ABSTRACT The overall goal of the proposed research is to understand how enhanced expression of MUC5B contributes to epithelial cell injury and lung fibrosis. Over the past decade, we have found that: 1) a gain- of-function MUC5B promoter variant rs35705950 is the dominant risk factor for the development of IPF which has been validated by multiple independent investigators; 2) among patients with IPF, MUC5B is misexpressed in bronchioles and alveolar epithelial type 2 (AEC2) cells; 3) IPF epithelia from distal airways (<2 mm airway caliber) have a unique migratory, pro-fibrotic phenotype in vitro that is replicated ex vivo in mice exposed to bleomycin; and 4) MUC5B appears to be involved in the pathogenesis of IPF, and the concentration of Muc5b is directly related to bleomycin-induced lung fibrosis in mice. Despite these findings, we don’t fully understand how excess MUC5B in the distal lung is mechanistically linked to the development of pulmonary fibrosis. Our preliminary findings combined with the established association between endoplasmic reticulum (ER) stress and both IPF and experimental models of lung fibrosis, and the recent observation that XBP1(S) appears to be necessary and sufficient for MUC5B expression induced by the MUC5B promoter variant, suggest that while overexpression of MUC5B places individuals at risk of developing IPF by causing persistent homeostatic ER stress of bronchiolar epithelia, fibroblast recruitment and pro-fibrotic programming requires second hits (such as aging, tobacco smoke, and/or inflammation) resulting in detrimental ER stress of bronchiolar epithelia and recruitment and activation of fibroblasts. Accordingly, we hypothesize that MUC5B overexpression in bronchiolar epithelia causes homeostatic ER stress that primes responses to subsequent injury, thereby leading to persistent activation of detrimental ER stress responses that cause epithelial dysfunction during injury/repair and lead to fibroblast activation. In Aim 1, we will characterize the airway epithelial cell populations most susceptible to variant-induced MUC5B overexpression and ER stress and map these expression changes to the heterogeneity of lung fibrosis in IPF and the MUC5B promoter variant, examining the relationship between ER stress, UPR, autophagy, cell senescence, and apoptosis. In Aim 2, we will use in vitro experimental models to test the role of MUC5B and ER stress on the biophysical properties of airway epithelia and epithelial-driven activation of fibroblasts. In Aim 3, we will use Muc5b and Xbp1(S) overexpression models, and pharmacologic and genetic IRE1 pathway inhibition approaches at baseline (first hit) and in response to aging and/or bleomycin (second hits) to investigate the relationship between Muc5b, ER stress, and lung fibrosis. This research will address a crucial question about the pathobiology of IPF: How does enhanced expression of MUC5B promote epithelial injury and lung fibrosis?