Session Type: Abstract Submissions (ACR)
Background/Purpose: 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 various types of cellular stress including oxidative stress. Here, we analyzed ATF3 as a downstream mediator of TGF- β signaling in fibroblast activation and tissue fibrosis in systemic sclerosis (SSc).
Activation of ATF3 expression in the skin and dermal fibroblasts was determined by real-time PCR, Western blot and immunohistochemistry. To investigate the role of ATF3 in fibrosis, ATF3 knock-out mice and wildtype littermates were 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). The content was determined by haematoxylin-eosin and trichrome stainings, by immunohistostaining for aSMA and hydroxyproline assays respectively. In vitro cultured fibroblasts were used and measure collagen release by SirCol and study target genes by RT-PCR. Co-immunoprecipitation (Co-IP) and Smad reporter assay were performed to study physical and functional interactions between ATF3 and Smad3.
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 with increases of 292% (p= 0.043). TGF-β induces ATF3 with an increase of 256% (p= 0.01). ATF3 knock-out 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. In the model of bleomycin-induced fibrosis, dermal thickening was decreased by 70% (p= 0.02), the hydroxyproline content by 73% (p= 0.035) and the myofibroblast counts by 80% (p= 0.0003) in AFT3 knockout mice compared to wild type littermates. ATF3 knockout mice were also protected from TBR induced fibrosis with significant decreases in dermal thickening (p= 0.003), accumulation of collagen (p= 0.001) and myofibroblast differentiation (p= 0.002). Function studies demonstrated that ATF3 interacts with Smad3 to regulate the pro-fibrotic effects of TGF-β. Co-IP demonstrated that TGF-β induces binding of ATF3 to Smad3. Reporter study and analyses of the expression of classical Smad target genes such as PAI-1 demonstrated that the binding of ATF3 to Smad3 stimulates the transcriptional activity of Smad3. The activity in Smad3 reporter assays and the expression of PAI-1 upon stimulation with TGF-β were both strongly reduced in ATF3 knockout fibroblasts compared to control cells (decreases of 56 %, p= 0.004 and 85 %, p= 0.02, respectively).
We demonstrate for the first time a key-role of ATF3 in fibroblast activation and tissue fibrosis in SSc. Targeting of the ATF3 reduced the stimulatory effect of TGF-β on cultured fibroblasts by interfering with canonical Smad signaling. Moreover, knockdown of ATF3 protected from experimental fibrosis in different mouse models. Considering the potent anti-fibrotic effects observed in this study, ATF3 might be a candidate for molecular targeted therapies of SSc.
G. A. Schett,
J. H. W. Distler,
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/activating-transcription-factor-3-regulates-canonical-transforming-growth-factor-beta-signaling-in-experimental-fibrosis/