Evaluation of the proteasome as a trigger and target in idiopathic pulmonary fibrosis

Semren, Nora (2015): Evaluation of the proteasome as a trigger and target in idiopathic pulmonary fibrosis. Dissertation, LMU München: Fakultät für Chemie und Pharmazie

Abstract

Idiopathic pulmonary fibrosis (IPF) is an irreversible and progressive disease of the lungs, which is characterized by aberrant tissue remodeling and massive deposition of extracellular matrix proteins. This process is mainly conducted by myofibroblasts, an activated fibroblast phenotype. During the pathogenesis of IPF, the fine alveolar structure is destroyed and gas exchange declines, finally resulting in organ failure. So far, pharmacological treatment options are very limited and lung transplantation still remains the only curative therapy. Pathologic tissue remodeling in IPF is closely connected to altered cell and protein homeostasis. The ubiquitin-proteasome system is critical for degradation of polyubiquitinated proteins in a spatially and timely controlled manner, thereby regulating protein levels. The proteasome is a multicatalytic enzyme complex consisting of a barrel shaped 20S catalytic core particle (CP) and one or two 19S regulatory particles (RP), thus forming active 26S/30S proteasomes. Dysregulation of the proteasome has been reported for several chronic diseases of the heart, brain, and also lung. Furthermore, inhibition of the proteasome has been shown to provide antifibrotic effects in different organs, including the lung. As nothing is known about proteasome function in the pathogenesis of IPF, the first aim of the present study was to analyze proteasomal regulation during tissue remodeling and myofibroblast differentiation. For that, lung fibroblasts were treated with transforming growth factor-β (TGF-β) and proteasome activity as well as composition was examined. For in vivo testing, the bleomycin mouse model of lung fibrosis was used and human lung tissue of IPF patients was analyzed. It was found that induction of myofibroblast differentiation by TGF-β mediated assembly of 19S RPs with 20S CPs, thereby forming 26S/30S complexes, which was critically dependent on the regulatory particle non ATPase 6 subunit (Rpn6). In addition, silencing of Rpn6 in primary human lung fibroblasts counteracted TGF β induced myofibroblast differentiation. During bleomycin-induced fibrotic remodeling of mouse lungs, increased formation of 26S/30S proteasomes was accompanied by augmented expression of Rpn6 in fibrotic lungs. Here, Rpn6 was highly expressed in hyperplastic alveolar epithelial cells and Clara cells. Overexpression of Rpn6 was also observed in myofibroblasts and hyperplastic bronchiolar basal cells of fibrotic lung tissue of IPF patients and accompanied by enhanced polyubiquitination of proteins. As therapeutic application of proteasome inhibitors in pulmonary fibrosis showed controversial results including beneficial antifibrotic effects but also toxicity, the second aim of this study was to test whether site specific inhibition of the proteasome, using the novel second generation inhibitor oprozomib, provides antifibrotic effects in the absence of systemic side effects after local pulmonary application. Oprozomib was compared to the FDA-approved proteasome inhibitor bortezomib and tested on the human alveolar epithelial cancer cell line A549 and on primary mouse alveolar epithelial type II cells regarding its cytotoxic effects. Oprozomib was less toxic than bortezomib and provided high selectivity for the chymotrypsin-like active site of the proteasome. In primary mouse lung fibroblasts, oprozomib showed significant antifibrotic effects like reduction of collagen I and α-smooth muscle actin expression at non-toxic doses. When applied locally into the lungs of healthy mice via instillation, oprozomib was well tolerated and effectively reduced pulmonary proteasome activity. In bleomycin-challenged mice, however, locally applied oprozomib resulted in accelerated weight loss and increased mortality. Furthermore, oprozomib failed to reduce fibrosis in these mice, but rather augmented fibrotic lung remodeling in bleomycin-challenged animals. To conclude, this study identified a novel mechanism for fibrotic remodeling of the lungs involving 26S/30S proteasome activation via Rpn6 upon TGF-β-mediated myofibroblast differentiation. Increased levels of Rpn6 and polyubiquitinated proteins in IPF lungs further suggest an important contribution of the ubiquitin-proteasome system to the pathogenesis of this disease. Inhibition of the proteasome with the novel site-specific proteasome inhibitor oprozomib provided low toxicity and antifibrotic effects in alveolar epithelial cells and pulmonary fibroblasts. These results could not be confirmed in pulmonary fibrosis of bleomycin-treated mice, as oprozomib treatment showed high toxicity in fibrotic animals. In light of these data, current proteasome inhibitors, which block the catalytic core, might be too toxic as therapeutic agents for the treatment of fibrotic lung diseases. However, interference with the formation of 26S/30S proteasomes, as shown by Rpn6 knockdown, might provide a novel concept for therapeutic regulation of proteasome activity in lung fibrosis.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: Idiopathic Pulmonary Fibrosis, Proteasome, Fibroblasts
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 540 Chemie
• Fakultäten: Fakultät für Chemie und Pharmazie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 8. Oktober 2015
• 1. Bericht­erstatter:in: Wagner, Ernst