1-mediated alpha-SMA induction in a dose dependent manner. HDAC4 knock down was effective in inhibiting TGF-beta1 stimulated alpha-SMA expression as well as the phosphorylation of Akt. Moreover, the inhibitors of protein phosphotase 2A and 1 (PP2A and PP1) rescued TGF-beta1-mediated alpha-SMA induction from the inhibitory effect of TSA. We also observed that TGF-beta1 stimulates NOX4 mRNA and protein expression, and a subsequent increase in intracellular ROS in normal human lung fibroblasts, as well as, the export of HDAC4 from the nucleus, an event that is mediated by oxidation of HDAC4 disulfide bonds. The NOX family inhibitor, DPI, inhibited TGF-beta1 stimulated HDAC4-GFP cytoplasm translocation. In addressing the clinical relevance of our in vitro findings, lung tissue from IPF patients demonstrate higher NOX4 expression than that of COPD controls. Furthermore, immunohistochemistry results showed that lung alveolar epithelial cells have strong nuclear HDAC4 expression, whereas HDAC4 nuclear staining was absent in myofibroblasts. Taken together, these data demonstrate that TGF-beta1 stimulated NHLF to myofibroblast differentiation is HDAC4 dependent and requires phosphorylation of Akt. Moreover TGF-beta1 induces NOX4 to generate ROS, which in turn mediates myofibroblast differentiation at least in part through facilitating the translocation of HDAC4 into the cytoplasm.