ABSTRACT
Pleiotrophin (PTN) is a pleiotropic growth factor that exhibits angiogenic properties and is involved in tumor growth and metastasis. Although it has been shown that PTN is expressed in tumor cells, few studies have investigated its receptors and their involvement in cell migration and invasion. Neuropilin-1 (NRP-1) is a receptor for multiple growth factors that mediates cell motility and plays an important role in angiogenesis and tumor progression. Here we provide evidence for the first time that NRP-1 is crucial for biological activities of PTN. We found that PTN interacted directly with NRP-1 through its thrombospondin type-I repeat domains. Importantly, binding of PTN to NRP-1 stimulated the internalization and recycling of NRP-1 at the cell surface. Invalidation of NRP-1 by RNA interference in human carcinoma cells inhibited PTN-induced intracellular signaling of the serine-threonine kinase, mitogen-activated protein MAP kinase, and focal adhesion kinase pathways. Accordingly, NRP-1 silencing or blocking by antibody inhibited PTN-induced human umbilical vein endothelial cell migration and tumor cell invasion. These results suggest that NRP-1/PTN interaction provides a novel mechanism for controlling the response of endothelial and tumoral cells to PTN and may explain, at least in part, how PTN contributes to tumor angiogenesis and cancer progression.
Subject(s)
Carrier Proteins/metabolism , Cell Movement/physiology , Cytokines/metabolism , Neuropilin-1/metabolism , Signal Transduction/physiology , Animals , Binding Sites/genetics , CHO Cells , Carrier Proteins/genetics , Cell Line, Tumor , Cell Movement/drug effects , Cell Movement/genetics , Cells, Cultured , Cricetinae , Cricetulus , Cytokines/genetics , Endocytosis/drug effects , Endocytosis/genetics , Endocytosis/physiology , Glutathione Transferase/genetics , Glutathione Transferase/metabolism , Humans , Immunoblotting , Microscopy, Fluorescence , Neoplasm Invasiveness , Neuropilin-1/genetics , Protein Binding/genetics , RNA Interference , Recombinant Fusion Proteins/metabolism , Recombinant Fusion Proteins/pharmacology , Signal Transduction/drug effects , Signal Transduction/geneticsABSTRACT
Angiogenesis is the physiological process involving the growth of new blood vessels from pre-existing vessels. In normal or pathological angiogenesis, angiogenic growth factors activate cognate receptors on endothelial cells. Fibroblast growth factor-2 (FGF-2) and heparin affin regulatory peptide (HARP) are two heparin-binding growth factors and were described for their pro-angiogenic properties on human umbilical endothelial cells (HUVEC). We now show that HARP acts as a mediator of FGF-2's stimulatory effects, since it is able to inhibit the proliferation and migration of HUVEC induced by FGF-2. We demonstrate by ELISA and optical biosensor binding assay that HARP and FGF-2 interact through direct binding. We have adapted a previously developed structural proteomics method for the identification of residues involved in protein-protein interactions. Application of this method showed that two sequences in HARP were involved in binding FGF-2. One was in the C-thrombospondin type 1 repeat (C-TSR-1) domain and the other in the C-terminal domain of HARP. The identification of these regions as mediating the binding of FGF-2 was confirmed by ELISA using synthetic peptides, which are as well mediators of FGF-2-induced proliferation, migration and tubes formation on HUVEC in vitro. These results imply that besides a regulation of the proliferation, migration and angiogenesis of HUVEC by direct interaction of FGF-2 with its receptors, an alternative pathway exists involving its binding to growth factors such as HARP.
