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Abstract Number: 1967

Adenosine A2A Receptor Signals Through AMPK and SIRT1 to Increase Chondrocyte Homeostasis

Benjamin Friedman1 and Bruce Cronstein 2, 1Department of Medicine, Division of Rheumatology NYUSoM, NYC, 2NYU Langone, New York

Meeting: 2019 ACR/ARP Annual Meeting

Keywords: Adenosine receptors, cartilage, chondrocytes, sirt1 and ampk

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Session Information

Date: Tuesday, November 12, 2019

Session Title: Osteoarthritis & Joint Biology – Basic Science Poster

Session Type: Poster Session (Tuesday)

Session Time: 9:00AM-11:00AM

Background/Purpose: OA is characterized by loss of cartilage and chondrocyte dysfunction. Our lab has shown CGS21680 (CGS, 1µM) activation of adenosine A2AR leads to chondrocyte homeostasis. We have demonstrated in vitro and in vivo activation of FoxO1/3 in human TC28⍺2 chondrocytes and in obesity-OA mice treated with liposomal injections of adenosine or CGS. In this study, we sought to identify an upstream mechanism to explain A2AR related FoxO1/3 activation and cartilage homeostasis.

Methods: WB or IF was done for AMPK, phos-AMPK, LKB1, Sirt1, p53, Lys 382 Acetyl-p53, and Acetyl lysine in human TC28⍺2 cells ± CGS (1µM). Autophagy inhibitor hydroxychloroquine (HCQ, 25µM), Sirt1 inhibitor nicotinamide (NAM, 5mM) and peptide PKA inhibitor (15µM) were added in vitro as indicated. Sirt1 and acetyl lysine in vivo levels were measured by IHC in 1yr old A2ARKO, CD73KO, or WT mice.

Results: vs 1.0±0.2, p=0.02, n=4). Sirt1 is a homeostatic deacetylase that increases nuclear retention of deacetylated Foxo1/3. Further, it can indirectly activate AMPK via deacetylation of nucleus-sequestered LKB1 kinase so it can exit the nucleus to phosphorylate/activate AMPK. IF exhibited bimodal Sirt1 nuclear elevation based on average cell intensity from 3 experiments both at 10m (5.3±0.4 vs 1.0±0.2, p=0.001) and 30m (8.2±0.8 vs 1.0±0.2, p=0.01). LKB1 cytoplasmic fraction increased with in vitro CGS at 15m (1.34±0.22 vs 1.0±0.14, p< 0.01, n=3) and 30m (1.5±0.2 vs 1.0±0.14, p< 0.01, n=3). Sirt1 proved to be a key mediator in the signaling pathway as its inhibitor NAM blocked CGS-induced activation of Sirt1 and Foxo proteins. In joint sections, Sirt1 was visibly decreased in A2ARKO and CD73KO mice (lower extracellular adenosine) with an increase in total cartilage acetylated lysine. To assess Sirt1 function we evaluated known target p53, which when deacetylated undergoes proteasomal degradation. Prior results in our lab show CGS decreases p53 in vitro. This decrease may be related to a chondroprotective increase in autophagy that occurs with A2AR activation, but we found cells treated simultaneously with starvation to induce autophagy plus autophagy inhibitor HCQ exhibited much higher p53 levels by IF and CGS treatment dramatically reduced p53 despite +HCQ. To test the alternative hypothesis that Sirt1 deacetylates and degrades p53, we performed IF for Ac-Lys-382 p53 and noted clear decrease at 30m post-CGS. Lastly, we identified an in vitro role for PKA in this A2AR pathway by demonstrating a PKA inhibitor blocked normal CGS-mediated activation of Sirt1 and FoxO1/3 assessed by IF.

Conclusion: These early results provide a viable mechanism by which A2AR can activate Sirt1 and AMPK through PKA, explaining the A2AR-associated chondroprotective effect with activation of FoxO1/3 and related increase in autophagy. Both AMPK and Sirt1, which reciprocally activate each other, are important for maintenance of healthy cartilage as evidenced by the fact that knockdown of these proteins is associated with OA. This AMPK/Sirt1 mechanism lends further support to the importance of A2AR activation in chondrocyte homeostasis that our lab has demonstrated in vivo.


Disclosure: B. Friedman, None; B. Cronstein, AstraZeneca, 5, CanFite Biopharmaceuticals, 4, Horizon Pharmaceuticals, 5, Regenosine, Inc., 4.

To cite this abstract in AMA style:

Friedman B, Cronstein B. Adenosine A2A Receptor Signals Through AMPK and SIRT1 to Increase Chondrocyte Homeostasis [abstract]. Arthritis Rheumatol. 2019; 71 (suppl 10). https://acrabstracts.org/abstract/adenosine-a2a-receptor-signals-through-ampk-and-sirt1-to-increase-chondrocyte-homeostasis/. Accessed July 2, 2022.
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