Ecto-5'-nucleotidase/CD73 reduces COX-2 expression in activated macrophages

Extracellular ATP (eATP) is a significant contributor of inflammation through enhanced expression of cyclooxygenase 2 (COX-2). In this study, we explored whether overexpression of ectonucleotidases such as ecto-5'-nucleotidase (NT5E, or CD73), responsible for the hydrolysis of AMP to adenosine, can modulate COX-2 expression in macrophages. CD73 was subcloned in both bacterial and mammalian expression vector systems. In the first model, recombinant CD73 was exogenously added into the media of J774A.1 macrophage cells co-stimulated with LPS and AMP. In the second model, J774A.1 cells were transfected with CD73 cloned in pcDNA3.1 vector and checked for the expression of COX-2 following treatment with LPS and AMP. In both models, CD73 activity was assessed by the release of inorganic phosphate from AMP. We found that CD73, when added exogenously to the media, reduced COX-2 expression in LPS-activated J774A.1 cells stimulated with ATP or AMP, the substrate of CD73. Similar effect was seen in CD73-transfected J774A.1 cells treated with LPS and AMP, and this effect was abolished by AMP-CP, the inhibitor of CD73. The decrease in COX-2 expression involved NF-κB pathway but not through p42/44 MAPK. Our results show that overexpression of CD73 reduced COX-2 expression by increasing the formation of adenosine.