Background/Purpose: Ntn1 is a member of the family of axonal guidance proteins that plays a role in leukocyte function and inflammation and is critical for OC differentiation.  Because A2AR ligation, either directly or indirectly following methotrexate treatment, diminishes wear particle-induced inflammatory osteolysis we asked whether A2AR activation might regulate expression of Ntn1 at sites of inflammatory osteolysis.

Methods: 1cm midline sagittal incision was made over the calvaria in C57Bl/6 mice age 6-8 weeks. Mice received no particles (Control) or 3mg of UHMWPE wear particles with 20µl of saline 0.9% or CGS21680 (A2AR agonist, 1µM, n=4 each) at the surgical site every day for 14 days. After sacrifice, calvaria were prepared for immunostaining for Ntn1/Unc5b/DCC. Protein and mRNA expression were studied by RT-PCR and western blot in mouse primary bone marrow-derived OC and OB and in RAW264.7 cells (murine cell line) in the presence/absence of CGS21680 and ZM241385 (A_2AR antagonist) 1µM each.

Results: Wear particles stimulated increased expression of Ntn1 and Unc5b, but not DCC, in periosteal inflammatory infiltrates, an effect which was blocked by the A2AR agonist CGS21680. In in vitro studies RANKL induced a 25±4 and 3±0.5 fold change, respectively, in Ntn1 and Unc5b mRNA during OC differentiation, changes that were completely blocked by CGS21680 (1.17±0.1, p<0.001, n=4). In contrast, DCC mRNA expression did not significantly change during OC differentiation and DCC expression was unaffected by CGS21680 (1.16±0.2 fold increase vs 1.9±0.2 for RANKL, p=ns, n=4). Similar changes were observed in protein expression and secretion. When Ntn1 or Unc5b were knocked-down in RAW264.7 cells using selective shRNA, basal mRNA expression for PKA, EPAC1 and EPAC2 (cAMP signaling molecules activated by A2AR) were increased and vice versa, when PKA, EPAC1 or EPAC2 were knocked down with selective shRNA, Ntn1 and Unc5b basal mRNA levels were increased. NFkB nuclear translocation was decreased in the presence of CGS21680 or in the absence of Ntn1. There was no change in Ntn1 or Unc5b expression during OB differentiation.

Conclusion: Ntn1 expression on macrophages and OC plays a central role in wear particle-induced osteolysis and adenosine A_2AR stimulation downregulates Ntn1expression and inhibits bony destruction at sites of wear particle-induced osteolysis. Moreover, Ntn1-unc5b and A2AR stimulation reciprocally diminish signaling by each other. These results suggest that targeting Ntn1 directly or via stimulation of adenosine A_2AR may be a novel approach to prevent osteolysis and joint prosthesis loosening.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/stimulation-of-the-adenosine-a2a-receptor-a2ar-regulates-the-expression-of-netrin-1-ntn1-and-its-receptors-unc5b-dcc-and-inhibits-wear-particle-induced-inflammatory-osteolysis-in-a-model-o/