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Essential role for the ATG4B protease and autophagy in bleomycin-induced pulmonary fibrosis.

ABSTRACT: Autophagy is a critical cellular homeostatic process that controls the turnover of damaged organelles and proteins. Impaired autophagic activity is involved in a number of diseases, including idiopathic pulmonary fibrosis suggesting that altered autophagy may contribute to fibrogenesis. However, the specific role of autophagy in lung fibrosis is still undefined. In this study, we show for the first time, how autophagy disruption contributes to bleomycin-induced lung fibrosis in vivo using an Atg4b-deficient mouse as a model. Atg4b-deficient mice displayed a significantly higher inflammatory response at 7 d after bleomycin treatment associated with increased neutrophilic infiltration and significant alterations in proinflammatory cytokines. Likewise, we found that Atg4b disruption resulted in augmented apoptosis affecting predominantly alveolar and bronchiolar epithelial cells. At 28 d post-bleomycin instillation Atg4b-deficient mice exhibited more extensive and severe fibrosis with increased collagen accumulation and deregulated extracellular matrix-related gene expression. Together, our findings indicate that the ATG4B protease and autophagy play a crucial role protecting epithelial cells against bleomycin-induced stress and apoptosis, and in the regulation of the inflammatory and fibrotic responses.

SUBMITTER: Cabrera S

PROVIDER: S-EPMC4502665 | biostudies-literature | 2015 Apr

REPOSITORIES: biostudies-literature

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Essential role for the ATG4B protease and autophagy in bleomycin-induced pulmonary fibrosis.

Cabrera Sandra S Maciel Mariana M Herrera Iliana I Nava Teresa T Vergara Fabián F Gaxiola Miguel M López-Otín Carlos C Selman Moisés M Pardo Annie A

Autophagy 20150401 4

Autophagy is a critical cellular homeostatic process that controls the turnover of damaged organelles and proteins. Impaired autophagic activity is involved in a number of diseases, including idiopathic pulmonary fibrosis suggesting that altered autophagy may contribute to fibrogenesis. However, the specific role of autophagy in lung fibrosis is still undefined. In this study, we show for the first time, how autophagy disruption contributes to bleomycin-induced lung fibrosis in vivo using an Atg ...[more]

PMID: 25906080

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Project description:Pulmonary fibrosis is a group of progressive, fibrotic, and fatal lung diseases, and the role of autophagy in pulmonary fibrosis is controversial. In the current research, we dynamically observed a bleomycin-induced pulmonary fibrosis mouse model after 3, 7, 14, 21, and 28 days and investigated the expression of autophagy markers. We found that autophagy markers were not significantly changed on the indicated days in the mouse lung tissue. Then, RNA-Seq was used to analyze the gene expression and associated functions and pathways in fibrotic lung tissue on different days post-bleomycin. In addition, short time series expression miner (STEM) analysis was performed to explore the temporal post-bleomycin gene expression. Through STEM, continually up- or downregulated profiles did not demonstrate the critical role of autophagy in the development of fibrosis. Furthermore, gene ontology (GO) annotations showed that continually upregulated profiles were mainly related to fibrosis synthesis, extracellular space, and inflammation, while enriched pathways were mainly related to the PI3K-Akt signaling pathway, ECM-receptor interactions, and focal adhesion signaling pathway. For continually downregulated profiles, GO annotations mainly involved sarcomere organization, muscle contraction, and muscle fiber development. The enriched KEGG signaling pathways were the cAMP signaling pathway, cGMP-PKG signaling pathway, calcium signaling pathway, and cardiac muscle contraction. Moreover, we analyzed autophagy-related genes' expression in specific cells from a publicly available database of three human and one animal study of pulmonary fibrosis using single-cell sequencing technology. All results consistently demonstrated no critical role of autophagy in the pathogenesis of pulmonary fibrosis. In summary, autophagy may not critically and consistently change during the development of pulmonary fibrosis at different stages post-bleomycin in a mouse model. These continually up- or downregulated profiles, including gene profiles, and the corresponding functions and pathways may provide mechanistic insights into IPF therapy.

