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Characterize Disease-related Mutants of RAF Family Kinases by Using a Set of Practical and Feasible Methods
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20-HETE activates the Raf/MEK/ERK pathway in renal epithelial cells through an EGFR- and c-Src-dependent mechanism

Talha Akbulut1, Kevin R Regner, Richard J Roman

  • 1Department of Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.

American Journal of Physiology. Renal Physiology
|July 3, 2009
PubMed

Insights

20-Hydroxyeicosatetraenoic acid (20-HETE) activates key signaling pathways in renal cells. This study reveals 20-HETE activates the Raf/MEK/ERK and PI3K-Akt pathways via c-Src and EGFR.

Area of Science:

  • Cellular signaling
  • Renal physiology
  • Molecular biology

Background:

  • 20-Hydroxyeicosatetraenoic acid (20-HETE) is implicated in renal cell mitogenicity.
  • The precise signal transduction cascades initiated by 20-HETE remain incompletely elucidated.

Purpose of the Study:

  • To investigate the effects of 20-HETE and its analogs on the Raf/MEK/ERK and PI3K-Akt pathways in LLC-PK(1) renal epithelial cells.
  • To identify the upstream mediators of 20-HETE-induced signaling.

Main Methods:

  • LLC-PK(1) cells were treated with 20-HETE and its analogs (5,14-20-HEDE, 5,14-20-HEDGE).
  • Western blotting was used to assess phosphorylation of Raf-1, MEK1/2, ERK1/2, and Akt.
  • Effects of EGFR and c-Src inhibitors (EKB-569, SKI-606) and a PKC inhibitor (bisindolylmaleimide I) were evaluated.

Main Results:

  • 20-HETE significantly increased phosphorylation of Raf-1, MEK1/2, and ERK1/2.
  • 20-HETE analogs also activated ERK1/2 and increased Akt phosphorylation.
  • EGFR activation was observed, and its inhibition blocked 20-HETE effects.
  • Inhibition of EGFR and c-Src abolished 20-HETE-mediated activation of both pathways.
  • PKC inhibition did not affect 20-HETE-induced ERK1/2 activation.

Conclusions:

  • 20-HETE activates the Raf/MEK/ERK and PI3K-Akt signaling pathways in renal epithelial cells.
  • Activation of c-Src and EGFR are critical upstream events in 20-HETE signaling.
  • These findings clarify the molecular mechanisms underlying 20-HETE's effects on renal cells.

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