Session Information
Session Type: Abstract Submissions (ACR)
Background/Purpose:
Activation of Adenosine 2A Receptors (A2AR) promotes fibrosis and collagen synthesis. However, the underlying mechanism by which A2AR stimulate collagen synthesis is still unclear. Previous reports indicate that cAMP, principal effector of the A2AR, inhibits Transforming Growth Factor β1-induced collagen synthesis and has been proposed as an anti-fibrotic mediator.
Methods:
Primary Human Dermal Fibroblasts (NHDF) were stimulated with increasing concentrations of the A2AR agonist CGS21680 or the direct adenyl cyclase (AC) activator forskolin, and results were compared to the well-known profibrotic agent TGF-β1. cAMP intracellular levels and proliferation were measured and collagen1 (Col1), collagen3 (Col3), MAPK and Smad2/3 expression were assessed by western blotting. The role of MAPK and Smads in regulating collagen synthesis was studied by use of specific inhibitors and knock-downs with siRNA.
Results:
A2AR stimulation with CGS21680 elicited a modest cAMP increase (150.3±11% of control; P<0.01, n=5) which stimulated both Col1 (194.3±28% of control; P<0.01, n=8) and Col3 (178.4±9.8% of control; P<0.001, n=10) synthesis, but higher cAMP concentrations resulting from direct activation of AC by forskolin (15688±7038% of control; P<0.01, n=3) inhibited Col1 production (41±2% inhibition; P<0.001, n=3). Surprisingly, Col3 expression was increased at maximal cAMP levels (218.7±38.5% of control; P<0.05, n=3). Similar to Col1 expression, fibroblast proliferation increased following physiologic increases in cAMP levels induced by A2AR stimulation (164.7±20.8% of control; P<0.05, n=4) but was inhibited by greater cAMP increases resulting from forskolin incubation (26±2% inhibition; P<0.05, n=3). The A2AR-mediated increase of Col1 and Col3 was mediated by an AKT-dependent pathway as shown in a dose-response of CGS21680 in the presence of two AKT inhibitors LY294002 and wortmannin (two-way ANOVA Col1: P<0.001, n=5 for both inhibitors; Col3: LY294002 P<0.001, n=5 and wortmannin P<0.01, n=4) while Col3, but not Col1, expression was dependent on p38MAPK activation (p38 inhibitor SB20358: two-way ANOVA P<0.01, n=3) and repressed by ERK (ERK1/2 siRNA: two-way ANOVA P<0.05, n=4). Transforming Growth Factor β1 strongly induced phosphorylation of Smad2 (426.3±136% of control; P<0.001, n=3) and Smad3 (1530.3±268% of control; P<0.001, n=3) and increased Col3 expression was prevented by Smad3 depletion (after Smad3 silencing: control vs CGS21680 P=non significant, n=3), whereas CGS21680 did not induce phosphorylation of either Smad2 (P=non significant, n=3) or Smad3 (P=non significant, n=3), and Smad2/3 knockdown did not prevent CGS21680-induced Col1 or Col3 increases.
Conclusion:
Our results indicate that cAMP is an all-or-none intracellular switch for collagen production via the non-canonical Smad2/3-independent AKT-dependent pathway, which provides an attractive explanation for the paradox that A2AR induces pro-fibrotic signals via cAMP in dermal fibroblasts.
Disclosure:
M. Perez-Aso,
None;
P. Fernandez,
None;
A. Mediero,
Filed a patent on use of adenosine A2AR agonists to prevent prosthesis loosening (pending). ,
9;
E. S. Chan,
Patent on the use of adenosine A2A antagonists for the treatment of fibrosis.,
9;
B. N. Cronstein,
Canfite Pharma,
1,
NIH, Gilead, Takeda, AstraZeneca,
2,
NYU School of Medicine,
3,
Merck-SeronoBristol-Myers Squibb, Novartis, CanFite Biopharmaceuticals, Cypress Laboratories, Regeneron (Westat, DSMB), Endocyte, Protalex, Allos, Inc., Savient, Gismo Therapeutics, Antares Pharmaceutical, Medivector,
5,
Multiple patents on adenosine receptors and bone metabolism, pharmacology,
9.
ACR Meeting Abstracts - https://acrabstracts.org/abstract/adenosine-2a-receptor-promotes-collagen-production-by-human-fibroblasts-via-smad23-independent-pathways-involving-cyclic-amp-and-akt/