Engrailed 1 coordinates cytoskeletal re-organization to induce myofibroblast differentiation

Transforming growth factor-ß (TGFß) is a key mediator of fibroblast activation in fibrotic diseases including systemic sclerosis. Here we show that Engrailed 1 (EN1) is re-expressed in multiple fibroblast subpopulations in the skin of SSc patients. We characterize EN1 as a molecular amplifier of TGFß signaling in myofibroblast differentiation: TGFß induces EN1 expression in a SMAD3-dependent manner, and, in turn, EN1 mediates the pro-fibrotic effects of TGFß. RNA sequencing demonstrates that EN1 induces a pro-fibrotic gene expression profile functionally related to the cytoskeleton organization and ROCK activation. EN1 regulates gene expression by modulating the activity of SP1 and other SP-transcription factors, as confirmed by ChIP-seq experiments for EN1 and SP1. Functional experiments confirm the coordinating role of EN1 on ROCK activity and the re-organization of cytoskeleton during myofibroblast differentiation both in standard fibroblast culture systems and in in vitro skin models. Consistently, mice with fibroblast-specific knockout of En1 demonstrate impaired fibroblast-to-myofibroblast transition and are partially protected from experimental skin fibrosis. Overall design: EN1 and SP1 genomic targets in dermal fibroblasts stimulated with TGFß with siRNA-induced knockdown of EN1, with fibroblasts stimulated with TGFß and treated with non-targeting siRNA as controls