Activating Transcription Factor 6 (ATF6) Acts as a Rheostat To Balance UPR Signaling and The Proteostatic Response To Mutant SFTPC Substrates By The Alveolar Epithelium

Abstract

The alveolar type II (AT2) epithelial cell is a metabolically active, biosynthetically robust component of the distal lung responsible for the synthesis of pulmonary surfactant. Disruption of AT2 cell quality control provides a molecular signature found in Idiopathic Pulmonary Fibrosis (IPF), a progressive interstitial lung disease. Mutations in the surfactant protein C (SP-C) gene ( SFTPC) are a high effect size etiological cause of PF in a subset of these patients. Prior in vitro studies from our lab showed that mutations located within the SFTPC BRICHOS domain of the SP-C proprotein result in production of ER retained and aggregation prone conformers. Two critical aspects of the proteostasis network are ER Associated Degradation (ERAD) and the unfolded protein response (UPR). Misfolded conformers activate the UPR via one or more of 3 ER transmembrane sensing proteins (Protein kinase R-like ER kinase (PERK), the Inositol-Requiring kinase 1 alpha (IRE1α) and Activating Transcription Factor 6 (ATF6). We have shown in vitro and in mouse models that clinical IPF associated BRICHOS SFTPC conformers produce unbalanced, exuberant UPR signaling and AT2 dysfunction. The goal of this study was to probe AT2 proteostatic repertoires using BRICHOS SFTPC mutants as substrates. For this, we generated stable doxycycline-inducible Mouse Lung Epithelial (MLE-12) cell lines expressing either Wild Type (SP-CWT) or an aggregating mutant (SP-CC121G) which were interrogated using cycloheximide chase and / or treatments with activators or inhibitors of ATF6, IRE1, and PERK as well as ERAD (VCP/p97), the proteasome, or macroautophagy. Readouts included fluorescence microscopy, qPCR, luciferase reporters, and immunoblotting to assess SFPTC aggregate formation, UPR signaling, and downstream ER stress related events. Using this model system, we found that SP-CC121G was ER retained and activated the UPR within 8 hours of Dox induction (as BIP expression). Commensurate with this, IRE1α (as p-IREα and spliced XBP1 (sXBP1)), ATF6 (by luciferase assay), and PERK (as p-Eif2α, ATF4, CHOP) signals were activated by SP-CC121G. The clearance of mutant SP-CC121G as well as a portion of proSP-CWT was dependent on ERAD and the Ubiquitin-Proteasome System (UPS) as their disruption using either inhibitors of p97 (EEY) or the proteasome (MG132) induced formation of higher molecular weight aggregates. Bafilomycin failed to induce SP-CC121G aggregates or block their clearance. The ATF6 agonist AA147 administered during ERAD inhibition accelerated aggregate protein degradation. When SP-CC121G MLE12 cells were treated with an ATF6 inhibitor (CEAPIN7), we observed marked increases in IRE1α kinase activity with downstream activation of JNK accompanied by commensurate activation of PERK signals and initiation of caspase-3 cleavage. Collectively, these data demonstrate a primary role for ERAD in the AT2 proteostatic response to mutant SFTPC substrates and identify ATF6 as a critical UPR rheostat to provide protection from SFTPC mutant expression both by limiting SP-C aggregate formation and balancing exuberant UPR signals from IRE1α and PERK. NIH R01 HL145408 (MFB), VA Merit Review 2I01BX001176-09 (MFB), Pulmonary Fibrosis Foundation (LRR), NIH 2T32 HL007586-36 (SB), NIH K08 HL150226 (JBK). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.