The Amino-Terminal Oligomerization Domain of Angiopoietin-2 Affects Vascular Remodeling, Mammary Gland Tumor Growth, and Lung Metastasis in Mice.

Angiopoietin-2 (ANGPT2) is a context-dependent TIE2 agonistic or antagonistic ligand that induces diverse responses in cancer. Blocking ANGPT2 provides a promising strategy for inhibiting tumor growth and metastasis, yet variable effects of targeting ANGPT2 have complicated drug development. ANGPT2443 is a naturally occurring, lower oligomeric protein isoform whose expression is increased in cancer. Here, we use a knock-in mouse line (mice expressing Angpt2443), a genetic model for breast cancer and metastasis (MMTV-PyMT), a syngeneic melanoma lung colonization model (B16F10), and orthotopic injection of E0771 breast cancer cells to show that alternative forms increase the diversity of Angpt2 function. In a mouse retina model of angiogenesis, expression of Angpt2443 caused impaired venous development, suggesting enhanced function as a competitive antagonist for Tie2. In mammary gland tumor models, Angpt2443 differentially affected primary tumor growth and vascularization; these varying effects were associated with Angpt2 protein localization in the endothelium or in the stromal extracellular matrix as well as the frequency of Tie2-positive tumor blood vessels. In the presence of metastatic cells, Angpt2443 promoted destabilization of pulmonary vasculature and lung metastasis. In vitro, ANGPT2443 was susceptible to proteolytical cleavage, resulting in a monomeric ligand (ANGPT2DAP) that inhibited ANGPT1- or ANGPT4-induced TIE2 activation but did not bind to alternative ANGPT2 receptor α5ß1 integrin. Collectively, these data reveal novel roles for the ANGPT2 N-terminal domain in blood vessel remodeling, tumor growth, metastasis, integrin binding, and proteolytic regulation. SIGNIFICANCE: This study identifies the role of the N-terminal oligomerization domain of angiopoietin-2 in vascular remodeling and lung metastasis and provides new insights into mechanisms underlying the versatile functions of angiopoietin-2 in cancer.See related commentary by Kamiyama and Augustin, p. 35.

Angiopoietin-4-dependent venous maturation and fluid drainage in the peripheral retina.

The maintenance of fluid homeostasis is necessary for function of the neural retina; however, little is known about the significance of potential fluid management mechanisms. Here, we investigated angiopoietin-4 (Angpt4, also known as Ang3), a poorly characterized ligand for endothelial receptor tyrosine kinase Tie2, in mouse retina model. By using genetic reporter, fate mapping, and in situ hybridization, we found Angpt4 expression in a specific sub-population of astrocytes at the site where venous morphogenesis occurs and that lower oxygen tension, which distinguishes peripheral and venous locations, enhances Angpt4 expression. Correlating with its spatiotemporal expression, deletion of Angpt4 resulted in defective venous development causing impaired venous drainage and defects in neuronal cells. In vitro characterization of angiopoietin-4 proteins revealed both ligand-specific and redundant functions among the angiopoietins. Our study identifies Angpt4 as the first growth factor for venous-specific development and its importance in venous remodeling, retinal fluid clearance and neuronal function.

Functional organization of Golgi N- and O-glycosylation pathways involves pH-dependent complex formation that is impaired in cancer cells.

Glycosylation is one of the most common modifications of proteins and lipids and also a major source of biological diversity in eukaryotes. It is critical for many basic cellular functions and recognition events that range from protein folding to cell signaling, immunological defense, and the development of multicellular organisms. Glycosylation takes place mainly in the endoplasmic reticulum and Golgi apparatus and involves dozens of functionally distinct glycosidases and glycosyltransferases. How the functions of these enzymes, which act sequentially and often competitively, are coordinated to faithfully synthesize a vast array of different glycan structures is currently unclear. Here, we investigate the supramolecular organization of the Golgi N- and O-glycosylation pathways in live cells using a FRET flow cytometric quantification approach. We show that the enzymes form enzymatically active homo- and/or heteromeric complexes within each pathway. However, no complexes composed of enzymes that operate in different pathways, were detected, which suggests that the pathways are physically distinct. In addition, we show that complex formation is mediated almost exclusively by the catalytic domains of the interacting enzymes. Our data also suggest that the heteromeric complexes are functionally more important than enzyme homomers. Heteromeric complex formation was found to be dependent on Golgi acidity, markedly impaired in acidification-defective cancer cells, and required for the efficient synthesis of cell surface glycans. Collectively, the results emphasize that the Golgi glycosylation pathways are functionally organized into complexes that are important for glycan synthesis.