Probes for YAP

Abstract

RATIONALE:

The lung ECM in IPF mediates progression of fibrosis by decreasing fibroblast expression of microRNA-29 (miR-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-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 employed 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 micro-RNA processing machinery in IPF lung specimens revealed decreased Dicer1 expression in the procollagen-rich myofibroblastic core of fibroblastic foci compared to the focus perimeter and adjacent alveolar walls. Mechanistically, IPF ECM increased association of the Dicer1 transcript with RNA binding protein AUF1, 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.