Background/Purpose: Pathological fibrosis in the skin and other organs is the hallmark of scleroderma and other fibrosing diseases.  Adenosine, acting at A_2AR, plays a critical role in wound healing and fibrosis of the skin and previous studies have demonstrated that blockade or deletion of A_2A receptors prevents dermal fibrosis in response to bleomycin, a murine model of scleroderma.  Moreover, A_2AR blockade enhances the strength of scars by, in part, diminishing the collagen 3 (Col3) content of the scar relative to Col1 content.  Here we determined whether expression of Collagen-1 (Col1) and Col3 proceed by different signaling pathways.

Methods: Col1 and Col3 expression were determined by Western-Blot analysis of normal human dermal fibroblasts (NHDF). 

Results: Surprisingly the concentrations of the A_2AR agonist CGS21680 required to increase Col1 expression were significantly lower than those required to increase Col3 (0.1µM: 147±6% for Col1 [p<0.01, n=23] vs 1µM: 155±16% for Col3, [p<0.01, n=18) although increases in both were completely blocked by the A_2AR-antagonist SCH58261 (0.1µM). The selective Protein kinase A (PKA) activator 8-Cl-cAMP markedly increased Col1 expression (0.1µM: 280±74% [p<0.01, n=3), but inhibited Col3 expression by as much as 68±11% (p<0.01, n=4). PKA inhibition by PKAi prevented the CGS21680-stimulated increase in Col1 but elicited an increase of Col3 at lower concentrations than in the absence of PKAi (0.1µM, 110±10% versus 182±30% [p<0.05, n=4). In contrast, stimulation with the specific Epac activator 8-CPT-2’-O-Me-cAMP did not affect Col1, but increased Col3 production  by 362±85% (p<0.05, n=3). Inhibition of AKT, erk, p38 and JNK with LY294002 (10µM), U0126 (1µM), SB203580 (1µM) and SP600125 (20µM), respectively, demonstrated that increased expression of Col1 and Col3 depend on AKT and p38. However, CGS21680 0.1µM increased Col3 only upon ERK or JNK blockade, an effect similar to PKA inhibition. 

Conclusion: Our work strongly suggests that at nanomolar concentrations of CGS21680 PKA activity prevails over Epac, thereby activating Col1 expression and inhibiting Col3 but in the mmolar range, the PKA/ERK/JNK inhibition of Col3 is overcome by activation of Epac/p38 and Pi3K/AKT. These observations may explain the dramatic decrease of the Col1/Col3 ratio in hypertrophic and immature scars, where adenosine is present in µmolar ranges, when compared to normal skin, where adenosine concentration varies from 30 to 300nM.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/adenosine-a2a-receptor-a2ar-activation-stimulates-increased-expression-of-collagen-1-and-collagen-3-by-different-signaling-pathways-in-normal-human-dermal-fibroblasts/