The Ying and the Yang: Compensatory UPR Signaling Responses Observed In An In-Vitro Model Expressing Clinical Mutant Surfactant Protein C Isoforms.

Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease characterized by alveolar type II (AT2) cell dysfunction. Mutations in surfactant protein C (SP-C) are a high effect size, recognized etiological cause of IPF. The SP-C proprotein contains an ~100 KDa Bri2 chondromodulin-1 and prosurfactant protein C (BRICHOS) domain that acts as a molecular chaperone preventing protein aggregation. The presence of misfolded proteins in the ER can be sensed by three transmembrane sentinel proteins - activating transcription factor 6 (ATF6), protein kinase R-like ER kinase (PERK), and inositol-requiring enzyme 1α (IRE1α) - which trigger an intracellular signaling pathway called the unfolded protein response (UPR). Previous characterization of our in-vitro and in-vivo models has demonstrated that BRICHOS mutants activate all 3 arms of the UPR, activate a proinflammatory cascade, and drive AT2 cell death. In-vivo, their expression initiates pathological fibroproliferative lung remodeling, characteristic of human IPF. Using our in-vitro models of BRICHOS-driven ER stress, we explore potential IPF therapeutic intervention targeting two upstream sensors of the UPR, PERK and IRE-1. Mouse lung epithelial (MLE-12) cells were transfected with wildtype (WT) or BRICHOS mutant (C121G) SP-C plasmids in media containing inhibitors of either PERK signaling or IRE-1 RNase activity. Pathway readouts included: quantification of cell death via LDH cytotoxicity and flow cytometry, protein immunoblots to detect UPR activation and apoptotic signaling, QPCR of spliced X-Box Binding Protein 1 (XBP1) and UPR transcriptional targets, and high-resolution microscopy to observe altered SP-C trafficking. Within 48 hours of transfection of MLE-12 cells with SP-C we observed activation of the 3 arms of the UPR, upregulated downstream targets, and initiation of cell death. Inhibition of PERK resulted in down-regulation of PERK pathway proteins and transcriptional targets, increased SP-C protein expression, and promoted increased XBP1 splicing. Inhibition of IRE1 RNase inhibited splicing of XBP1, reduced expression of XBP1 transcriptional targets, and increased JNK phosphorylation. Inhibition of either pathway alone did not reduce cell death, though partial reduction of cell death was noted with either small molecule targeted caspase 3/8 or necroptosis inhibition. Our data suggests that while intervention directed at one arm of the UPR may block proinflammatory signaling or transcriptomic reprogramming, this strategy may not be sufficient to completely inhibit cell death. These findings highlight the interconnectivity and redundancy of the cell quality control machinery and the emerging role of necroptosis due to chronic UPR stress-mediated cell death. Further characterization of this pathway is needed to improve IPF therapeutic outcomes.