Activating Transcription Factor 6 (ATF6) Modulates The Proteostatic Response To Mutant Surfactant Protein C Conformers In Alveolar Epithelial Cells

Alveolar Type II (AT2) epithelial cell dysfunction is a hallmark of the pathogenesis of 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 SFTPC mutations located within the BRICHOS domain of the SP-C proprotein result in production of ER retained and aggregation prone conformers. A major aspect of the proteostasis network is the unfolded protein response (UPR), which is initiated when misfolded proteins activate one or more of 3 ER transmembrane sensing proteins (Protein kinase R-like ER kinase (PERK), the Inositol-Requiring kinase 1 (IRE1) and Activating Transcription Factor 6 (ATF6). Consistent with this, we published that clinical IPF associated BRICHOS SFTPC mutations activate all 3 arms of UPR/ER stress signaling in mice in vivo. The goal of this study was to define AT2 proteostatic responses to SFTPC expression and the contribution of ATF6 to the UPR. 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 (vp97) or autophagy. Readouts included qPCR, luciferase reporter assays, and immunoblotting to assess UPR signaling and apoptosis, coupled with fluorescence microscopy to localize SP-C expression. Using this model system we found that SP-CC121G was ER retained and activated the UPR within 8 hours of Dox induction expression as measured by BIP expression. Commensurate with this both IRE1a (as spliced XBP1 (sXBP1)) and ATF6 were activated by SP-CC121G. The clearance of both mutant SP-CC121G as well as a portion of proSP-CWT was dependent on ERAD as its disruption using the vp97 inhibitor Eeyarestatin I induced formation of higher molecular weight aggregates while bafilomycin failed to induce SP-CC121G aggregates or block their clearance. ATF6 and IRE1 activation during ERAD inhibition accelerated aggregate protein degradation. When SP-CC121G MLE12 cells were treated with either an ATF6 inhibitor or an IRE1 activator, marked increases in levels of C/EBP Homologous Protein (CHOP) occur while either ATF6 agonism or IRE1 inhibition decreased CHOP expression. Collectively, these our data demonstrate a primary role for ERAD in the proteostatic response to SP-C expression while ATF6 agonism appears to provide protection from SFTPC mutant expression in part by accelerating degradation of SP-C aggregates in the face of IRE1 UPR signaling. NIH R01 HL145408 (MFB), VA Merit Review 2I01BX001176 (MFB) and NIH F32HL160011 (LRR) This is the full abstract presented at the American Physiology Summit 2023 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.