FRI0523 Activating Transcription Factor 3 Regulates Canonical Tgf-[beta] Signaling in Systemic Sclerosis

Background Activating transcription factor 3 (ATF3), a member of the activating transcription factor/cAMP-responsive element binding protein (ATF/CREB) family of transcription factors is induced by cellular stress including oxidative stress. Objectives The aim of this study was to analyze ATF3 in fibroblast activation and tissue fibrosis in systemic sclerosis (SSc). Methods Activation of ATF3 expression in skin tissue and in human dermal fibroblasts was determined by real-time PCR, Western blot and immunohistochemistry. ATF3 knockout fibroblasts were used to evaluate the effect of ATF3 on fibroblast activation and collagen release. Smad reporter assay were performed to study functional interactions between ATF3 and Smad signaling. The outcome of ATF3 knock-out mice and wildtype littermates was evaluated in the mouse models of bleomycin-induced dermal fibrosis and dermal fibrosis induced by overexpression of a constitutively active TGF-β receptor I (TBR). Results An increased expression of ATF3 was detected in the upper layer of the dermis of SSc patients on fibroblasts double stained for ATF3 and anti-prolyl-4-hydroxylase (p=0.0016). The overexpression of ATF3 persisted in cultured SSc fibroblasts. The upregulation of ATF3 was mediated by TGF-β. ATF3 expression was induced by TGF-β in cultured fibroblasts and in murine skin and treatment with the TBR inhibitor SD-208 prevented the induction of ATF3 in experimental fibrosis. ATF3 deficient fibroblasts were less sensitive to the pro-fibrotic effects of TGF-β with impaired induction of collagen mRNA and protein upon stimulation with TGF-β decreases of 64% (p=0.04) and 51% (p=0.03), respectively. Knockdown of ATF3 also protected from experimental fibrosis. In the model of bleomycin-induced fibrosis, dermal thickening was decreased by 70% (p=0.02), hydroxyproline content by 73% (p=0.035) and myofibroblast counts by 80% (p=0.0003) in AFT3 knockout mice compared to wildtype littermates. ATF3 knockout mice were also protected from TBR induced fibrosis. Function studies demonstrated that ATF3 is induced by Smad3 and regulates the pro-fibrotic effects of TGF-β. In addition reporter studies and analyses of the expression of classical Smad target genes such as PAI-1 demonstrated that binding of ATF3 to Smad3 stimulates the transcriptional activity of Smad3. Consistently, Smad3 reporter activity and the expression of PAI-1 upon stimulation with TGF-β were both strongly reduced in ATF3 knockout fibroblasts compared to c