Research Article: Autophagy in Idiopathic Pulmonary Fibrosis

Date Published: July 18, 2012

Publisher: Public Library of Science

Author(s): Avignat S. Patel, Ling Lin, Alexander Geyer, Jeffrey A. Haspel, Chang Hyeok An, Jiaofei Cao, Ivan O. Rosas, Danielle Morse, Oliver Eickelberg. http://doi.org/10.1371/journal.pone.0041394

Abstract

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.

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.

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.

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.

Partial Text

Autophagy is a catabolic process by which components of the cytoplasm are degraded in lysosomes. During this process, cytoplasmic cargo is sequestered within double-membraned autophagosomes and then degraded after fusion of autophagosomes with lysosomes. Although this morphological process was originally described half a century ago [1], the molecular mechanisms of autophagy have been only lately described, leading to recent interest in the role of autophagy in disease. Autophagy was first understood as a process by which organelles and macromolecules could be catabolized to remove damaged structures or to provide energy under conditions of nutrient starvation. The importance of autophagy to homeostasis and development has been underscored by numerous studies in yeast and mice [2], [3], [4], [5]. The function of autophagy in human disease and pre-clinical models of disease appears to be highly pleiotropic, however, and it is not always possible to predict the outcome of interventions that affect autophagy. In some instances, autophagy has been shown to be an adaptive pro-survival response, whereas under other circumstances autophagy appears to promote cell death and morbidity [6], [7]. Relatively few studies have addressed the role of autophagy in the lung; hence the role of autophagy in adult lung disease remains largely undefined. Autophagy in chronic obstructive lung disease (COPD) is greatly enhanced in the epithelium and appears to precede apoptosis and contribute to progression of disease [8], [9]. In alveolar macrophages of COPD patients, however, there is an impairment in autophagy [10]. In cystic fibrosis, there is evidence that mutant CFTR protein impairs autophagosome formation via depletion of beclin-1 [11]. Recently, it was demonstrated that tumorigenesis in tuberous sclerosis complex (TSC) which includes pulmonary lymphangioleiomyomatosis (LAM) is autophagy dependent [12], and Mi et.al. found that IL-17 antagonism induces autophagy and is protective in a mouse model of fibrosis [13]. Whether autophagy influences the outcome of idiopathic pulmonary fibrosis (IPF), a disease involving multiple cell types and pathogenic mechanisms, is unknown.

Our findings provide evidence that autophagy is not activated in the setting of IPF despite well described elevations in (ER) stress [14], [15], oxidative stress [19], and (HIF)-1α [23], which are all known to induce autophagy. We have shown that TGF-β1 inhibits autophagy in fibroblasts in vitro, and it is therefore possible that elevated TGF-β1 activity in the IPF lung accounts for this disconnect. Our studies using inhibitors of mTORC1 and PI3K suggest that the effect of TGF-β1 on autophagy is mediated at least in part via activation of PI3K, Akt and mTORC1, although the increased expression of TIGAR we observed may also play a role. In the bleomycin model of pulmonary fibrosis, administration of rapamycin, an inducer of autophagy [43], significantly reduced the degree of lung fibrosis. This is consistent with the findings of a previous study using a TGF-α overexpression model of pulmonary fibrosis; rapamycin was also shown to have a protective effect against fibrosis [44]. Other studies evaluating rapamycin in a rat bleomycin model and a murine systemic sclerosis model have discovered similar anti-fibrotic effects [45], [46], [47]. Finally, we have shown that inhibition of autophagic proteins LC3 and Beclin-1 leads to increased α-SMA and fibronectin expression by fibroblasts in response to TGF-β1, and that rapamycin has the opposite effect.

Source:

http://doi.org/10.1371/journal.pone.0041394