MKK4 is a novel target for the inhibition of tumor necrosis factor-α-induced vascular endothelial growth factor expression by myricetin

Tumor necrosis factor-α (TNF-α) is a mediator of multiple inflammatory diseases. Vascular endothelial growth factor (VEGF) plays a critical role in TNF-α-mediated diseases. We investigated the inhibitory effects of 3,3′,4′,5,5′,7-hexahydroxyflavone (myricetin), an abundant natural flavonoid, on TNF-α-induced VEGF upregulation and the underlying molecular mechanism. Myricetin is a direct inhibitor of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase 1 (MEK1) and inhibits neoplastic cell transformation. We found that myricetin inhibited TNF-α-induced VEGF expression in JB6 P+ mouse epidermal cells by targeting MAPK kinase 4 (MKK4), as well as MEK1. The activation of activator protein-1 by TNF-α was inhibited by myricetin in a dose-dependent manner. The phosphorylation of c-Jun N-terminal kinase (JNK) and ERK was inhibited by myricetin, but not the phosphorylation of their upstream kinases MKK4 and MEK1. TNF-α-induced VEGF expression was inhibited by SP600125 and U0126, which are inhibitors of JNK and MEK, respectively. Myricetin inhibited TNF-α-induced MKK4 activity and bound glutathione S-transferase-MKK4 directly by competing with ATP. Computer modeling suggested that myricetin docks onto the ATP-binding site in MKK4, which is located between the N- and C-lobes of the kinase domain. Overall, our results indicate that myricetin has potent chemopreventive effects against TNF-α-related disease, mainly by targeting MKK4 and MEK1.