Alveolar Type 2 Epithelial Cell Quality Control Responses to Pulmonary Fibrosis Related SFTPC Mutations Are Dysfunctional And Substrate Specific
Authors: Beers Michael F. , Rodriguez Luis , Hawkins Arie , Russo Scott J. , Tomer Yaniv , Mulugeta Surafel , Katzen Jeremy 
Published: April 2020

Mutations in the alveolar type 2 cell (AT2) restricted Surfactant Protein C gene [SFTPC] gene are associated with pulmonary fibrosis (PF) in humans. Two classes of disease related SFTPC mutations present distinct challenges to AT2 proteostasis: aggregation‐prone BRICHOS mutants (SFTPCBRICHOS) are endoplasmic reticulum retained while Linker mutations such as SftpcI73T cause proSP‐C mistrafficking. The ubiquitin proteasome system (UPS) and macroautophagy (autophagy) are the main cellular quality control (QC) pathways for regulation of aberrant protein levels. The goal of this study was to leverage novel in vivo mouse models and human AT2 (hAT2) cells expressing Sftpc mutations to characterize proteostastic responses to mutant SP‐C substrates. In vitro primary hAT2 and mAT2 cells each display a complete proteostatic repertoire with significant degrees of chymotrypsin‐like proteasomal activity as well as mTOR dependent macroautophagy. UPS inhibition by MG132 also resulted in increased LC3‐II suggesting cross‐talk between the two pathways. In vivo, expression of BRICHOS mutations (SftpcC121G; SftpcC185G) activated AT2 ER stress pathways, whereas the SftpcI73T linker mutant inhibited autophagy. The AT2 proteostatic response to Sftpc isoforms was both distinct and overlapping: (i) Transcriptional changes in AT2 from the Sftpc murine models interrogated by bulk RNAseq revealed an enrichment in chaperones limited to BRICHOS mutants and autophagy genes enriched only in the SftpcI73T mice. There was a shared enrichment in proteasome core genes; (ii) Functional studies demonstrated that in either mAT2 isolated from Sftpc mice or hAT2 cells transiently expressing mutant proSP‐C isoforms, the SftpcI73T mutant was not a substrate for the proteasome. In contrast, at baseline, levels of SftpcBRICHOS mutant proproteins were significantly lower than wild‐type SP‐C in both transfected hAT2 and mouse SftpcBRICHOS AT2 but which were dramatically increased by MG132 UPS inhibition. Conversely, autophagy inhibition raised SftpcI73T levels but had minimal effects on BRICHOS proSP‐C levels; (iii) In AT2 from either SftpcI73T or SftpcC121G mice, the increased proteasome transcripts were accompanied by increases in both proteasome mass and activity but this failed to compensate for accumulation of mutant BRICHOS or Linker isoforms (iv) AT2 from SftpcC121G mice also demonstrated marked increases in polyubiquitinated substrates suggesting a global disruption of proteostasis by these isoforms. These results indicate that the proteasome represents the primary protein QC pathway in AT2 cells in the distal lung epithelium but this pathway is partially substrate restricted. We have also identified a functional limitation of the AT2 cell in managing malformed protein conformers associated with fibrotic lung disease in humans.Support or Funding InformationNIH R01 HL145408 and VA Merit 2I01BX001176‐05