AP-1 (Fra-1/c-Jun)-mediated Induction of Expression of Matrix Metalloproteinase-2 Is Required for 15(S)-Hydroxyeicosatetraenoic Acid-induced Angiogenesis

Nikhlesh K Singh,Dong Van Quyen,Venkatesh Kundumani-Sridharan,Peter C Brooks,Gadiparthi N Rao

doi:10.1074/jbc.m110.106187

Abstract

To understand the involvement of matrix metalloproteinases (MMPs) in 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE)-induced angiogenesis, we have studied the role of MMP-2. 15(S)-HETE induced MMP-2 expression and activity in a time-dependent manner in human dermal microvascular endothelial cells (HDMVECs). Inhibition of MMP-2 activity or depletion of its levels attenuated 15(S)-HETE-induced HDMVEC migration, tube formation, and Matrigel plug angiogenesis. 15(S)-HETE also induced Fra-1 and c-Jun expression in a Rac1-MEK1-JNK1-dependent manner. In addition, 15(S)-HETE-induced MMP-2 expression and activity were mediated by Rac1-MEK1-JNK1-dependent activation of AP-1 (Fra-1/c-Jun). Cloning and site-directed mutagenesis of MMP-2 promoter revealed that AP-1 site proximal to the transcriptional start site is required for 15(S)-HETE-induced MMP-2 expression, and Fra-1 and c-Jun are the essential components of AP-1 that bind to MMP-2 promoter in response to 15(S)-HETE. Hind limb ischemia led to an increase in MEK1 and JNK1 activation and Fra-1, c-Jun, and MMP-2 expression resulting in enhanced neovascularization and recovery of blood perfusion in wild-type mice as compared with 12/15-Lox(-/-) mice. Together, these results provide the first direct evidence for a role of 12/15-Lox-12/15(S)-HETE axis in the regulation of ischemia-induced angiogenesis.

Highlights

Angiogenesis has been implicated in the pathogenesis of cancer, diabetic retinopathy, and atherosclerosis [1]
To understand the involvement of matrix metalloproteinases (MMPs) in 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE)induced angiogenesis, we have studied the role of MMP-2. 15(S)-HETE induced MMP-2 expression and activity in a timedependent manner in human dermal microvascular endothelial cells (HDMVECs)
Many studies have focused on the role of 12/15-Lox, a murine ortholog of 15-Lox1, in the pathogenesis of atherosclerosis and restenosis (24 –26, 54), underscoring the importance of 15-Lox/15-HETE in these diseases

Summary

MATERIALS AND METHODS

Reagents—15(S)-HETE was bought from Cayman Chemicals (Ann Arbor, MI). Growth factor-reduced Matrigel was obtained from BD Biosciences (Bedford, MA). Cells were quiesced by incubating in medium 131 without microvascular growth supplements for 24 h and used to perform the experiments unless otherwise indicated. When the effect of a pharmacological inhibitor was tested on 15(S)-HETE-induced HDMVEC migration, cells were incubated with the inhibitor for 30 min first and added to the upper chamber. When the effect of a pharmacological inhibitor was tested on 15(S)-HETE-induced HDMVEC tube formation, cells were incubated with the inhibitor for 30 min first and plated. A and B, quiescent HDMVECs were treated with and without 0.1 ␮M 15(S)-HETE for the indicated time periods, and either total cellular RNA was isolated and analyzed for MMP-2 and ␤-actin mRNA levels by QRT-PCR (A) or medium was collected and analyzed for MMP-2 activity by gelatin zymography (B). In the case of double immunofluorescence staining, ChIP analysis, EMSA, gelatin zymography, and Western blotting, one representative set of data is shown

RESULTS

Findings

DISCUSSION