Background/Purpose: Osteoarthritis (OA) is a debilitating and costly condition caused by loss of articular cartilage secondary to chondrocyte dysfunction. Autophagy is a homeostatic pathway that is upregulated in cellular stress and it is decreased in OA. Recent work demonstrates that the Forkhead transcription factors FoxO1 and FoxO3 regulate chondrocyte autophagy and their deficiency leads to the development of OA. We have previously reported that A2ARKO mice develop severe OA and rats with post-traumatic OA (PTOA) have lower OARSI scores when treated with liposomal preparations of adenosine by activating A2AR (Corciulo, et al NComms, 2017).

Methods: Active (non-phos FoxO) and inactive (Akt-phos FoxO) levels were evaluated in the human TC28⍺2 chondrocyte cell line by IF and WB after stimulation with the A2AR agonist CGS21680 (CGS, 1mM) +/- A2AR antagonist ZM241385 (ZM, 1mM) at 30 min. Autophagy proteins p62/SQTSM1, Gabarapl1, Beclin-1 and apoptosis proteins p53 and caspase 3 were analyzed by IF and WB at 1h. An in vivo obesity OA mouse model treated with joint injections of liposomal-CGS or lipo-adenosine was assessed by IHC for FoxO1/3 or phos-FoxO1/3, p62, Gabarapl1, and Beclin-1.

Results: FoxO1/3 both translocated to the nucleus in TC28⍺2 cells 30m post-CGS, while phos-FoxO1/3 translocated to the cytoplasm; these changes were all reversed with ZM pre-treatment. Total cellular FoxO1 increased significantly after A2AR activation by WB (1.6±0.08 vs 1.0±0.08, p< 0.001, n=4) and FoxO3 levels trended up (2.0±0.7 vs 1.0±0.3, p=0.11, n=3). There was p62 reduction in TC28⍺2 cells over a 3-hour time course (0.74±0.17 vs 1.0±0.24, p=0.01, n=3), which indicates active autophagy as p62 is degraded with the cargo. However, in the presence of 25µM hydroxychloroquine (HCQ) to inhibit autophagosome-lysosome fusion, there was a clear increase in p62 levels in A2AR-stimulated chondrocytes (78±7% vs 9±1.4% cells, p=0.003, n=3) consistent with increased autophagic flux. Similarly, autophagosome-associated Gabarapl1 and upstream regulator Beclin-1 formed punctate autophagy nucleation complexes after CGS treatment. While autophagy is generally pro-survival, it can be associated with apoptosis. We found that A2AR-stimulated autophagy was associated with decreased p53 level by WB at 1h (0.66±0.12 vs 1.0±0.11, p=0.02, n=3) and a visible reduction of p53 and caspase 3 by IF. The in vitro results were reflected by in vivo experiments from obese mouse knee joints, in which we observed an increase in cellular levels and nuclear localization of FoxO1/3 with a concomitant decrease in phos-FoxO1 in mice treated with the A2AR agonist. Similar to our in vitro results, the treated obese mice joints displayed decreased p62 and increase in Gabarapl1 and Beclin-1.

Conclusion: These results demonstrate that A2AR stimulation increases activation and nuclear localization of FoxO1 and FoxO3 and promotes an increase in autophagic flux without increasing apoptosis in vitro. More importantly, similar changes were observed in vivo in a murine model of obesity-induced OA. These findings provide a mechanism by which A2AR stimulation maintains chondrocyte homeostasis and reduces OA.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/adenosine-a2a-receptor-signaling-activates-foxo1-and-foxo3-and-promotes-cartilage-autophagy/