ZEB1 Serves as a Key Regulator of Pro-fibrotic Signaling in Lung Fibrosis by Modulating miR-96-Dependent Regulation of FoxO3a

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Abstract Rationale: Fibroblast apoptosis is normally triggered by interactions with a collagen-rich matrix, but this homeostatic response is suppressed in idiopathic pulmonary fibrosis (IPF) fibroblasts. Increased miR-96 in IPF fibroblasts leads to the reduction of FoxO3a, a transcriptional activator that promotes fibroblast apoptosis. We found a consensus binding site for ZEB1, a transcription factor that is increased in IPF mesenchymal cells, in the promoter region of miR-96. Additionally, a ZEB1 binding site was found in the lysyl oxidase homolog 2 (LOXL2, a collagen cross-linking enzyme), suggesting a role for ZEB1 in the regulation of the biomechanical properties of the extracellular matrix (ECM). We hypothesize that ZEB1 acts as a central nexus of pro-fibrotic signaling by regulating both fibroblast apoptosis resistance and ECM crosslinking. The goal of this study is to examine the role of ZEB1 in the pathogenesis of lung fibrosis and explore the potential of targeting ZEB1 as a novel anti-fibrotic approach. Methods: Control and IPF fibroblasts (n=5 each) were cultured on decellularized matrices from non-IPF and IPF patients, and miR-96 and FoxO3a levels were measured. Fibroblasts with transfected with siRNA targeting ZEB1 (or non-targeting siRNA) were then cultured in the presence/absence of TGF-β1, on 3D collagen matrices, on silicone gels of varying stiffness (0.5 & 64 KPa), or on methacrylated gelatin (GelMA) hydrogels. ZEB1, FoxO3a, LOXL2, and caspase 3/7 levels were assessed. Finally, the potential anti-fibrotic role of a ZEB1 inhibitor and antimiR-96 was assessed using an in vivo model. Results: Review of transcriptomic data available via the IPF Cell Atlas confirmed that ZEB1 is increased in IPF mesenchymal cells. We found that ZEB1 was induced by TGF-β.ZEB1 and miR-96 expression were significantly elevated in IPF fibroblasts compared to normal fibroblasts, while FoxO3a and its target proteins were reduced when cells were cultured on decellularized ECM, stiff silicone gels, and GelMA hydrogels. A similar expression pattern was observed in the lungs of bleomycin-induced fibrotic mice, where ZEB1 and miR-96 levels were elevated, and FoxO3a levels were decreased. In vitro inhibition of ZEB1 reduced miR-96, collagen, and LOXL2 expression but increased FoxO3a and enhanced fibroblast apoptosis. In vivo, ZEB1 or miR-96 targeting significantly reduced lung fibrosis in bleomycin-treated mice. Conclusions: Our findings suggest that ZEB1 is a central regulator of pro-fibrotic signaling in lung fibrosis, governing both fibroblast resistance to apoptosis and ECM crosslinking. Targeting ZEB1 may represent a novel therapeutic approach for treating fibrotic lung diseases.

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