Background/Purpose: Uncontrolled activation of fibroblasts releasing large amounts of extracellular matrix proteins is a key feature of systemic sclerosis (SSc). The nuclear receptor coactivators (NCOA) are named after their role as coactivators for nuclear hormone receptors, but are indeed modulating a large number of signaling pathways. In this study, we aimed to analyze whether targeting of NCOA3 may ameliorate fibrosis in preclinical models of SSc.

Methods: Fibroblasts were exposed to chronically high levels of TGF-β to mimic the situation in fibrotic diseases. The expression of different transcriptional cofactors in response to TGF-β stimulation was analyzed by quantitative real-time PCR. Protein levels were analyzed by Western Blot. Knockdown of NCOA3 was achieved via transfection with siRNA in vitro. For knockdown of NCOA3 in vivo, siRNA was premixed with atelocollagen for stabilization and injected subcutaneously at the site of bleomycin injections. Additionally, NCOA3 was targeted with the SRC3 inhibitor-2 (SI-2) in different murine models. The interaction of NCOA3 and TGF-β/SMAD signaling was analyzed by SMAD-responsive reporter as well as CoIP assays.

Results:

SiRNA-mediated knockdown of NCOA3 strongly decreased the pro-fibrotic effects of TGF-β, with significant reductions in collagen protein, in the mRNA levels of COL1A1 and ACTA2 as well as significant decreases in the prototypical TGF-β target genes PAI1 and CTGF. Knockdown of NCOA3 also ameliorated fibrosis in the bleomycin-induced dermal fibrosis model of SSc, with reductions of dermal thickening, of myofibroblast counts and of hydroxyproline content. Furthermore, pharmacological targeting of NCOA3 by the inhibitor SI-2 was able to ameliorate fibrosis in bleomycin-induced pulmonary fibrosis and in dermal fibrosis induced by overexpression of constitutively active TGF-β receptor type I (TBRI^act). Mechanistically, NCOA3 directly interacts with SMAD3 to promote TGF-β/SMAD3 signaling as shown by reporter studies and CoIP assays.

When we analyzed the expression levels of NCOA3 in SSc and in matched healthy individuals, we observed a modest, but statistically significant downregulation of NCOA3 expression, a phenotype that persisted also in cultured SSc fibroblasts. Stimulation of normal fibroblasts with chronically high levels of TGF-β also decreased the mRNA and protein levels of NCOA3. Given the profibrotic effects of NCOA3, the downregulation of NCOA3 in fibrotic SSc skin may be an endogenous regulatory attempt to counteract the increased activity of TGF-β/SMAD3 signaling.

Conclusion: We demonstrate in our study that inhibition of NCOA3 has profound anti-fibrotic effects and serves as a coactivator for the profibrotic effects of TGF-β/SMAD. Antagonizing NCOA3 augments an endogenous regulatory loop to reduce persistent fibroblast activation and tissue fibrosis in SSc.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/inhibition-of-nuclear-receptor-coactivator-3-attenuates-fibrosis-in-murine-models-of-systemic-sclerosis/