Abstract
RATIONALE: The lung extracellular matrix (ECM) in idiopathic pulmonary fibrosis (IPF) mediates progression of fibrosis by decreasing fibroblast expression of miR-29 (microRNA-29), a master negative regulator of ECM production. The molecular mechanism is undefined. IPF-ECM is stiffer than normal. Stiffness drives fibroblast ECM production in a YAP (yes-associated protein)-dependent manner, and YAP is a known regulator of miR-29. Therefore, we tested the hypothesis that negative regulation of miR-29 by IPF-ECM was mediated by mechanotransduction of stiffness. OBJECTIVES: To determine how IPF-ECM negatively regulates miR-29. METHODS: We decellularized lung ECM using detergents and prepared polyacrylamide hydrogels of defined stiffness by varying acrylamide concentrations. Mechanistic studies were guided by immunohistochemistry of IPF lung and used cell culture, RNA-binding protein assays, and xenograft models. MEASUREMENTS AND MAIN RESULTS: Contrary to our hypothesis, we excluded fibroblast mechanotransduction of ECM stiffness as the primary mechanism deregulating miR-29. Instead, systematic examination of miR-29 biogenesis revealed a microRNA processing defect that impeded processing of miR-29 into its mature bioactive forms. Immunohistochemical analysis of the microRNA processing machinery in IPF lung specimens revealed decreased Dicer1 expression in the procollagen-rich myofibroblastic core of fibroblastic foci compared with the focus perimeter and adjacent alveolar walls. Mechanistically, IPF-ECM increased association of the Dicer1 transcript with RNA binding protein AUF1 (AU-binding factor 1), and Dicer1 knockdown conferred primary human lung fibroblasts with cell-autonomous fibrogenicity in zebrafish and mouse lung xenograft models. CONCLUSIONS: Our data identify suppression of fibroblast Dicer1 expression in the myofibroblast-rich IPF fibroblastic focus core as a central step in the mechanism by which the ECM sustains fibrosis progression in IPF.