Steady laminar blood flow protects vessels from atherosclerosis. We showed that flow decreased tumor necrosis factor-alpha (TNF)-mediated VCAM1 expression in endothelial cells (EC) by inhibiting JNK. Here, we determined the relative roles of MEK1, MEK5 and their downstream kinases ERK1/2 and BMK1 (ERK5) in flow-mediated inhibition of JNK activation. Steady laminar flow (shear stress = 12 dyn/cm(2)) increased BMK1 and ERK1/2 activity in EC. Pre-exposing EC for 10 min to flow inhibited TNF activation of JNK by 58%. A key role for BMK1, but not ERK1/2 was shown. (1) Incubation of EC with PD184352, at concentrations that blocked ERK1/2, but not BMK1, had no effect on flow inhibition of TNF-mediated JNK activation. (2) BIX02188, a MEK5-selective inhibitor, completely reversed the inhibitory effects of flow. These findings indicate that flow inhibits TNF-mediated signaling events in EC by a mechanism dependent on activation of MEK5-BMK1, but not MEK1-ERK1/2. These results support a key role for the MEK5-BMK1 signaling pathway in the atheroprotective effects of blood flow. (c) 2008 Elsevier Inc. All rights reserved.