POS0328\u2005ENGRAILED 1 COORDINATES CYTOSKELETAL ORGANIZATION TO PROMOTE MYOFIBROBLAST DIFFERENTIATION AND FIBROTIC TISSUE REMODELING

Abstract

Engrailed 1 (EN1) is a homeodomain-containing transcription factor with essential roles in embryonic development. In most cell types, the expression of EN1 is restricted to embryonic development. However, under pathological conditions, EN1 can be re-expressed to promote phenotypical adaptation. En1 is transiently expressed in the developing dermis of murine embryos in a distinct fibroblast lineage and silenced before birth (1). Former EN1-expressing cells give rise to a subpopulation of fibroblasts that has a high capacity for extracellular matrix production in adult murine skin. The role of EN1 in systemic sclerosis (SSc) was previously not explored.To study the role of EN1 in the pathological activation of fibroblasts in tissue fibrosis.Bulk RNA-Seq and EN1 or SP1 ChIP-Seq were performed from cultured human dermal fibroblasts. The expression of EN1 was inhibited by siRNA. Cytoskeletal drugs paclitaxel, vinblastin and ROCK inhibitor (Y27632) were used to modulate the cytoskeleton in EN1 knockdown or overexpressing dermal fibroblasts. The role of EN1 in fibroblast activation was evaluated by functional experiments with EN1 knockdown or overexpression in standard 2D culture systems as well as in 3D skin equivalent models. The role of EN1 in skin fibrosis was further studied in En1fl/fl X Col6Cre mice, with fibroblast-specific knockout of En1 in three complementary mouse models: overexpression of a constitutively active TGFß-receptor I (TBRICA), bleomycin-induced skin fibrosis and TSK1 mice.Pathologically activated dermal fibroblasts from SSc patients express higher levels of EN1 compared with age and sex matched healthy individuals in the skin and in vitro. TGFβ induces EN1 expression in fibroblasts in a SMAD3-dependent manner both in cultured fibroblasts and in murine skin. Knockdown of EN1 prevents TGFβ-induced fibroblast activation, whereas overexpression of EN1 fosters the pro-fibrotic effects of TGFβ with increased expression of αSMA, stress fibers and collagen. RNA sequencing demonstrates that EN1 induces a pro-fibrotic gene expression profile functionally related to cytoskeleton organization and ROCK activation. In silico analyses of the promoters of En1 target genes coupled with siRNA-mediated knockdown demonstrated that EN1 regulates these pro-fibrotic target genes by modulating the activity of regulatory modules that contain transcription factors of the specificity protein (SP) family. Functional experiments with selective modulators of ROCK and of microtubule polymerization confirm the coordinating role of EN1 on ROCK activity and the re-organization of cytoskeleton during myofibroblast differentiation in both conventional culture systems and 3D skin equivalents. Consistently, mice with fibroblast-specific knockout of En1 demonstrate impaired fibroblast-to-myofibroblast transition, reduced dermal thickening and impaired collagen deposition in the TBRICA, bleomycin-induced and TSK1 models.We characterize the homeodomain transcription factor EN1 as a molecular amplifier of TGFβ signaling in myofibroblast differentiation that coordinates cytoskeletal organization in a SP-dependent manner. EN1 might thus be a novel candidate for molecular targeted therapies to interfere with myofibroblast differentiation in fibrotic diseases.[1]Rinkevich Y, Walmsley GG, Hu MS, Maan ZN, Newman AM, Drukker M, et al. Skin fibrosis. Identification and isolation of a dermal lineage with intrinsic fibrogenic potential. Science. 2015;348(6232):aaa2151.Andrea-Hermina Györfi: None declared, Alexandru-Emil Matei: None declared, Maximilian Fuchs: None declared, Aleix Rius Rigau: None declared, Xuezhi Hong: None declared, ZHU Honglin: None declared, Markus Luber: None declared, Christina Bergmann: None declared, Clara Dees: None declared, Ingo Ludolph: None declared, Raymund Horch: None declared, Oliver Distler Consultant of: Actellion, AbbVie, Acceleron Pharma, Anamar, Amgen, Blade Therapeutics, CSL Behring, ChemomAb, Ergonex, Glenmark Pharma, GSK, Inventiva, Italfarmaco, iQvia, Medac, Medscape, Lilly, Sanofi, Target BioScience, UCB, Bayer, Boehringer Ingelheim, Catenion, iQone, Menarini, Mepha, Novartis, Mitsubishi, MSD, Roche, Pfizer, Georg Schett: None declared, Meik Kunz: None declared, Jörg H.W. Distler Consultant of: Actelion, Active Biotech, Anamar, ARXX, Bayer Pharma, Boehringer Ingelheim, Celgene, Galapagos, GSK, Inventiva, JB Therapeutics, Medac, Pfizer, RuiYi and UCB., Grant/research support from: Anamar, Active Biotech, Array Biopharma, aTyr, BMS, Bayer Pharma, Boehringer Ingelheim, Celgene, Galapagos, GSK, Inventiva, Novartis, Sanofi-Aventis, RedX, UCB