Alveolar Type 2 Epithelial Cell Quality Control Responses to Pulmonary Fibrosis Related SFTPC Mutations Are Dysfunctional And Substrate Specific

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 ( SFTPC BRICHOS ) are endoplasmic reticulum retained while Linker mutations such as Sftpc I73T 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 ( Sftpc C121G ; Sftpc C185G ) activated AT2 ER stress pathways, whereas the Sftpc I73T 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 Sftpc I73T 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 Sftpc I73T mutant was not a substrate for the proteasome. In contrast, at baseline, levels of Sftpc BRICHOS mutant proproteins were significantly lower than wild‐type SP‐C in both transfected hAT2 and mouse Sftpc BRICHOS AT2 but which were dramatically increased by MG132 UPS inhibition. Conversely, autophagy inhibition raised Sftpc I73T levels but had minimal effects on BRICHOS proSP‐C levels; (iii) In AT2 from either Sftpc I73T or Sftpc C121G 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 Sftpc C121G 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 Information NIH R01 HL145408 and VA Merit 2I01BX001176‐05