This Article Full Text Full Text (PDF) Data Supplement Correction (v29,pe132) All Versions of this Article: 29/6/909    most recent ATVBAHA.108.178541v1 Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Google Scholar Articles by Hayashi, H. Articles by Kaneda, Y. PubMed PubMed Citation Articles by Hayashi, H. Articles by Kaneda, Y.

(Arteriosclerosis, Thrombosis, and Vascular Biology. 2009;29:909.)
© 2009 American Heart Association, Inc.

Cell Biology/Signaling

FHL-2 Suppresses VEGF-Induced Phosphatidylinositol 3-Kinase/Akt Activation via Interaction With Sphingosine Kinase-1

Hiroki Hayashi; Hironori Nakagami; Yoichi Takami; Hiroshi Koriyama; Masaki Mori; Katsuto Tamai; Jianxin Sun; Kaori Nagao; Ryuichi Morishita; Yasufumi Kaneda

From the Division of Gene Therapy Science (H.H., H.N., Y.T., H.K., M.M., K.T., Y.K.), the Department of Geriatric Medicine (Y.T., H.K.), and the Division of Clinical Gene Therapy and Graduate School of Medicine (K.N., R.M.), Osaka University, Japan; and the Department of Cell Biology and Molecular Medicine (J.S.), UMDNJ-New Jersey Medical School, Newark.

Correspondence to Hironori Nakagami, MD, PhD, Division of Gene Therapy Science, Osaka University Graduate School of Medicine, 2-2 Yamada-oka, Suita 565-0871, Japan. E-mail nakagami{at}gts.med.osaka-u.ac.jp

Objective— In the functional screening of a human heart cDNA library to identify a novel antiangiogenic factor, the prime candidate gene was "four-and-a-half LIM only protein-2" (FHL-2). The goal of this study is to clear the mechanism of antiangiogenic signaling of FHL-2 in endothelial cells (ECs).

Methods and Results— Overexpressed FHL-2 strongly inhibited vascular endothelial growth factor (VEGF)-induced EC migration. In the angiogenic signaling, we focused on sphingosine kinase-1 (SK1), which produces sphingosine-1-phosphate (S1P), a bioactive sphingolipid, as a potent angiogenic mediator in ECs. Immunoprecipitation and immunostaining analysis showed that FHL-2 might bind to SK1. Importantly, overexpression of FHL-2 in ECs inhibited VEGF-induced SK1 activity, phosphatidylinositol 3-kinase activity, and phosphorylation of Akt and eNOS. In contrast, overexpression of FHL-2 had no effect on S1P-induced Akt phosphorylation. Interestingly, VEGF stimulation decreased the binding of FHL-2 and SK1. Depletion of FHL-2 by siRNA increased EC migration accompanied with SK1 and Akt activation, and increased the expression of VEGF receptor-2 which further enhanced VEGF signaling. Furthermore, injection of FHL-2 mRNA into Xenopus embryos resulted in inhibition of vascular network development, assessed by in situ hybridization with endothelial markers.

Conclusions— FHL-2 may regulate phosphatidylinositol 3-kinase/Akt via direct suppression of the SK1-S1P pathway in ECs.

In the functional screening of a novel antiangiognic factor, the prime candidate gene was "four-and-a-half LIM only protein-2" (FHL-2). Overexpression of FHL-2 in ECs inhibited VEGF-induced SK1 activation, phosphatidylinositol 3-kinase/Akt activation, cell migration, and vascular network in Xenopus embryos. FHL-2 may directly suppress the SK1-S1P pathway in ECs.

Key Words: FHL-2 • VEGF • sphingosine kinase-1 • endothelial cells • Akt pathway