Urotensin II promotes secretion of LTB through 5-lipoxygenase via the UT-ROS-Akt pathway in RAW264.7 macrophages.

INTRODUCTION

Urotensin II (UII) is an important vasoactive peptide involved in the pathogenesis of atherosclerosis. Monocytes/macrophages play important roles in every step of atherosclerosis. Although UII has a chemoattractant effect on monocytes, it is unclear whether UII regulates inflammatory responses in macrophages. The present study sought to explore whether UII can promote leukotriene B (LTB) production by macrophages.

MATERIAL AND METHODS

The mRNA expression level of LTB and 5-lipoxygenase were determined by real-time polymerase chain reaction. The protein level of LTB and 5-lipoxygenase expression was assayed by enzyme-linked immunosorbent assay and Western blot, respectively. Western blot analysis was also employed to determine the phosphorylated forms of Akt. Reactive oxygen species (ROS) level was detected by the fluorescent probe 2',7'-dichlorofluorescin diacetate and fluorescence intensity was measured with a multiwell fluorescence plate reader.

RESULTS

Urotensin II promoted LTB release and increased 5-lipoxygenase expression in a concentration- and time-dependent manner in RAW264.7 cells. Leukotriene B4 production and 5-lipoxygenase expression were decreased by blocking the UII receptor (UT) with urantide, eliminating ROS with N-acetylcysteine and diphenyliodonium, and inhibiting Akt phosphorylation with LY294002. UII significantly elevated ROS production, whereas urantide, N-acetylcysteine and diphenyliodonium substantially attenuated this effect. UII also enhanced Akt phosphorylation significantly, and this effect was potently inhibited by urantide, N-acetylcysteine, diphenyliodonium and LY294002.

CONCLUSIONS

Urotensin II may promote 5-lipoxygenase expression and LTB release in RAW264.7 macrophages via UT-ROS-Akt pathways. These results indicate that UII may participate in macrophage activation and suggest a potential new mechanism underlying atherosclerosis.