Tubastatin ameliorates pulmonary fibrosis by targeting the TGFβ-PI3K-Akt pathway.

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-β1-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-β1 ± 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-β1-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-β1-induced S6K phosphorylation, HIF-1α expression, and VEGF expression. Tubastatin also repressed TGF-β1-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-β1-induced collagen expression in lung fibroblasts.

CONCLUSIONS

HDAC6 expression is altered during lung fibrogenesis. Tubastatin represses TGF-β1-induced collagen expression, by diminishing Akt phosphorylation and regulating downstream targets such as HIF-1α-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 TGFβ-PI3K-Akt pathway, likely via an HDAC6-independent mechanism.