Collagen and matrix deposition by fibroblasts is an essential part of wound healing but also contributes to pathologic remodeling of organs leading to substantial morbidity and mortality. Adenosine, a small molecule generated extracellularly from adenine nucleotides as a result of direct stimulation, hypoxia, or injury, acts via a family of classical sevenpass G protein-coupled protein receptors, A 2A and A 2B , leading to generation of cAMP and activation of downstream targets such as PKA and Epac. These effectors, in turn, lead to fibroblast activation and collagen synthesis. The regulatory actions of these receptors likely involve multiple interconnected pathways, and one of the more interesting aspects of this regulation is opposing effects at different levels of cAMP generated. Additionally, adenosine signaling contributes to fibrosis in organ-specific ways and may have opposite effects in different organs. The development of drugs that selectively target these receptors and their signaling pathways will disrupt the pathogenesis of fibrosis and slow or arrest the progression of the important diseases they underlie.

Adenosine A 2A receptor stimulation promotes the synthesis of collagen type I and type III (Col1 and Col3), mediators of fibrosis and scarring. The A 2A receptor modulates collagen balance via cAMP/PKA/p38-MAPK/Akt pathways. Wnt signalling is important in fibrosis and the cAMP and Wnt pathways converge. Because the A 2A receptor is Gs-linked and increases cAMP, we determined whether A 2A receptors and Wnt signalling interact. EXPERIMENTAL APPROACHTotal β-catenin, de-phosphorylated β-catenin (canonical activation, de-phospho β-catenin) and phosphorylated β-catenin at Ser 552 (non-canonical activation, p-Ser 552 β-catenin) levels were determined in primary human dermal fibroblasts, cytosol and nucleus, by western blot analysis and fluorescence microscopy, before and after stimulation by A 2A receptor-selective agonist CGS21680, with/without A 2A receptor-selective antagonist (SCH56261) pretreatment. β-Catenin was knocked down by transfection with scrambled-siRNA or specific-siRNA, and Col1 and Col3 levels determined by western blots. KEY RESULTSCGS21680 stimulation rapidly (15 min) increased cellular β-catenin levels. Both de-phospho β-catenin and p-Ser 552 β-catenin levels were also increased. CGS21680 stimulated the translocation of total de-phospho and p-Ser 552 β-catenin to the nucleus. A 2A receptor-stimulation increased Col1 synthesis similarly in β-catenin knockeddown and scrambled cells. However, β-catenin knockdown abolished the increase in Col3 synthesis induced in A 2A receptor-stimulated fibroblasts. CONCLUSIONS AND IMPLICATIONSA 2A receptor stimulation promotes Col3 synthesis via the activation of canonical and non-canonical β-catenin, consistent with a role for A 2A receptors in dermal fibrosis and scarring. AbbreviationsCol1, collagen type I; Col3, collagen type III; CBP, cofactor CREB-binding protein; CREB, cAMP response element binding protein; CTGF, connective tissue growth factor; NHDFs, normal human dermal fibroblasts

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