Fortilin binds IRE1β to facilitate mucin 5AC expression via the IRE1β/XBP1 signaling pathway

RATIONALE: Airway mucus is a complex process influenced by various factors and signaling pathways. A key player is mammalian inositol-requiring enzyme 1 beta (IRE1β), a paralog of IRE1 alpha (IRE1α), found only in epithelial cells lining the mucosal surfaces of the gastrointestinal and respiratory tracts. IRE1β processes X-box binding protein 1 (XBP1) mRNA via its endoribonuclease (RNase) domain, generating the active XBP1 spliced form (XBP1s). XBP1s is crucial for mucin production, the main component of mucus. IRE1β is upregulated in human bronchial epithelial (HBE) cells from individuals with cystic fibrosis and asthma. Fortilin binds to IRE1α, blocking its kinase/RNase functions and preventing cell death. However, the interaction between fortilin and IRE1β, and its effects on airway mucus under basal conditions, remain unknown. OBJECTIVES: We investigate whether fortilin binds IRE1β, regulates its RNase activity, and is associated with IRE1β-mediated mucin expression. METHODS: The interaction between fortilin and IRE1β was examined by immunoprecipitation and microscale thermophoresis. IRE1β RNase and kinase activities were assessed using in vitro RNase and kinase assays in the presence of fortilin and the IRE1 inhibitor KIRA8. Functional effects of fortilin were evaluated in CRISPR/Cas9-mediated fortilin-knockout HBE cells cultured under air-liquid interface conditions. IRE1β-mediated mucin expression was assessed by RT-qPCR and western blot analyses. MEASUREMENTS AND MAIN RESULTS: We find that fortilin binds to the cytosolic domain of IRE1β, significantly increasing its RNase and kinase activities. Furthermore, fortilin depletion significantly attenuates mucin 5AC (MUC5AC) expression by reducing XBP1 splicing and AKT phosphorylation in differentiated HBE cells under air-liquid interface culture (ALI-HBE). IRE1 inhibitor KIRA8 blunts IRE1β kinase/RNase activities in ALI-HBE cells, inhibiting both XBP1 splicing and AKT phosphorylation regardless of fortilin presence. CONCLUSIONS: These data suggest that fortilin promotes IRE1β-mediated MUC5AC expression primarily via the IRE1β/XBP1 signaling pathway. The IRE1β-fortilin complex holds promise for developing innovative therapies to regulate mucin production in conditions characterized by airway mucus hypersecretion, including chronic obstructive pulmonary disease, asthma, bronchiectasis, and cystic fibrosis.