BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive and fatal disease. Histone deacetylase 6 (HDAC6) alters function and fate of various proteins via deacetylation of lysine residues, and is implicated in TGF-beta1-induced EMT (epithelial-mesenchymal transition). However, the role of HDAC6 in pulmonary fibrosis is unknown. METHODS: HDAC6 expression in IPF and control lungs was assessed by quantitative real-time PCR (qRT-PCR) and immunoblots. Lung fibroblasts were treated with TGF-beta1 +/- HDAC6 inhibitors (Tubacin, Tubastatin, ACY1215, or MC1568), and fibrotic markers such as type I collagen were assessed using qRT-PCR and immunoblots. Mice were treated with bleomycin (oropharyngeal aspiration; single dose) +/- Tubastatin (intraperitoneally injection; daily for 21 days), and lung collagen expression was gauged using immunoblots and trichrome staining. In a separate experiment, HDAC6 wild-type (WT) and knockout (KO) mice were administered bleomycin, and lungs were evaluated in the same manner. RESULTS: HDAC6 expression was deregulated in IPF lungs. Among the HDAC6 inhibitors tested, only Tubastatin significantly repressed TGF-beta1-induced expression of type-1 collagen in lung fibroblasts, and this finding was coupled with decreased Akt phosphorylation and increased Akt-PHLPP (PH domain and Leucine rich repeat Protein Phosphatase) association. Tubastatin repressed TGF-beta1-induced S6K phosphorylation, HIF-1alpha expression, and VEGF expression. Tubastatin also repressed TGF-beta1-induced inhibition of LC3B-II (a marker of autophagosome formation). In bleomycin-treated mouse lungs, HDAC6 expression was increased, and Tubastatin repressed type-1 collagen expression. However, in HDAC6 KO mice, bleomycin-induced type-1 collagen expression was not repressed compared to WT mice. Knockdown of HDAC6, as well as HDAC10, another potential Tubastatin target, did not inhibit TGF-beta1-induced collagen expression in lung fibroblasts. CONCLUSIONS: HDAC6 expression is altered during lung fibrogenesis. Tubastatin represses TGF-beta1-induced collagen expression, by diminishing Akt phosphorylation and regulating downstream targets such as HIF-1alpha-VEGF axis and autophagy. Tubastatin-treated WT mice are protected against bleomycin-induced fibrosis, but HDAC6 KO mice are not. Our data suggest that Tubastatin ameliorates pulmonary fibrosis, by targeting the TGFbeta-PI3K-Akt pathway, likely via an HDAC6-independent mechanism.
- Saito, S.
- Zhuang, Y.
- Shan, B.
- Danchuk, S.
- Luo, F.
- Korfei, M.
- Guenther, A.
- Lasky, J. A.
Keywords
- Aged
- Aged, 80 and over
- Animals
- Autophagosomes/drug effects/metabolism
- Autophagy/drug effects
- Bleomycin
- Collagen Type I/metabolism
- Female
- Fibroblasts/drug effects/metabolism
- Histone Deacetylase 6
- Histone Deacetylases/genetics/metabolism
- Humans
- Hydroxamic Acids/pharmacology/*therapeutic use
- Hypoxia-Inducible Factor 1, alpha Subunit/metabolism
- Idiopathic Pulmonary Fibrosis/*drug therapy/genetics/*metabolism/pathology
- Indoles/pharmacology/*therapeutic use
- Lung/metabolism/pathology
- Male
- Mechanistic Target of Rapamycin Complex 1
- Mice, Knockout
- Middle Aged
- Multiprotein Complexes/metabolism
- Nuclear Proteins/metabolism
- Phosphatidylinositol 3-Kinases/*metabolism
- Phosphoprotein Phosphatases/metabolism
- Phosphorylation/drug effects
- Proto-Oncogene Proteins c-akt/*metabolism
- RNA, Messenger/genetics/metabolism
- Ribosomal Protein S6 Kinases/metabolism
- Signal Transduction/*drug effects
- TOR Serine-Threonine Kinases/metabolism
- Transforming Growth Factor beta/*metabolism/pharmacology
- Tubulin/metabolism
- Vascular Endothelial Growth Factor A/metabolism