Idiopathic pulmonary fibrosis (IPF) is a progressive and end-stage lung disease of unknown etiology and no cure. It is likely that genetic changes increase a person's risk of developing IPF, and then exposure to certain environmental factors and/or aging trigger the onset of the disease. The MUC5B promoter variant rs35705950 is present in ∼50% of individuals with IPF and is recognized as the strongest known risk factor (genetic and otherwise) for the development of IPF. This variant leads to overexpression of MUC5B mRNA and protein in both distal airway epithelia and honeycomb cysts in the peripheral lung. These observations raise the question of why excessive MUC5B expression in distal airways is associated with IPF? Recently, our laboratory identified for the first time that the ER stress transcription factor XBP1S is highly expressed in the epithelium lining the distal airways and honeycomb cysts of IPF lung, activates MUC5B gene expression by direct binding to its promoter. Further, XBP1S differentially regulating the MUC5B promoter variant. These data connected activation of ER stress with excessive MUC5B expression in a promoter variant-dependent model likely impairs mucociliary clearance and function of distal airway stem cells that increase susceptibility of development of pulmonary fibrosis. We hypothesize that activation of XBP1S induces MUC5B expression in the distal airways that promotes pulmonary fibrosis. To test this central hypothesis, we propose the following aims: 1) XBP1S- mediated MUC5B secretion in distal airway epithelium enhances susceptibility to development of pulmonary fibrosis in vivo. We will assess the role of XBP1S by exposing Xbp1 airway epithelium-specific overexpression or deletion mice to bleomycin induced respiratory epithelial injury. 2) XBP1S-mediated MUC5B secretion impairs distal airway stem cell function to repair peripheral lung epithelia after injury. We will utilize mouse airway epithelium-lineage tracing system and in vitro 2-D and 3-D differentiation assays to evaluate the role of XBP1S and mucin in maintenance of the airway stem cell homeostasis in response to injury. 3) Activation of XBP1S and presence of MUC5B promoter variant cause abnormal mucus secretion, impaired mucociliary clearance and activation of myofibroblast differentiation. We will analyze the biochemical and biophysical properties of the secreted mucus and electrophysiology of the DAE carrying the MUC5B promoter variant rs35705950 at baseline and after activation of ER stress. We will also test whether XBP1S-expressing DAE carrying the MUC5B promoter variant and/or hypoxia-induced epithelial injury directly promotes myofibroblast differentiation. Completing the aims proposed in this application will provide novel mechanisms underlying ER stress-mediated excessive mucin secretion by DAE promotes pulmonary fibrosis. These mechanistic studies will likely identify novel biomarkers for early diagnosis as well as therapeutic targets (e.g., s...