Project Summary/Abstract Idiopathic pulmonary fibrosis (IPF) is a disease of progressive interstitial fibrosis, which leads to severe debilitation and eventually respiratory failure and death. Recent studies have implicated endoplasmic reticulum stress (ER stress) and the resulting unfolded protein response (UPR) in the pathophysiology of pulmonary fibrosis, including findings of increased UPR signaling and epithelial cell apoptosis in patients with hereditary and sporadic IPF. Despite these observations, it remains unclear how UPR activation leads mechanistically to fibrosis. The laboratory of Dr. Feroz Papa (co-sponsor) has developed KIRA8, a highly specific small molecule inhibitor of IRE1α, the most deeply-conserved mediator of the UPR. In unpublished work, the Sheppard and Papa laboratories (sponsor and co-sponsor, respectively) have shown that KIRA8 decreases markers of fibrosis in mice exposed to bleomycin. TGFβ is a well-established driver of tissue fibrosis. Mice lacking integrin αvβ6, a critical activator of extracellular, latent TGFβ, exhibit decreased UPR signaling and are correspondingly protected from fibrosis. In the MLE12 lung epithelial cell line, KIRA8 inhibition of IRE1α decreases SMAD2 phosphorylation, an early step in the TGFβ signaling pathway. Together, these data suggest the central model of the proposal: that TGFβ and UPR signaling conspire to promote excessive collagen deposition and pathological fibrosis. The proposed research will dissect the molecular mechanisms by which the UPR enhances TGFβ signaling and fibrosis. The first aim of the study seeks to identify and characterize the relevant cell types undergoing ER stress in the lungs of mice exposed to bleomycin, using immunofluorescence staining and cell-type specific purification of messenger RNA (mRNA) and microRNA (miRNA) from epithelial cells and fibroblasts. The second aim of the study will probe interactions between TGFβ signaling and the UPR in cell lines and mice. Because IRE1α is known to modulate miRNAs that are thought to regulate components of the TGFβ signaling pathway, modulation of miRNAs is likely to be a mechanism by which the UPR enhances TGFβ signaling. In cell lines, TGFβ signaling will be evaluated after the UPR has been activated or inactivated by chemical and genetic techniques. Levels of miRNAs, particularly miR-17, miR-200, and miR-150, will be measured by quantitative PCR, and regulation of candidate miRNA targets evaluated by dual luciferase assay. These results will be extended in vivo by analyzing mRNA and miRNA purified from epithelial cells and fibroblasts. The proposed studies will advance the fundamental understanding of the mechanisms of pulmonary fibrosis and offer novel targets for therapy of this devastating disease.