Expression Of The Surfactant Protein C Trafficking Mutant SP‐CI73T In Vivo Results In Alterations In Alveolar Type 2 Cell Quality Control and Parenchymal Remodeling In Mice
Authors: Beers Michael Francis , Nureki Shin‐Ichi , Hawkins Arie , Russo Scott , Mulugeta Surafel 
Published: November 2023

Mutations in the gene encoding for the alveolar type 2 cell (AT2) specific protein, Surfactant Protein C (SFTPC; SP‐C), have been linked to both familial and sporadic variants of diffuse parenchymal lung disease (DPLD) in adults and children. The most common DPLD‐associated SFTPC mutation, 218T>C, results in a missense substitution of isoleucine to threonine at position 73 [I73T] in the SP‐C proprotein [proSP‐C]). Using cell lines stably expressing SP‐C isoforms, we had previously shown that in contrast to wild‐type SP‐C, the mutant SP‐CI73T proprotein is mistrafficked to the plasma membrane, accumulates in endosomal compartments, and produces a late block in macroautophagy. To define the consequences of expression of SP‐CI73T for AT2 cells in vivo, recombineering strategies were employed to selectively knock‐in an HA‐tagged mouse SP‐CI73T sequence into the sftpc locus. The founder line, whose alleles retained the PGK‐neomycin (PGK‐neo) cassette in intron 1 used for ES cell selection, demonstrates hypomorphic expression of mutant sftpc mRNA (~ 10% of wild‐type levels) permitting the animals to be born alive but subjected to the accumulation of mutant HA‐proSP‐CI73T in a time dependent fashion. Morphologically, these animals exhibit normal post‐natal alveolarization; however, by 16 weeks of age, they develop age‐dependent diffuse parenchymal lung remodeling characterized by a cellular and mesenchymal expansion of alveolar septae. By fluorescence IHC, abnormal trafficking of mutant HA‐SP‐CI73T proprotein was manifested by its prominent localization in plasma membrane and sub‐plasma membrane vesicular compartments. Subcellular fractionation and Western blotting confirmed that proSP‐CI73T was misprocessed and excluded from lamellar bodies of AT2 cells. Autophagic flux assays using AT2 cells isolated from 16 and 32 week old SP‐CI73T mice revealed aberrant LC3 (atg8) interconversion accompanied by increases in p62 expression both of which were more sensitive to bafilomycin A1 treatment than wild‐type AT2 cells. Ultrastructurally, transmission EM showing hypertrophic SP‐CI73T AT2 cells with large autophagosomal vacuoles containing mitochondria and proteinacious debris confirmed the late block in macroautophagy. Despite a 2 fold compensatory increase in total 26S proteasomal (chymotrypsin‐like) enzymatic activity and biomass, treatment of SP‐CI73T expressing cells with MG132 did not alter the level of mutant proSP‐CI73T. We conclude that expression of aberrantly trafficked and misprocessed SP‐CI73T in AT2 cells in vivo results in its exclusion from proteasomal degradation and inhibition of macroautophagy. These results offer proof of concept that disruption of epithelial cell quality control could contribute to parenchymal lung remodeling through alterations in AT2 cell homeostasis and function.Support or Funding InformationSupported by NIH HL 119436, VA Merit Award BX001176, and an Albert Rose Career Development Award from the Pulmonary Fibrosis Foundation (all to MFB).