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

<div><p>Background</p><p>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.</p><p>Methods</p><p>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.</p><p>Results</p><p>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.</p><p>Conclusions</p><p>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.</p></div

HDAC6 knockout mice are not protected against bleomycin-induced pulmonary fibrosis.

<p>(A, B) HDAC6 wild-type (WT) and knockout (KO) mice were treated with PBS or bleomycin (2units/kg; by oropharyngeal aspiration) on Day 0 and then lungs were harvested on Day 21. (A) Collagen expression was assessed using trichrome staining of lung sections. Representative images (taken at 40x magnification) are shown. Scale bars: 3mm (left panels), 300μm (middle panels), 200μm (right panels). (B) Protein expression levels were assessed using immunoblots of lung homogenates.</p

HDAC6 knockdown does not ameliorate TGF-β1-induced type-1 collagen expression in NHLFs.

<p>Subconfluent NHLFs were pretreated with siRNA for 24 hours and then co-treated with TGF-β1 for 48hours. ++ denotes double concentration. The immunoblots show that knockdown of HDAC6 and/or HDAC10 failed to repress TGF-β1-induced type-1 collagen expression in NHLFs.</p

Tubastatin decreases TGF-β1-induced expression of type-1 collagen.

<p>(A-E) Subconfluent NHLFs (A, C, D, E) or IPF fibroblasts (B) were pretreated with an HDAC6 inhibitor (Tubacin, Tubastatin, ACY1215, or MC1568) for 6 hours and then co-treated with TGF-β1 and the HDAC6 inhibitor. (A, B) Protein expression levels were assessed using immunoblots at 48 hours. D: DMSO, Tc: Tubacin, Ts: Tubastatin, A: ACY1215, M: MC1568; Col-1: type-1 collagen, α-SMA: α-smooth muscle actin (C) Gene expression levels for collagen-1 (col1a1) were assessed using qRT-PCR at 24 hours. * p<0.05 compared to the control; # p<0.05 compared to the TGF-β1. (D) Correlation between the degree of α-tubulin acetylation and collagen-1 protein expression in experiments involving Tubastatin treatment. There was a modest inverse correlation between the degree of α-tubulin acetylation and collagen-1 expression (r² = 0.7602 [p<0.0001] for pooled results of six independent experiments.). (E) An MTT assay showing that Tubastatin does not affect NHLF viability. Values for cell viability were normalized to the control group (= DMSO treatment).</p

Tubastatin decreases TGF-β1-induced phosphorylation of Akt, likely by restoring the association of Akt and PHLPP.

<p>(A) Subconfluent NHLFs were pretreated with Tubastatin for 6 hours and then co-treated with TGF-β1 and Tubastatin. Protein expression levels were assessed using immunoblots at the designated time points. Tubastain repressed TGF-β1-induced phosphorylation of Akt, but not Smad2 or Smad3. (B) Subconfluent NHLFs were pretreated with Tubastatin for 6 hours and then co-treated with TGF-β1 and Tubastatin for 12 hours. <i>Left</i>: Cell lysates were immunoprecipitated with an anti-Akt antibody, and protein expression levels for PHLPP and Akt were assessed using immunoblots. IP denotes immunoprecipitation. The fourth lane (*) represents IP using isotype control IgG antibody and the combined samples from the three treatment groups (i.e. an equal amount of the sample from each treatment group was mixed). <i>Right</i>: “Input” refers immunoblots without IP. TGF-β1 disrupted Akt-PHLPP association, and this was ameliorated by Tubastatin.</p

Tubastatin-treated mice are protected against bleomycin-induced pulmonary fibrosis.

<p>Mice were treated with PBS or bleomycin (2units/kg; by oropharyngeal aspiration) on Day 0, followed by daily intraperitoneal injection of DMSO or Tubastatin (80 mg/kg/day) on Day 1 through Day 21 (n = 4-5/group). (A) Collagen expression was assessed using trichrome staining of lung sections. Representative images (taken at 40x magnification) are shown. Scale bars: 3mm (left panels), 300μm (middle panels), 200μm (right panels). (B) Protein expression levels were assessed using immunoblots of lung homogenates. Densitometry analysis of type-1 collagen expression is shown in the bar graphs. * p<0.05 compared to the control (= PBS+DMSO); # p<0.05 compared to Bleomycin+DMSO.</p