Autophagy in Idiopathic Pulmonary Fibrosis

Background: Autophagy is a basic cellular homeostatic process important to cell fate decisions under conditions of stress. Dysregulation of autophagy impacts numerous human diseases including cancer and chronic obstructive lung disease. This study investigates the role of autophagy in idiopathic pulmonary fibrosis. Methods: Human lung tissues from patients with IPF were analyzed for autophagy markers and modulating proteins using western blotting, confocal microscopy and transmission electron microscopy. To study the effects of TGF-β1 on autophagy, human lung fibroblasts were monitored by fluorescence microscopy and western blotting. In vivo experiments were done using the bleomycin-induced fibrosis mouse model. Results: Lung tissues from IPF patients demonstrate evidence of decreased autophagic activity as assessed by LC3, p62 protein expression and immunofluorescence, and numbers of autophagosomes. TGF-β1 inhibits autophagy in fibroblasts in vitro at least in part via activation of mTORC1; expression of TIGAR is also increased in response to TGF-β1. In the bleomycin model of pulmonary fibrosis, rapamycin treatment is antifibrotic, and rapamycin also decreases expression of á-smooth muscle actin and fibronectin by fibroblasts in vitro. Inhibition of key regulators of autophagy, LC3 and beclin-1, leads to the opposite effect on fibroblast expression of á-smooth muscle actin and fibronectin. Conclusion: Autophagy is not induced in pulmonary fibrosis despite activation of pathways known to promote autophagy. Impairment of autophagy by TGF-β1 may represent a mechanism for the promotion of fibrogenesis in IPF

APPA Increases Lifespan and Stress Resistance via Lipid Metabolism and Insulin/IGF-1 Signal Pathway in <i>Caenorhabditis elegans</i>

Animal studies have proven that 1-acetyl-5-phenyl-1H-pyrrol-3-yl acetate (APPA) is a powerful antioxidant as a novel aldose reductase inhibitor independently synthesized by our laboratory; however, there is no current information on APPA’s anti-aging mechanism. Therefore, this study examined the impact and mechanism of APPA’s anti-aging and anti-oxidation capacity using the Caenorhabditis elegans model. The results demonstrated that APPA increases C. elegans’ longevity without affecting the typical metabolism of Escherichia coli OP50 (OP50). APPA also had a non-toxic effect on C. elegans, increased locomotor ability, decreased the levels of reactive oxygen species, lipofuscin, and fat, and increased anti-stress capacity. QRT-PCR analysis further revealed that APPA upregulated the expression of antioxidant genes, including sod-3, gst-4, and hsp-16.2, and the critical downstream transcription factors, daf-16, skn-1, and hsf-1 of the insulin/insulin-like growth factor (IGF) receptor, daf-2. In addition, fat-6 and nhr-80 were upregulated. However, the APPA’s life-prolonging effects were absent on the daf-2, daf-16, skn-1, and hsf-1 mutants implying that the APPA’s life-prolonging mechanism depends on the insulin/IGF-1 signaling system. The transcriptome sequencing also revealed that the mitochondrial route was also strongly associated with the APPA life extension, consistent with mev-1 and isp-1 mutant life assays. These findings aid in the investigation of APPA’s longevity extension mechanism

Deletion of caveolin-1 protects hyperoxia-induced apoptosis via survivin-mediated pathways

Hyperoxia-induced lung injury is an established model that mimics human acute respiratory distress syndrome. Cell death is a prominent feature in lungs following prolonged hyperoxia. Caveolae are omega-shaped invaginations of the plasma membrane. Caveolin-1 (cav-1), a 22-kDa transmembrane scaffolding protein, is the principal structural component of caveolae. We have recently shown that deletion of cav-1 (cav-1−/−) protected against hyperoxia-induced cell death and lung injury both in vitro and in vivo; however, the mechanisms remain unclear. Survivin, a member of the inhibitor of apoptosis protein family, inhibits apoptosis in tumor cells. Although emerging evidence suggests that survivin is involved in wound healing, especially in vascular injuries, its role in hyperoxia-induced lung injury has not been investigated. Our current data demonstrated that hyperoxia induced apoptosis via suppressing survivin expression. Deletion of cav-1 abolished this suppression and subsequently protected against hyperoxia-induced apoptosis. Using “gain” and “loss” of function assays, we determined that survivin protected lung cells from hyperoxia-induced apoptosis via the inhibition of apoptosis executor caspase-3. Overexpression of survivin by deletion of cav-1 was regulated by Egr-1. Egr-1 functioned as a negative regulator of survivin expression. Deletion of cav-1 upregulated survivin via decreased Egr-1 binding of the survivin promoter region. Together, this study illustrates the effect of hyperoxia on survivin expression and the role of survivin in hyperoxia-induced apoptosis. We also demonstrate that deletion of cav-1 protects hyperoxia-induced apoptosis via modulation of survivin expression

Autophagy is not increased in IPF.

