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).

Methods:

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.

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 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).

Conclusion:

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.

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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/