Submitted on March 11, 2009
Revised on June 1, 2009
Accepted on June 27, 2009
20-HETE activates the Raf/MEK/ERK pathway in renal epithelial cells through EGFR- and c-Src-dependent mechanisms
Talha Akbulut1, Kevin R. Regner1, Richard J. Roman1, Ellis D. Avner, John R. Falck2, and Frank Park1*
1 Medical College of Wisconsin
2 UT Southwestern Medical Center @ Dallas
* To whom correspondence should be addressed. E-mail: fpark{at}mcw.edu.
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 more stable agonist analogs, 20-hydroxyeicosa-5(Z),14(Z)-dienoic acid (HEDE) and N-[20-hydroxyeicosa-5(Z),14(Z)-dienoyl]glycine (HEDGE), on the Raf/MEK/ERK and PI3-K-Akt pathway in LLC-PK1 renal epithelial cells. 20-HETE (20 µM) increased the 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 to vehicle-treated cells. Similarly, the 20-HETE analogs also strongly activated ERK1/2 in a Raf-1 and MEK1/2-dependent manner. Moreover, HEDE increased the phosphorylation of Akt by 2.2 ± 0.3-fold. 20-HETE and HEDE also promoted activation of 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 both the Raf/MEK/ERK and PI3-K-Akt pathways. Blockade of Protein Kinase C (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.