<p>A) IPF whole lung homogenate demonstrates increased ER stress (elevated XBP1 expression) and increased phosphorylation of AMPK, factors which should drive autophagy. B) LC3-II (lower band) expression in IPF whole lung homogenate is decreased relative to control lung tissue C) Densitometry of Western blots demonstrating LC3-II level is lower in IPF than in control lung (*p = 0.05). D) Increased p62 in IPF lung suggests decreased autophagy. E) Immunofluorescence confocal microscopy of control and IPF lung tissue for p62 (green), aggresome (red), DAPI (blue) demonstrates increased p62 expression and aggresomes. F) Representative electron microscopy images from IPF (panels A, B), control (Panel C), and COPD (panel D); white arrows indicate autophagosomes. G) Quantitation of autophagic vacuoles in control, IPF, and COPD lung by EM demonstrates significantly higher numbers in COPD (*p<0.05 for IPF vs. COPD).</p

TGF-β₁ activates mTOR and TIGAR.

<p>A) In human lung fibroblasts, TGF-β₁ inhibits LC3-II formation, even in the presence of IFN-γ which induces autophagy. B) TGF-β₁ is unable to inhibit LC3-II formation in presence of mTOR inhibitor rapamycin. C) TGF-β₁ is able to activate mTORC1 which results in increased phospho-S6 but this activation does not occur in the presence of rapamycin. D) TGF-β₁ appears to activate mTORC1 by activating upstream PI3K/AKT and treatment with PI3K inhibitor LY294002 prevents TGF-β₁ induced mTOR activation. E) Western blot of phospho-mTOR (Ser2448) showing increased phospho-mTOR with TGF-β₁ in fibroblasts and inhibition by rapamycin. F) Western blot of phospho-S6 from mouse lung tissue treated with bleomycin and rapamycin. G) Densitometry of blot from 4E. (*p = 0.03 for controls vs. bleomycin, **p = 0.003 for bleomycin vs rapamycin + bleomycin). H) phospho-S6 protein levels in human lung tissue is higher in IPF patients compared with COPD patients and healthy controls. I) TIGAR is induced by TGF-β₁ in fibroblasts in a dose-responsive manner. J) Western blot demonstrating TIGAR protein levels in lung homogenate from human tissue is higher in IPF patients compared with COPD patients and healthy controls.</p

Inhibition of autophagy potentiates fibroblast activation.

<p>A) Rapamycin inhibits fibronectin and α-SMA expression in fibroblasts, and abrogates TGF-β₁ suppression of Id-1. B) Beclin and LC3 siRNA effectively inhibit protein expression. C) α-SMA and fibronectin expression increases in fibroblasts with beclin and LC3 silencing whereas collagen is unchanged. D) When LC3 is silenced, Id-1 expression is lower than control. E) Rapamycin does not modulate phosphorylation of Smad3 in response to TGF-β₁.</p

TGF-β₁ inhibits autophagy <i>in vitro</i>.

<p>A) Human lung fibroblasts were cultured and treated with varying concentrations of TGF-β₁. TGF-β₁ inhibited activation of LC3, decreasing the intensity of the lower band on the western blot, and B) increased p62. C) Inhibition of LC3 activation by TGF-β₁ with varying serum conditions. D) Fibroblasts treated with TGF-β₁ for 24 hours and measurement of autophagic flux by LC3 western blotting (using lysosomal acidification inhibitor chloroquine). E) Densitometry of western blot shown in D (p = 0.045). F) Fluorescence microscopy of fibroblasts transfected with GFP-LC3 construct and stimulated with TGF-β₁ showing that TGF-β₁ inhibits formation of LC3 puncta. G) Confocal microscopy of type II alveolar epithelial cells isolated from GFP-LC3 transgenic mice and stimulated with TGF-β₁ showing that TGF-β₁ inhibits formation of LC3 puncta (green = GFP-LC3, red = SP-C, blue = DAPI). H) Quantification of GFP puncta per cell from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041394#pone-0041394-g002" target="_blank">figure 2G</a>.</p

Effects of rapamycin on bleomycin induced fibrosis and autophagy.

<p>Hydroxyproline assay measuring lung collagen content demonstrates co-administration of rapamycin and bleomycin protects against fibrosis (*p = 0.003 for control vs. bleomycin; **p = 0.008 for bleomycin vs. rapamycin + bleomycin).</p