Background/Purpose: Fibrosis of skin and other organs is a hallmark of Scleroderma.  We and others have previously reported that A2AR plays a role in skin and organ fibrosis in murine models of scleroderma. A2AR stimulation induces collagen type I and type III (Col1 and Col3) synthesis, mediators of fibrosis and scarring, via a mechanism involving cAMP/PKA/p38-MAPK/AKT. Wnt signaling is important in fibrosis and cAMP and Wnt signaling pathways converge. We therefore asked whether A2AR stimulates Wnt signaling pathways to promote collagen synthesis.

Methods: Total β-catenin, de-phosphorylated β-catenin (canonical activation, de-phospho β-catenin), and phosphorylated β-catenin at Ser552 (non-canonical activation, p-Ser552 β-catenin) levels were determined in primary human dermal fibroblast cytosol and nucleus by western blot and fluorescence microscopy after stimulation by A2AR-selective agonist CGS21680, with / without A2AR-selective antagonist (SCH582611) pretreatment. β-catenin was knocked down by lentiviral transfection with scrambled-siRNA or specific-siRNA, and Col1 and Col3 levels determined by western blot. In vivo effects were studied in a bleomycin-induced dermal fibrosis model using Wnt signaling reporter mice, Tcf/lef1-GFP homogenous mice, and mice were treated with A2AR antagonist (Istradefylline). Morphometric features and levels of hydroxyproline were determined as measures of dermal fibrosis. Fibrosis was confirmed by Picrosirius red stain. Immunohistochemistry for GFP and β-catenin were determined as measures of β-catenin translocation. 

Results: CGS21680 stimulation rapidly (15min) increased cellular β-catenin to 176 ± 16 % of control (n = 6, P < 0.05); Both de-phospho β-catenin (168 ± 30% of control, n = 6, P > 0.05) and p-Ser552 β-catenin (220 ± 22 % of control, n = 6, P < 0.05) were increased. CGS21680 stimulated translocation of total, de-phospho, and p-Ser552 β-catenin to the nucleus. A2AR-stimulation increased Col1 synthesis similarly in β-catenin knockdown and scrambled cells. However, β-catenin knockdown abrogated the A2AR-stimulated increments in Col3 synthesis by 73% (66 ± 14% vs. 18 ± 16% increase of Col3, n = 8, P < 0.05).  A2AR antagonist–treated mice were protected from developing bleomycin-induced dermal fibrosis (Figure 1) and there was diminished nuclear translocation of β-catenin in the affected skin (Figure 2).

Conclusion: A2AR stimulation promotes Col3 synthesis via canonical and non-canonical β-catenin activation, leading to dermal fibrosis and scarring. Selectively modifying this pathway represents an attractive therapeutic target in fibrotic diseases such as scleroderma.