Project description:As an interstitial fibrosis disease characterized by diffuse alveolitis and structural alveolar disorders, idiopathic pulmonary fibrosis (IPF) has high lethality but lacks limited therapeutic drugs. A hospital preparation used for the treatment of viral pneumonia, Qingfei Tongluo mixture (QFTL), is rumored to have protective effects against inflammatory and respiratory disease. This study aims to confirm whether it has a therapeutic effect on bleomycin-induced IPF in rats and to elucidate its mechanism of action. Male SD rats were randomly divided into the following groups: control, model, CQ + QFTL (84 mg/kg chloroquine (CQ) + 3.64 g/kg QFTL), QFTL-L, M, H (3.64, 7.28, and 14.56 g/kg, respectively) and pirfenidone (PFD 420 mg/kg). After induction modeling and drug intervention, blood samples and lung tissue were collected for further detection. Body weight and lung coefficient were examined, combined with hematoxylin and eosin (H&E) and Masson staining to observe lung tissue lesions. The enzyme-linked immunosorbent assay (ELISA) and the hydroxyproline (HYP) assay kit were used to detect changes in proinflammatory factors (transforming growth factor-β (TGF-β), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β)) and HYP. Immunohistochemistry and Western blotting were performed to observe changes in proteins related to pulmonary fibrosis (α-smooth muscle actin (α-SMA) and matrix metalloproteinase 12 (MMP12)) and autophagy (P62 and mechanistic target of rapamycin (mTOR)). Treatment with QFTL significantly improved the adverse effects of bleomycin on body weight, lung coefficient, and pathological changes. Then, QFTL reduced bleomycin-induced increases in proinflammatory mediators and HYP. The expression changes of pulmonary fibrosis and autophagy marker proteins are attenuated by QFTL. Furthermore, the autophagy inhibitor CQ significantly reversed the downward trend in HYP levels and α-SMA protein expression, which QFTL improved in BLM-induced pulmonary fibrosis rats. In conclusion, QFTL could effectively attenuate bleomycin-induced inflammation and pulmonary fibrosis through mTOR-dependent autophagy in rats. Therefore, QFTL has the potential to be an alternative treatment for IPF in clinical practice.

Project description:Idiopathic pulmonary fibrosis is a progressive, fatal disease with limited treatment options. Protease-mediated transforming growth factor-? (TGF-?) activation has been proposed as a pathogenic mechanism of lung fibrosis. Protease activity in the lung is tightly regulated by protease inhibitors, particularly secretory leukocyte protease inhibitor (SLPI). The bleomycin model of lung fibrosis was used to determine the effect of increased protease activity in the lungs of Slpi(-/-) mice following injury. Slpi(-/-), and wild-type, mice received oropharyngeal administration of bleomycin (30?IU) and the development of pulmonary fibrosis was assessed. Pro and active forms of matrix metalloproteinase (MMP)-2 and MMP-9 were measured. Lung fibrosis was determined by collagen subtype-specific gene expression, hydroxyproline concentration, and histological assessment. Alveolar TGF-? activation was measured using bronchoalveolar lavage cell pSmad2 levels and global TGF-? activity was assessed by pSmad2 immunohistochemistry. The active-MMP-9 to pro-MMP-9 ratio was significantly increased in Slpi(-/-) animals compared with wild-type animals, demonstrating enhanced metalloproteinase activity. Wild-type animals showed an increase in TGF-? activation following bleomycin, with a progressive and sustained increase in collagen type I, alpha 1 (Col1?1), III, alpha 1(Col3?1), IV, alpha 1(Col4?1) mRNA expression, and a significant increase in total lung collagen 28 days post bleomycin. In contrast Slpi(-/-) mice showed no significant increase of alveolar TGF-? activity following bleomycin, above their already elevated levels, although global TGF-? activity did increase. Slpi(-/-) mice had impaired collagen gene expression but animals demonstrated minimal reduction in lung fibrosis compared with wild-type animals. These data suggest that enhanced proteolysis does not further enhance TGF-? activation, and inhibits sustained Col1?1, Col3?1, and Col4?1 gene expression following lung injury. However, these changes do not prevent the development of lung fibrosis. Overall, these data suggest that the absence of Slpi does not markedly modify the development of lung fibrosis following bleomycin-induced lung injury.

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Essential role for the ATG4B protease and autophagy in bleomycin-induced pulmonary fibrosis.

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Essential role for the ATG4B protease and autophagy in bleomycin-induced pulmonary fibrosis.

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