Background/Purpose:

Osteoarthritis (OA) is a debilitating condition characterized by chondrocyte dysfunction and loss of cartilage. Autophagy, a homeostatic process that occurs in times of cellular stress and starvation, is decreased in chondrocytes with aging and OA. The adenosine A2A receptor (A2AR) maintains chondrocyte homeostasis; A2AR knockout (KO) mice spontaneously develop OA. Moreover, intra-articular injection of liposomal preparations of adenosine or an A2AR-specific agonist (CGS12680 (CGS)) in rats with post-traumatic OA (PTOA) or mice with obesity-induced OA prevents development of PTOA and reverses obesity-induced OA. We sought to determine whether adenosine signaling maintains chondrocyte homeostasis, in part, by regulating autophagic flux in chondrocytes.

Methods:

Autophagy proteins Beclin-1, p62/SQSTM1, and LC3 were analyzed by western blotting and IF in TC28⍺2 human chondrocytes ±1µM CGS for various times in normal culture and starvation (FBS 10% vs 1%). Chondrocytes were pretreated with autophagy inhibitors hydroxychloroquine (prevents autophagosome destruction by lysosomes, 25µM, HCQ) and/or 3-methyladenine (blocks autophagy initiation, 3MA, 5mM). Knee joint sections from obese mice treated with adenosine or CGS were analyzed by fluorescent IHC. Autophagy gene expression was determined in differential display analyses comparing wildtype (WT) vs A2AR KO mice and rats with established PTOA treated with intra-articular injections of liposomal CGS or liposomes alone.

Results:

Consistent with enhanced autophagy, CGS reduced p62 levels in starved chondrocytes by western blot (4.1 ± 0.7 vs 5.7 ± 0.8 p62/actin, p<0.02). More cells stained diffusely positive for p62 without punctae (less autophagy) in the control vs CGS (67 ± 6% vs. 19 ± 3%, p<0.0002). With HCQ added, punctate p62 was more marked and brighter on CGS treatment (78 ± 5% vs 13 ± 4%, p<0.0001). Chondrocyte pre-treatment with 3-MA (±HCQ) followed by starvation ±CGS for 1h demonstrated almost complete absence of either p62 or Beclin-1. When studied in vivo in OA mouse knees, Beclin-1 was not visible in chondrocytes in normal and obese (OA) mouse controls whereas it was present in all of the chondrocytes in obese mice treated with either CGS or adenosine (p<0.0001). Differential expression analysis of WT vs A2AR KO murine chondrocytes demonstrated significant changes in numerous autophagy genes; e.g. p62 was elevated in WT mice. Similarly, there were significant changes in autophagy gene expression in PTOA rats given liposomal-CGS vs liposome. Of note the autophagy Atg8-family member Gabarapl1 that integrates into autophagosomes was increased with CGS-liposome injections (FC +1.75, adj p=0.0049).

Conclusion:

These results demonstrate, both in vitro and in vivo, that A2AR stimulation promotes autophagy, evidenced by decreased p62, increased Beclin-1 and reversal of these effects with autophagy inhibitors. Although the precise mechanism is not known, it is possible that A2AR signaling increases Beclin-1 activity, thereby increasing downstream autophagic flux. This increase in autophagy may contribute to maintaining chondrocyte homeostasis and reversing OA.

« Back to 2018 ACR/ARHP Annual Meeting

ACR Meeting Abstracts - https://acrabstracts.org/abstract/adenosine-a2a-receptor-stimulation-regulates-autophagy-in-chondrocytes/