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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2008;28:835.)
© 2008 American Heart Association, Inc.

Integrated Physiology/Experimental Medicine

Suppression of the Raf/MEK/ERK Signaling Cascade and Inhibition of Angiogenesis by the Carboxyl Terminus of Angiopoietin-Like Protein 4

Ying-Hua Yang; Yu Wang; Karen S.L. Lam; Ming-Hon Yau; Kenneth K.Y. Cheng; Jialiang Zhang; Weidong Zhu; Donghai Wu; Aimin Xu

From the Departments of Medicine (Y.-H.Y., K.S.L.L., M.-H.Y., K.K.Y.C., J.Z., W.Z., A.X.), Research Center of Heart, Brain, Hormone, and Healthy Aging (Y.-H.Y., Y.W., K.S.L.L., M.-H.Y., K.K.Y.C., J.Z., W.Z., A.X.), and Genome Research Center (Y.W.), and the Department of Pharmacology (A.X.), The University of Hong Kong, and the Guang Zhou Institute of Biomedicine and Health (D.W., A.X.), Chinese Academy of Sciences, China. Current affiliation for Y.-H.Y.: Department of Biology, Hai Nan Normal University, Hai Kou, China.

Correspondence to Aimin Xu, Department of Medicine, The University of Hong Kong, L8-40, New Laboratory Block, 21 Sassoon Road, Hong Kong. E-mail amxu{at}hkucc.hku.hk

Abstract

Objectives— Angiopoietin-like protein 4 (Angptl4) is a secreted glycoprotein that has recently been implicated in the regulation of angiogenesis and metastasis. This study aimed to investigate the structural and cellular basis underlying the biological actions of Angptl4.

Methods and Results— Circulating Angptl4 was proteolytically cleaved into NH2-terminal coiled-coil domain (N-Angptl4) and COOH-terminal fibrinogen-like domain (C-Angptl4). Using amino acid sequencing analysis, we identified a major cleavage site between Lys¹⁶⁸ and Leu¹⁶⁹ and a minor cleavage site between Lys¹⁷⁰ and Met¹⁷¹ in mouse Angptl4. C-Angptl4, but not N-Angptl4, potently inhibited both bFGF- and VEGF-induced cell proliferation, migration, and tubule formation in endothelial cells, and prevented neovascularization in mice. Treatment of C-Angptl4 with PNGase F (an N-glycosidase) ablated its N-linked glycosylation, and also significantly attenuated its antiangiogenic activities. C-Angptl4 blocked bFGF-induced activation of ERK1/2 MAP kinase, but had no obvious effect on Akt and P38 MAP kinase. Furthermore, C-Angptl4 abrogated bFGF-induced phosphorylation of Raf-1 and MEK1/2, whereas neither auto-phosphorylation of FGF receptor-1 nor activation of Ras was affected, suggesting that the blockage occurs at the level of Raf-1 activation.

Conclusions— The carboxyl terminus of Angptl4 alone is sufficient to suppress angiogenesis, possibly through inhibiting the Raf/MEK/ERK1/2 MAP kinase pathway in endothelial cells.

This study comprehensively investigated the structural and cellular basis underlying the antiangiogenic activities of angiopoietin-like protein-4 (Angptl4). We found that the carboxyl terminus of Angptl4 alone is sufficient to suppress neovascularization in an N-glycosylation–dependent manner. Furthermore, our results showed that Angplt4 inhibits the Raf-1/ERK1/2 MAPK pathway in endothelial cells.

Key Words: angiogenesis • angiopoietin-like proteins • glycosylation • MAP kinase • neovascularization