Neuropilin-1 Signaling through p130^Cas Tyrosine Phosphorylation Is Essential for Growth Factor-Dependent Migration of Glioma and Endothelial Cells

Abstract

Neuropilin-1 (NRP1) is a receptor for vascular endothelial growth factor (VEGF) and plays an important role in mediating cell motility. However, the NRP1 signaling pathways important for cell motility are poorly understood. Here we report that p130Cas tyrosine phosphorylation is stimulated by hepatocyte growth factor and platelet-derived growth factor in U87MG glioma cells and VEGF in endothelial cells and is dependent on NRP1 via its intracellular domain. In endothelial cells, NRP1 silencing reduced, but did not prevent, VEGF receptor 2 (VEGFR2) phosphorylation, while expression of a mutant form of NRP1 lacking the intracellular domain (NRP1⌬C) did not affect receptor phosphorylation in U87MG cells or human umbilical vein endothelial cells (HUVECs). In HUVECs, NRP1 was also required for VEGF-induced phosphorylation of proline-rich tyrosine kinase 2, which was necessary for p130Cas phosphorylation. Importantly, knockdown of NRP1 or p130Cas or expression of either NRP1⌬C or a nontyrosine-phosphorylatable substrate domain mutant protein (p130 Cas15F ) was sufficient to inhibit growth factor-mediated migration of glioma and endothelial cells. These data demonstrate for the first time the importance of the NRP1 intracellular domain in mediating a specific signaling pathway downstream of several receptor tyrosine kinases and identify a critical role for a novel NRP1-p130Cas pathway in the regulation of chemotaxis.Neuropilin-1 (NRP1) is a coreceptor for vascular endothelial growth factor (VEGF) in endothelial cells and is essential for embryonic angiogenesis and vascular development (10,29). Though the precise cellular functions of NRP1 have yet to be elucidated, there is a growing body of evidence supporting a key role for NRP1 in the migration of both endothelial and tumor cells (9,11,15,19). NRP1 is thought to act as a coreceptor for VEGF by forming complexes with the VEGF receptor tyrosine kinase (RTK) VEGFR2. Complexation between NRP1 and VEGFR2 enhances VEGF binding, and inhibition of complex formation is associated with reduced VEGFR2 phosphorylation, intracellular signaling, mitogenesis, cell migration, and angiogenesis (16,18,28,34,35). However, the precise role of NRP1 in VEGF signaling remains unclear. Recent evidence indicates that NRP1 also regulates tumor and vascular cell functions stimulated by other growth factors, such as hepatocyte growth factor (HGF) and plateletderived growth factor (PDGF). Overexpression of NRP1 promotes tumor progression by potentiating the effect of the HGF/c-Met pathway, and tumor cell invasion mediated by the HGF/c-Met pathway is dependent on NRP1 through an association with c-Met (11, 15). Furthermore, NRP1 and NRP2 can bind HGF and mediate HGF stimulation of endothelial cell migration and proliferation (30). A recent report showed that NRP1 is also required for tumor cell-derived PDGF-mediated migration of smooth muscle cells (2). While these results indicate that NRP1 is required for optimal growth factor signaling important for cell motility, it remains unclear whether NR...

Neuropilin-2 regulates α6β1 integrin in the formation of focal adhesions and signaling

“…Of note, a recent study concluded that NRP1 does not localize to focal adhesions, establishing a key difference between these two receptors (Evans et al, 2011). Importantly, we also implicate FAK activation as the prime consequence of VEGF-NRP2 regulation of a6b1 integrin and demonstrate the significance of this mechanism in tumor cells isolated from invasive breast carcinomas.…”

Protein kinase LKB1 promotes RAB7-mediated neuropilin-1 degradation to inhibit angiogenesis

“…Enhanced NRP-1 expression during tumorigenesis may represent an angiogenic switch, and the receptor remains an attractive therapeutic target. NRP-1 expression in endothelial and tumor cells has been associated with regulating VEGFR2 trafficking (28)(29)(30)(31). However, the factors that regulate NRP-1 trafficking and the fate of the internalized receptor are less understood.…”

“…The expression of more than 60 different mammalian RAB family members (31,35) highlights complex but highly specific functions. While certain RABs are ubiquitously expressed, others demonstrate tissue-or functionspecific expression (32).…”

Occludin downregulation in high glucose is regulated by SSTR2 via the VEGF/NRP1/Akt signaling pathway in RF/6A cells

“…This was consistent with a previous study that demonstrated NRP1 to be a required component for the regulation of vascular permeability, induced by VEGF (40). Evans et al (44) suggested that treatment of small interfering RNA on NRP1 in HUVEC cells did not affect the phosphorylation of AKT and ERK mediated by VEGF. This was in accordance with the results of the present study.…”

“…NRP1 may promote VEGF binding and potentiate VEGFR2 activation and intracellular signaling (44). To illustrate whether NRP1 was involved in the high glucose-induced VEGF autocrine response and occludin reduction, a NRP1 inhibitor, ATWLPPR was administered to RF/6A cells.…”