Suppression of the Raf/MEK/ERK signaling cascade and inhibition of angiogenesis by the carboxyl terminus of angiopoietin-like protein 4

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 Lys(168) and Leu(169) and a minor cleavage site between Lys(170) and Met(171) 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.