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A2199 - The Ubiquitin-Proteasome System Regulates Mitochondria Quality Control in Alveolar Epithelial Type II Cells After Bleomycin
Author Block: H. Shaghaghi1, D. Schriner1, X. Hong2, D. Sales1, V. Desai3, A. Bhushan3, M. I. Ramirez1, R. S. Summer1, F. Romero1; 1Center for Translational Medicine and The Jane and Leonard Korman Institute, Thomas Jefferson University, Philadelphia, PA, United States, 2Medical Research Center, North China University of Science and Technology, Thangshan City, China, 3Department of Pharmaceutical Sciences, Thomas Jefferson University, Philadelphia, PA, United States.
Rationale: Mitochondria are highly dynamic organelles that undergo frequent biogenesis and clearance in order to maintain cellular homeostasis. Recently, large dysmorphic mitochondria have been shown to accumulate in the alveolar epithelium of patients with idiopathic pulmonary fibrosis (IPF), in part, because of a decrease in mitochondrial clearance, also called mitophagy. However, the underlying mechanisms leading to these cellular changes remain unknown. Objectives: To determine whether the ubiquitin-proteasome system (UPS), which has been implicated in regulating mitophagy in other cell systems, plays a role in controlling mitochondrial homeostasis in AE2 cells. Methods: Male C57BL/6 mice (2-3 months) were exposed to a one-time oropharyngeal dose of bleomycin (0.075 U) and whole lung tissues and AE2 cells were harvested at baseline, 3, and 7 days after injury. For in vitro studies, alveolar epithelial MLE12 cells were exposed to bleomycin (50-200 μg/ml) for 24h. In some studies, MLE12 cells were exposed to MG-132 (10 μM) in order to inhibit the UPS. Gene expression profiling was performed to evaluate the expression of mitochondrial biogenesis genes (PGC1, NRF-1 TFAM) and to measure TGF-β levels. Western blot analysis was performed to evaluate levels of various UPS proteins including RPN6, RPN5, 20S and FXB011. Apoptotic cell death was assessed by caspase 3 cleavage. Mitophagy was determined by quantifying levels of LC3B and p62 proteins and mitochondrial function was assessed using the Seahorse XFp Bioanalyzer. Results: We found that levels of UPS proteins were markedly increased in whole lung tissues and freshly isolated AE2 cells at 3 and 7 days after exposure to bleomycin. These findings also associated with an increase in mitophagy, as demonstrated by the conversion of LC3BI to LC3BII and the decrease in p62 levels; the expression of mitochondrial biogenesis genes was reduced at both time points. Similarly, we found that levels of UPS proteins were increased in MLE12 cells after exposure to bleomycin and comparable changes were observed in the expression of mitophagy proteins and mitochondrial biogenesis genes. Treatment with MG-132 reduced mitophagy in MLE12 exposed to bleomycin and was associated with a decline in mitochondria oxygen consumption and an increase in apoptotic cell death. Conclusions: Our data indicate that the UPS regulates mitochondrial homeostasis after bleomycin treatment and suggest that functional decline in this system could compromise the ability of AE2 cells to clear dysfunctional mitochondria and recover from genotoxic stress.