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


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.
Yang
Ying-Hua
YH
Department of Medicine, The University of Hong Kong, L8-40, New Laboratory Block, 21 Sassoon Road, Hong Kong.
Wang
Yu
Y
Lam
Karen S L
KS
Yau
Ming-Hon
MH
Cheng
Kenneth K Y
KK
Zhang
Jialiang
J
Zhu
Weidong
W
Wu
Donghai
D
Xu
Aimin
A
D000595:Amino Acid Sequence; D000075803:Angiopoietin-Like Protein 4; D042682:Angiopoietins; D000818:Animals; D001798:Blood Proteins / Q000502:physiology*; D002460:Cell Line; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D004730:Endothelium, Vascular / Q000187:drug effects / Q000473:pathology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D005260:Female; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D006801:Humans; D020930:MAP Kinase Kinase Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008819:Mice, Nude; D008969:Molecular Sequence Data; D018919:Neovascularization, Physiologic / Q000502:physiology*; D010766:Phosphorylation / Q000187:drug effects; D015398:Signal Transduction; D042461:Vascular Endothelial Growth Factor A / Q000494:pharmacology; D048490:raf Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism
D000075803:Angiopoietin-Like Protein 4; D042682:Angiopoietins; C407088:Angptl4 protein, mouse; D001798:Blood Proteins; D042461:Vascular Endothelial Growth Factor A; D016222:Fibroblast Growth Factor 2; D048490:raf Kinases; D048049:Extracellular Signal-Regulated MAP Kinases; D020930:MAP Kinase Kinase Kinases