Feghali‐Bostwick, Carol A.

ABSTRACT Myofibroblasts play key roles in wound healing and pathological fibrosis. Here, we used an RNAi screen to characterize myofibroblast regulatory genes, using a high-content imaging approach to quantify α-smooth muscle actin stress fibers in cultured human fibroblasts. Screen hits were validated on physiological compliance hydrogels, and selected hits tested in primary fibroblasts from patients with idiopathic pulmonary fibrosis. Our RNAi screen led to the identification of STAT3 as an essential mediator of myofibroblast activation and function. Strikingly, we found that STAT3 phosphorylation, while responsive to exogenous ligands on both soft and stiff matrices, is innately active on a stiff matrix in a ligand/receptor-independent, but ROCK- and JAK2-dependent fashion. These results demonstrate how a cytokine-inducible signal can become persistently activated by pathological matrix stiffening. Consistent with a pivotal role for this pathway in driving persistent fibrosis, a STAT3 inhibitor attenuated murine pulmonary fibrosis when administered in a therapeutic fashion after bleomycin injury. Our results identify novel genes essential for the myofibroblast phenotype, and point to STAT3 as an important target in pulmonary fibrosis and other fibrotic diseases.

Pathological fibrosis is driven by a feedback loop in which the fibrotic extracellular matrix is both a cause and consequence of fibroblast activation. However, the molecular mechanisms underlying this process remain poorly understood. Here we identify yes-associated protein (YAP) (homolog of drosophila Yki) and transcriptional coactivator with PDZ-binding motif (TAZ) (also known as Wwtr1), transcriptional effectors of the Hippo pathway, as key matrix stiffness-regulated coordinators of fibroblast activation and matrix synthesis. YAP and TAZ are prominently expressed in fibrotic but not healthy lung tissue, with particularly pronounced nuclear expression of TAZ in spindle-shaped fibroblastic cells. In culture, both YAP and TAZ accumulate in the nuclei of fibroblasts grown on pathologically stiff matrices but not physiologically compliant matrices. Knockdown of YAP and TAZ together in vitro attenuates key fibroblast functions, including matrix synthesis, contraction, and proliferation, and does so exclusively on pathologically stiff matrices. Profibrotic effects of YAP and TAZ operate, in part, through their transcriptional target plasminogen activator inhibitor-1, which is regulated by matrix stiffness independent of transforming growth factor-β signaling. Immortalized fibroblasts conditionally expressing active YAP or TAZ mutant proteins overcome soft matrix limitations on growth and promote fibrosis when adoptively transferred to the murine lung, demonstrating the ability of fibroblast YAP/TAZ activation to drive a profibrotic response in vivo. Together, these results identify YAP and TAZ as mechanoactivated coordinators of the matrix-driven feedback loop that amplifies and sustains fibrosis.

Abstract Rationale Transforming growth factor (TGF)-β has a central role in driving many of the pathological processes that characterize pulmonary fibrosis. Inhibition of the integrin αvβ6, a key activator of TGF-β in lung, is an attractive therapeutic strategy, as it may be possible to inhibit TGF-β at sites of αvβ6 up-regulation without affecting other homeostatic roles of TGF-β. Objectives To analyze the expression of αvβ6 in human pulmonary fibrosis, and to functionally test the efficacy of therapeutic inhibition of αvβ6-mediated TGF-β activation in murine bleomycin–induced pulmonary fibrosis. Methods Lung biopsies from patients with a diagnosis of systemic sclerosis or idiopathic pulmonary fibrosis were stained for αvβ6 expression. A range of concentrations of a monoclonal antibody that blocks αvβ6-mediated TGF-β activation was evaluated in murine bleomycin–induced lung fibrosis. Measurements and Main Results αvβ6 is overexpressed in human lung fibrosis within pneumocytes lining the alveolar ducts and alveoli. In the bleomycin model, αvβ6 antibody was effective in blocking pulmonary fibrosis. At high doses, there was increased expression of markers of inflammation and macrophage activation, consistent with the effects of TGF-β inhibition in the lung. Low doses of antibody attenuated collagen expression without increasing alveolar inflammatory cell populations or macrophage activation markers. Conclusions Partial inhibition of TGF-β using αvβ6 integrin antibodies is effective in blocking murine pulmonary fibrosis without exacerbating inflammation. In addition, the elevated expression of αvβ6, an activator of the fibrogenic cytokine, TGF-β, in human pulmonary fibrosis suggests that αvβ6 monoclonal antibodies could represent a promising new therapeutic strategy for treating pulmonary fibrosis.