Laminar Shear Stress Up‐regulates Endothelial CD73 Expression by Activating Calmodulin‐dependent Kinase Kinase

Abstract

Endothelial cell (EC) surface CD73, ecto‐5′ nucleotidase, extracellularly metabolizes AMP to adenosine, a potent anti‐inflammatory molecule. Since athero‐protective high laminar shear stress (LS) rises intracellular calcium levels and induces the release of adenine nucleotides in ECs, we hypothesized that LS increases CD73 expression by activating calmodulin‐dependent kinase kinase (CaMKK), leading to the extracellular production of adenosine in ECs.Cultured human coronary artery ECs were subjected to static, LS (15 dyne/cm2), or oscillatory shear (OS; ± 5 dyne/cm2) condition for 24 hours. Real‐time PCR showed that LS increased CD73 mRNA expression (2.1±0.3‐fold increase vs. static condition, p<0.05, n=10–17), whereas OS unchanged the mRNA expression (0.9±0.2 fold, p=ns, n=6). STO‐609 (a CaMKK inhibitor, 10μg/ml) suppressed LS‐induced increase in CD73 mRNA expression (1.1±0.2 fold, n=5). In contrast, U‐73122 (a phospholipase C inhibitor, 1μM) and KN‐62 (a calcium/calmodulin‐dependent kinase inhibitor, 10 μM) had no inhibitory effect (2.7±0.5 fold, n=3 and 2.1±0.3, n=8). HPLC analysis revealed that LS resulted in the accumulation of adenosine in the conditioned medium.These results indicate that LS elicits the generation of extracellular adenosine from ECs by upregulating CD73 expression via activating CaMKK. Thus, CD73 might contribute to the athero‐protective effects of LS in ECs.