DCC-dependent Phospholipase C Signaling in Netrin-1-induced Neurite Elongation

Abstract

Netrins, a family of secreted molecules, play important roles in axon pathfinding during nervous system development. Although phosphatidylinositol signaling has been implicated in this event, how netrin-1 regulates phosphatidylinositol signaling remains poorly understood. Here we provide evidence that netrin-1 stimulates phosphatidylinositol bisphosphate hydrolysis in cortical neurons. This event appears to be mediated by DCC (deleted in colorectal cancer), but not neogenin or Unc5h2. Netrin-1 induces phospholipase C␥ (PLC␥) tyrosine phosphorylation. Inhibition of PLC activity attenuates netrin-1-induced cortical neurite outgrowth. These results suggest that netrin-1 regulates phosphatidylinositol turnover and demonstrate a crucial role of PLC signaling in netrin-1-induced neurite elongation.Proper wiring in developing brains requires neurite outgrowth and growth cone navigation. Netrins, a family of secreted factors, promote axon outgrowth (1-4). In addition, netrins are able to guide neuronal growth cones and regulate neuronal branching in the developing nervous system (1-4). Netrins act through two classes of receptors: DCC and Unc5. The DCC family includes DCC and neogenin in vertebrates (5), Unc40 in Caenorhabditis elegans (6), and Frazzled in Drosophila (7,8). DCC is required for the attractive response (9). Unc5 in C. elegans and Unc5A, -5B, and -5C in vertebrates belong to the Unc5 family, which appears to mediate the repulsive response (10 -14).DCC and Unc5 proteins are transmembrane proteins without any obvious catalytic activity, and thus it remains unknown exactly how they initiate downstream signaling to mediate or regulate axonal outgrowth and guidance. Nevertheless, perturbation of Rho family GTPases inhibits netrin-induced neurite outgrowth (15). Pharmacological inhibition of extracellular signal-regulated kinase (ERK) attenuates netrin-1-induced neurite outgrowth and growth cone turning (16,17). Inhibition of focal adhesion kinase (FAK), 3 a major tyrosine kinase localized in focal adhesions and implicated in cell spreading and migration, blocks netrin-1-induced neurite elongation and growth cone guidance (18 -20). Treatment of wortmannin, an inhibitor of phosphatidylinositol 3-kinase, attenuates netrin-1-induced growth cone turning in Xenopus spinal neurons (27).Phosphoinositides are quantitatively minor phospholipids of cell membranes, but their metabolism is highly active and tightly regulated. They (e.g. PIP 2 ) function either as precursors of second messengers such as inositol 1,4,5-trisphosphate (IP 3 ) and diacylglycerol or by directly interacting with both actin-binding and pleckstrin homology domaincontaining proteins to regulate their spatiotemporal distribution and/or activity. In addition, PIP 2 functions as a cofactor for small GTP-binding proteins (e.g. Arf) and phospholipase D (21, 22). In the nervous system, PIP 2 plays an important role in membrane trafficking at the synapse. Synaptic vesicle exocytosis and endocytosis require phosphatidylinositol 4,5-bisphosphate (23-26). Usin...

Opposing roles of netrin-1 and the dependence receptor DCC in cancer cell invasion, tumor growth and metastasis

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Overexpression of Netrin-1 Induces Neovascularization in the Adult Mouse Brain

“…Similar to in vitro studies, the infected cells (neurons and astrocytes) expressed and secreted functional netrin-1 and acted on cells in brain tissue, including endothelial cells. Endogenous netrin-1, predominantly expressed by the floor plate cells in the central nervous system, induces axonal outgrowth, axonal orientation, and neuronal migration during neuronal development (Barallobre et al, 2005;Xie et al, 2006). Besides the aforementioned roles it plays in the central nervous system, NT-1 has also been implicated in regulating salivary gland migration (Kolesnikov and Beckendorf, 2005), regulating invasion and migration of mouse mammary epithelial cells (Strizzi et al, 2005), promoting lung branching morphogenesis (Liu et al, 2004), and mediating pancreatic epithelial cell adhesion (Hebrok and Reichardt, 2004).…”

Calcium signaling in axon guidance

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