期刊
AMERICAN JOURNAL OF PHYSIOLOGY-LUNG CELLULAR AND MOLECULAR PHYSIOLOGY
卷 308, 期 1, 页码 L33-L47出版社
AMER PHYSIOLOGICAL SOC
DOI: 10.1152/ajplung.00217.2014
关键词
surfactant protein; autophagy; pulmonary fibrosis; alveolar epithelium; amphisome
资金
- National Institutes of Health [HL119436, HL090732, HL059959]
- Department of Veterans Affairs [BX001176]
- American Heart Association [13GRNT17070104]
- Cystic Fibrosis Foundation [CF GORALS12L0]
- NIH [2T32HL007586-26, T32HL007748-20]
- NATIONAL HEART, LUNG, AND BLOOD INSTITUTE [T32HL007748, R56HL059959, R01HL059959, R01HL090732, T32HL007586, R01HL119436, P01HL019737] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES [P30ES013508] Funding Source: NIH RePORTER
Mutation of threonine for isoleucine at codon 73 (I73T) in the human surfactant protein C (hSP-C) gene (SFTPC) accounts for a significant portion of SFTPC mutations associated with interstitial lung disease (ILD). Cell lines stably expressing tagged primary translation product of SP-C isoforms were generated to test the hypothesis that deposition of hSP-C-I73T within the endosomal system promotes disruption of a key cellular quality control pathway, macroautophagy. By fluorescence microscopy, wildtype hSP-C (hSP-C-WT) colocalized with exogenously expressed human ATP binding cassette class A3 (hABCA3), an indicator of normal trafficking to lysosomal-related organelles. In contrast, hSP-C-I73T was dissociated from hABCA3 but colocalized to the plasma membrane as well as the endosomal network. Cells expressing hSP-C-I73T exhibited increases in size and number of cytosolic green fluorescent protein/microtubule-associated protein 1 light-chain 3 (LC3) vesicles, some of which colabeled with red fluorescent protein from the gene dsRed/hSP-C-I73T. By transmission electron microscopy, hSP-C-I73T cells contained abnormally large autophagic vacuoles containing organellar and proteinaceous debris, which phenocopied ultrastructural changes in alveolar type 2 cells in a lung biopsy from a SFTPC I73T patient. Biochemically, hSP-C-I73T cells exhibited increased expression of Atg8/LC3, SQSTM1/p62, and Rab7, consistent with a distal block in autophagic vacuole maturation, confirmed by flux studies using bafilomycin A1 and rapamycin. Functionally, hSP-C-I73T cells showed an impaired degradative capacity for an aggregation-prone huntingtin-1 reporter substrate. The disruption of autophagy-dependent proteostasis was accompanied by increases in mitochondria biomass and parkin expression coupled with a decrease in mitochondrial membrane potential. We conclude that hSP-C-I73T induces an acquired block in macroautophagy-dependent proteostasis and mitophagy, which could contribute to the increased vulnerability of the lung epithelia to second-hit injury as seen in ILD.
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