Background Systemic sclerosis (SSc) is a chronic fibrotic connective disease of unknown etiology that affects the skin and internal organs. Transforming growth factor-β (TGFβ) has been characterized as a key-mediator of fibroblast activation in SSc. However, the intracellular signaling cascades that control TGFβ signaling and the TGFβ-induced activation of fibroblasts are still incompletely understood. Homeobox containing transcription factor, Engrailed-1 plays a key role in embryonic development and has also been linked to disease, including cancer. EN-1 is induced by proinflammary cytokines and oxidative stress which play an important role in Systemic Sclerosis (SSc).
Objectives The aim of this study was to determine the role of the embryonic homeoprotein EN-1 in fibroblast activation and tissue fibrosis in systemic sclerosis (SSc).
Methods The expression of EN-1 in skin tissue and in human dermal fibroblasts was determined by real-time PCR, Western blot and immunofluorescence. Knock-down and overexpression strategies were used to evaluate the effect of EN-1 on fibroblast activation. The outcome of mice with fibroblast-specific knockout of EN-1 (EN1 fibKO) was evaluated in bleomycin-induced skin fibrosis; fibrosis induced by overexpression of a constitutively active TGF-β receptor I (TBRIact) and in the Tsk model which resembled the later stages of SSc. Co-IP and CAGA Reporter assay were performed to study the physical and functional interaction between EN-1 and Smad3.
Results A five-fold increased expression of EN-1 was detected in the upper layer of the dermis of SSc patients on fibroblasts double stained for EN-1 and anti-prolyl-4-hydroxylase. EN-1 expression was induced by TGF-β in cultured fibroblasts and treatment with the TBR inhibitor SD-208 prevented the induction of EN-1 by two-fold decrease in experimental fibrosis. Fibroblasts lacking EN-1 were less sensitive to the pro-fibrotic effects of TGF-β with impaired induction of target genes mRNA and protein. Additionally, overexpression of EN-1 enhanced the profibrotic effect of TGF-β with myofibroblast differentiation and increased collagene release as well as mRNA and protein levels of target genes. Function studies demonstrated that EN-1 interacts with Smad3 to regulate the pro-fibrotic effects of TGF-β. Co-IP demonstrated that TGF-β induces binding of EN-1 to Smad3. Reporter study and analyses of the expression of classical Smad target genes such as PAI-1 demonstrated that the binding of EN-1 to Smad3 stimulates the transcriptional activity of Smad3. In the model of bleomycin-induced fibrosis dermal thickening, hydroxyproline content and myofibroblast counts were significantly decreased in EN1 fibKO mice as compared to wildtype littermates. EN1 fibKO also protected from TBRIact-induced fibrosis and ameliorated fibrosis in the Tsk1 model.
Conclusions We demonstrate for the first time a role of EN-1 in fibroblast activation and tissue fibrosis. Deficiency in EN-1 reduced the stimulatory effect of TGF-β on fibroblasts by interfering with canonical Smad and protected from experimental fibrosis in different mouse models. Considering the potent anti-fibrotic effects observed in this study, EN-1 might be a candidate for molecular targeted therapies of SSc.
Disclosure of Interest None declared