Deciphering the Dual Roles of IRE1α RNase and Kinase Activities in AT2 Cell Proteostasis and Pulmonary Fibrosis

RATIONALE: The UPR regulator inositol-requiring enzyme 1 alpha (IRE1α) plays a central role in the fate of alveolar type 2 (AT2) epithelial cells with emerging evidence from our lab and others highlighting the potential of IRE1α inhibition to mitigate pathological AT2 states associated with lung fibrosis. IRE1α possesses dual enzymatic functions: 1) RNase activity, which splices X-box binding protein 1 (XBP1) mRNA into its active transcription factor form to promote adaptive responses, and 2) kinase activity, which can activate downstream stress signaling pathways such as JNK and caspase-3 cleavage, contributing to apoptosis. This study investigates specific roles of IRE1α RNase and kinase activity in AT2 proteostasis and their impact on injury and repair processes in models of pulmonary fibrosis. Methods: To interrogate IRE1α RNase and kinase activities, we used selective pharmacologic inhibitors in MLE12 cells under proteostatic stress: ORIN1001, an RNase-specific inhibitor, and SP600125, a JNK kinase inhibitor. Genetic validation was performed using kinase-dead/RNase-dead mutants overexpressed in IRE1α deficient MLE12s. IRE1α's RNase mediated activity was tested in vivo using a mouse model with AT2 cell-specific knockout of XBP1 subjected to bleomycin-induced lung injury. At day 21 post-bleomycin exposure we assayed for lung injury and physiology, inflammatory infiltrates, and collagen deposition. RNA and protein lysates from isolated AT2 cells were analyzed for markers of UPR pathways and XBP1-targets. The effects of XBP1 depletion on AT2 progenitor function was assessed using XBP1-deficient primary AT2 cells placed in feeder-free 3D cultures. Results: Pharmacologic or geneticinhibition of IRE1α kinase activity in MLE12 in vitro reduced pro-apoptotic signaling. Overexpression of constitutively active (spliced) XBP1 in MLE12 cells reduced terminal UPR signaling. Compared to wild-type controls, mice with AT2-specific XBP1 deletion exhibited increased susceptibility to bleomycin, as evidenced by impaired lung function, elevated BAL total protein and inflammatory cells, increased fibrillar and soluble collagen content as well as decreased total AT2 cell numbers. Ex vivo , XBP1-deficient AT2 cells demonstrated impaired proliferation, accompanied by reduced expression of molecular chaperones such as Erdj4, indicative of impaired proteostasis. Conclusions: While IRE1α's kinase activity promotes pro-apoptotic signaling via JNK activation, its RNase activity supports AT2 cell survival and proteostasis mediated by splicing of XBP1 mRNA. These findings underscore the importance of the IRE1α-XBP1 axis in AT2 cell health and suggest that selective modulation of this pathway may offer therapeutic potential for pulmonary fibrosis. Understanding distinct kinase and RNase activities of IRE1α is critical to developing targeted therapies that preserve adaptive UPR signaling while mitigating pro-fibrotic stress responses. NIH U01 HL145408, NIH T32 HL007586, and VA Merit 2I01BX001176-05 This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.