Netrin-4 inhibits angiogenesis via binding to neogenin and recruitment of Unc5B

Abstract

Netrins are secreted molecules with roles in axon guidance and angiogenesis. We identified Netrin-4 as a gene specifically overexpressed in VEGF-stimulated endothelial cells (EC) in vitro as well as in vivo. Knockdown of Netrin-4 expression in EC increased their ability to form tubular structures on Matrigel. To identify which receptor is involved, we showed by quantitative RT-PCR that EC express three of the six Netrin-1 cognate receptors: neogenin, Unc5B, and Unc5C. In contrast to Netrin-1, Netrin-4 bound only to neogenin but not to Unc5B or Unc5C receptors. Neutralization of Netrin-4 binding to neogenin by blocking antibodies abolished the chemotactic effect of Netrin-4. Furthermore, the silencing of either neogenin or Unc5B abolished Netrin-4 inhibitory effect on EC migration, suggesting that both receptors are essential for its function in vitro. Coimmunoprecipitation experiments demonstrated that Netrin-4 increased the association between Unc5B and neogenin on VEGF-or FGF-2-stimulated EC. Finally, we showed that Netrin-4 significantly reduced pathological angiogenesis in Matrigel and laser-induced choroidal neovascularization models. Interestingly, Netrin-4, neogenin, and Unc5B receptor expression was up-regulated in choroidal neovessel EC after laser injury. Moreover, Netrin-4 overexpression delayed tumor angiogenesis in a model of s.c. xenograft. We propose that Netrin-4 acts as an antiangiogenic factor through binding to neogenin and recruitment of Unc5B.

Dependence receptors: a new paradigm in cell signaling and cancer therapy

“…It should be noted that the question of neogenin's ligand remains tricky: despite its higher affinity for RGM, neogenin seems to be able to mediate netrin-1 signaling in axon attraction (Wilson and Key, 2006) or cell adhesion (Srinivasan et al, 2003). Moreover, neogenin has also been proposed to mediate netrin-3 signaling during myotube formation (Kang et al, 2004), as well as netrin-4 signaling in angiogenesis (Lejmi et al, 2008).…”

Discovery of a functional protein complex of netrin-4, laminin γ1 chain, and integrin α6β1 in mouse neural stem cells

“…olfactory system ͉ phage display ͉ merogenesis ͉ rostral migratory stream ͉ astrocytes N etrins are a family of secreted proteins initially described as developmental axon attractants (1). Yet, with critical roles in tissue morphogenesis (2)(3)(4), vascular patterning (5), and angiogenesis (6)(7)(8), netrins have become recognized to mediate functions far beyond axonal guidance. Netrin-4 is expressed in both neural and nonneural tissues (8)(9)(10)(11).…”

“…Yet, with critical roles in tissue morphogenesis (2)(3)(4), vascular patterning (5), and angiogenesis (6)(7)(8), netrins have become recognized to mediate functions far beyond axonal guidance. Netrin-4 is expressed in both neural and nonneural tissues (8)(9)(10)(11). In the nervous system, it promotes neurite extension from olfactory bulb (OB) explants but the molecular mechanisms underlying this phenomenon remain unknown.…”

“…Integrins and netrins, along with the established netrin-1 and netrin-4 receptors DCC and UNC5, appear to cooperate to regulate multiple aspects of development (3,12,13). However, certain netrin-4 functions are mediated by neogenin (8) and likely by other, as yet unidentified receptors (14,15).…”

Netrin-4 regulates thalamocortical axon branching in an activity-dependent fashion

“…We studied the expression in the thalamus of putative receptors that reportedly bind to NTN4: deleted in colorectal carcinoma (dcc), neogenin-1 (neo1), and unc5 family (36,42). At P7, dcc, neo1, and unc5b were strongly expressed in the ventrobasal (VB) thalamus and the dorsal part of the lateral geniculate nucleus (dLGN) (Fig.…”