Background/Purpose:
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).

Methods:
The expression of EN-1 in skin tissue and in human dermal fibroblasts was
determined by real-time PCR, Western blot and immunohistochemistry. 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 collagen mRNA and protein. Additionally, overexpression
of EN-1 enhanced the profibrotic effect of TGF-β with myofibroblast differentiation
and increased collagene release. 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.

Conclusion:
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.