Abstract 5472: Nox4 is a Novel Inducible Source of Intracellular Reactive Oxygen Species in Human Monocyte-Derived Macrophages and Mediates Oxidized LDL-induced Macrophage Death

Abstract

Nox2/gp91 phox is the large subunit of the integral membrane component of the phagocytic NADPH oxidase (Nox) complex and was believed to be the sole Nox isoform present in monocytes and macrophages. Previously, we reported that increased intracellular ROS formation is required for OxLDL-induced cell death in human monocyte-derived macrophage (HMDM), but the source of these intracellular ROS was unknown (Asmis, Circ.Res. 2003). We now identified Nox4, not Nox2, as the source of OxLDL-induced ROS generation. Nox4 mRNA was detected in human blood monocytes and HMDM. Using a novel rabbit monoclonal antibody that detects Nox4 as a single 66 kDa band, we localized Nox4 to the ER, the nucleus and focal adhesion sites of HMDM, suggesting that Nox4 may play a role in macrophage redox signaling. After stimulation of HMDM with OxLDL, but not native LDL, Nox4 mRNA and protein expression were up-regulated in a dose- and time-dependent manner. Nox4 protein expression was maximal after 6 h and coincided with both increased p22 expression and increased intracellular ROS formation, suggesting that the Nox4/p22 complex is responsible for OxLDL-induced ROS formation in HMDM. Inhibition of MEK1 and ERK1/2 phosphorylation blocked both the induction of Nox4 expression and intracellular ROS formation induced by OxLDL, and protected HMDM from OxLDL-mediated cytotoxicity. In contrast, inhibitors of p38 MAPK or JNK affect neither Nox4 induction nor ROS formation, and showed no cytoprotection. Knockdown of Nox4 with adenovirus-delivered siRNA decreased OxLDL-induced ROS formation and protected macrophages from OxLDL-induced cell injury. In summary, we identified Nox4 as a novel, inducible source of intracellular ROS in human monocytes and macrophages. Nox4 is likely to play important roles in redox signaling and macrophage function. However, if overinduced Nox4-derived ROS promote macrophage death, a mechansim which may have important implications for the pathogenesis of atherosclerosis. This research has received full or partial funding support from the American Heart Association, South Central Affiliate (Arkansas, New Mexico, Oklahoma & Texas).