Homocysteine induces COX-2 expression in macrophages through ROS generated by NMDA receptor-calcium signaling pathways

Homocysteine (Hey) at elevated levels is a putative risk factor for many cardiovascular disorders including atherosclerosis. In the present study, we investigated the effect of Hey on the expression of cyclooxygenase (COX)-2 in murine macrophages and the mechanisms involved. Hey increased the expression of COX-2 mRNA and protein in dose- and time-dependent manners, but did not affect COX-1 expression. Hey-induced COX-2 expression was attenuated not only by the calcium chelators, EGTA and BAPTA-AM, but also by an antioxidant, N-acetylcysteine. Calcium chelators also attenuated Hey-induced reactive oxygen species (ROS) production in macrophages, indicating that Hey-induced COX-2 expression might be mediated through ROS generated by calcium-dependent signaling pathways. In another series of experiments, Hey increased the intracellular concentration of calcium in a dose-dependent manner, which was attenuated by MK-801, an N-methyl-D-aspartate (NMDA) receptor inhibitor, but not by bicuculline, a gamma-aminobutyric acid receptor inhibitor. Molecular inhibition of NMDA receptor using small interfering RNA also attenuated Hey-induced increases in intracellular calcium. Furthermore, both ROS production and Hey-induced COX-2 expression were also inhibited by MK-801 as well as by molecular inhibition of NMDA receptor. Taken together, these findings suggest that Hey enhances COX-2 expression in murine macrophages by ROS generated via NMDA receptor-mediated calcium signaling pathways.