20-HETE activates the Raf/MEK/ERK pathway in renal epithelial cells through an EGFR- and c-Src-dependent mechanism

Abstract

20-Hydroxyeicosatetraenoic acid (20-HETE) has been reported to promote mitogenicity in a variety of cell types, including renal epithelial cells. However, the signal transduction pathways activated by 20-HETE have not been fully defined. The present study evaluated the effects of 20-HETE and its more stable agonist analogs 20-hydroxyeicosa-5(Z),14(Z)-dienoic acid (5,14-20-HEDE) and N-[20-hydroxyeicosa-5(Z),14(Z)-dienoyl]glycine (5,14-20-HEDGE) on the Raf/MEK/ERK and phosphatidylinositol 3-kinase (PI3K)-Akt pathway in LLC-PK1renal epithelial cells. 20-HETE (20 μM) increased phosphorylation of Raf-1 (2.5 ± 0.2-fold), MEK1/2 (6.3 ± 1.6-fold), and ERK1/2 (5.8 ± 0.3-fold) compared with vehicle-treated cells. Similarly, the 20-HETE analogs also strongly activated ERK1/2 in a Raf-1- and MEK1/2-dependent manner. Moreover, 5,14-20-HEDE increased Akt phosphorylation by 2.2 ± 0.3-fold. 20-HETE and 5,14-20-HEDE also promoted activation (Y1086) of epidermal growth factor receptor (EGFR; Y1086) by 1.9 ± 0.2- and 2.5 ± 0.2-fold, respectively. These effects were completely blocked by the EGFR inhibitor EKB-569 (0.1 μM). Moreover, EKB-569 (0.1 μM), as well as a c-Src inhibitor, SKI-606 (0.05 μM), completely abolished the 20-HETE-mediated activation of the Raf/MEK/ERK and PI3K-Akt pathways. Blockade of PKC with bisindolylmaleimide I had no effect on 20-HETE-induced ERK1/2 activation. This study demonstrated that 20-HETE activated the Raf/MEK/ERK and Akt pathways in renal epithelial cells secondary to the activation of c-Src and EGFR.