A novel role for activating transcription factor-2 in 15(S)-hydroxyeicosatetraenoic acid-induced angiogenesis

Tieqiang Zhao,Dong Wang,Sergey Y Cheranov,Manjula Karpurapu,Koteswara R Chava,Venkatesh Kundumani-Sridharan,Dianna A Johnson,John S Penn,Gadiparthi N Rao

doi:10.1194/jlr.m800388-jlr200

Abstract

To investigate the mechanisms underlying 15(S)-HETE-induced angiogenesis, we have studied the role of the small GTPase, Rac1. We find that 15(S)-HETE activated Rac1 in human retinal microvascular endothelial cells (HRMVEC) in a time-dependent manner. Blockade of Rac1 by adenovirus-mediated expression of its dominant negative mutant suppressed HRMVEC migration as well as tube formation and Matrigel plug angiogenesis. 15(S)-HETE stimulated Src in HRMVEC in a time-dependent manner and blockade of its activation inhibited 15(S)-HETE-induced Rac1 stimulation in HRMVEC and the migration and tube formation of these cells as well as Matrigel plug angiogenesis. 15(S)-HETE stimulated JNK1 in Src-Rac1-dependent manner in HRMVEC and adenovirus-mediated expression of its dominant negative mutant suppressed the migration and tube formation of these cells and Matrigel plug angiogenesis. 15(S)-HETE activated ATF-2 in HRMVEC in Src-Rac1-JNK1-dependent manner and interference with its activation via adenovirus-mediated expression of its dominant negative mutant abrogated migration and tube formation of HRMVEC and Matrigel plug angiogenesis. In addition, 15(S)-HETE-induced MEK1 stimulation was found to be dependent on Src-Rac1 activation. Blockade of MEK1 activation inhibited 15(S)-HETE-induced JNK1 activity and ATF-2 phosphorylation. Together, these findings show that 15(S)-HETE activates ATF-2 via the Src-Rac1-MEK1-JNK1 signaling axis in HRMVEC leading to their angiogenic differentiation.

Highlights

To investigate the mechanisms underlying 15(S)HETE-induced angiogenesis, we have studied the role of the small GTPase, Rac1
Toward understanding the mechanisms of 15(S)-HETE-induced angiogenesis, here we report that 15(S)-HETE-induced JNK1 activation requires Src-dependent Rac1-mediated MEK1 stimulation and that Src-Rac1-MEK1-JNK1 signaling targets activating transcription factor-2 (ATF-2) in influencing human retinal microvascular endothelial cell (HRMVEC) angiogenic differentiation
The important findings of the present study are that 1) 15(S)-HETE activated Src, Rac1 and ATF-2 in a timedependent manner in human retinal microvascular endothelial cells (HRMVEC); 2) 15(S)-HETE-induced ATF-2 activation was dependent on Src-Rac1-MEK1-JNK1

Summary

MATERIALS AND METHODS

Arachidonic acid and 15(S)-HETE were bought from Cayman Chemicals (Ann Arbor, MI). Growth factor-reduced Matrigel The beads were heated in 50 ml of Laemmeli sample buffer for 10 min, and the released proteins were resolved by 0.1% SDS-12% PAGE followed by Western blot analysis for Rac using its specific antibodies. When the effect of dominant negative Src, Rac, JNK1, and ATF-2 mutants was tested on 15(S)-HETE-induced HRMVEC migration, cells were infected with Ad-GFP, Ad-dnSrc, Ad-dnRac, Ad-dnJNK1, or Ad-dnATF-2 at a moi of 80 and quiesced before they were subjected to the migration assay. When the effect of dominant negative Src, Rac, JNK1, and ATF-2 mutants was tested on 15(S)HETE-induced HRMVEC tube formation, cells were infected with Ad-GFP, Ad-dnSrc, Ad-dnRac, Ad-dnJNK1 or Ad-dnATF-2 at a moi of 80 and quiesced before they were subjected to tube formation. In the case of Western blotting, one representative set of data is shown

RESULTS

DISCUSSION