Neuropilin-1-mediated Vascular Permeability Factor/Vascular Endothelial Growth Factor-dependent Endothelial Cell Migration

Abstract

Neuropilin-1 (NRP-1) has been found to be expressed by endothelial cells and tumor cells as an isoform-specific receptor for vascular permeability factor/vascular endothelial growth factor (VEGF). Previous studies were mainly focused on the extracellular domain of NRP-1 that can bind to VEGF165 and, thus, enables NRP-1 to act as a co-receptor for VEGF165, which enhances its binding to VEGFR-2 and its bioactivity. However, the exact functional roles and related signaling mechanisms of NRP-1 in angiogenesis are not well understood. In this study we constructed a chimeric receptor, EGNP-1, by fusing the extracellular domain of epidermal growth factor receptor to the transmembrane and intracellular domains of NRP-1 and transduced it into HUVECs with a retroviral expression vector. We observed that NRP-1/EGNP-1 mediates ligand-stimulated migration of human umbilical vein endothelial cells (HUVECs) but not proliferation. Our results show that NRP-1 alone can mediate HUVEC migration through its intracellular domain, and its C-terminal three amino acids (SEA-COOH) are essential for the process. We demonstrate that phosphatidylinositol 3-kinase inhibitor Ly294002 and the p85 dominant negative mutant can block NRP-1-mediated HUVEC migration. NRP-1-mediated migration can be significantly reduced by overexpression of the dominant negative mutant of RhoA (RhoA-19N). In addition, Gq family proteins and Gβγ subunits are also required for NRP-1-mediated HUVEC migration. These results show for the first time that NRP-1 can independently promote cell signaling in endothelial cells and also demonstrate the importance of last three amino acids of NRP-1 for its function. Neuropilin-1 (NRP-1) has been found to be expressed by endothelial cells and tumor cells as an isoform-specific receptor for vascular permeability factor/vascular endothelial growth factor (VEGF). Previous studies were mainly focused on the extracellular domain of NRP-1 that can bind to VEGF165 and, thus, enables NRP-1 to act as a co-receptor for VEGF165, which enhances its binding to VEGFR-2 and its bioactivity. However, the exact functional roles and related signaling mechanisms of NRP-1 in angiogenesis are not well understood. In this study we constructed a chimeric receptor, EGNP-1, by fusing the extracellular domain of epidermal growth factor receptor to the transmembrane and intracellular domains of NRP-1 and transduced it into HUVECs with a retroviral expression vector. We observed that NRP-1/EGNP-1 mediates ligand-stimulated migration of human umbilical vein endothelial cells (HUVECs) but not proliferation. Our results show that NRP-1 alone can mediate HUVEC migration through its intracellular domain, and its C-terminal three amino acids (SEA-COOH) are essential for the process. We demonstrate that phosphatidylinositol 3-kinase inhibitor Ly294002 and the p85 dominant negative mutant can block NRP-1-mediated HUVEC migration. NRP-1-mediated migration can be significantly reduced by overexpression of the dominant negative mutant of RhoA (RhoA-19N). In addition, Gq family proteins and Gβγ subunits are also required for NRP-1-mediated HUVEC migration. These results show for the first time that NRP-1 can independently promote cell signaling in endothelial cells and also demonstrate the importance of last three amino acids of NRP-1 for its function. Angiogenesis, the formation of vascular networks by endothelial cells (ECs) 1The abbreviations used are: ECendothelial cellVPFvascular permeability factorVEGFvascular endothelial growth factorEGFepidermal growth factorEGFREGF receptor HUVEC, human umbilical vein endothelial cellNRP-1neuropilin-1EGNP-1the fusion protein with the extracellular domain of the EGF receptor and the transmembrane/intracellular domains of NRP-1EGDRthe fusion protein with the extracellular domain of the EGF receptor and the transmembrane/intracellular domain of VEGFR-2EGLTthe fusion protein with the extracellular domain of the EGF receptor and the transmembrane/intracellular domains of VEGFR-1EGNP-1ΔSEAthe mutant of EGNP-1 by deleting the C-terminal three amino acids of NRP-1 (SEA-COOH)PI3Kphosphatidylinositol 3-kinaseFACSfluorescence-activated cell sortingNIPNRP-1-interacting proteinEBMEC basic mediumPBSphosphate-buffered salineGSTglutathione S-transferaseTRBDGST-Rhotekin Rho binding domainDNdominant negative.1The abbreviations used are: ECendothelial cellVPFvascular permeability factorVEGFvascular endothelial growth factorEGFepidermal growth factorEGFREGF receptor HUVEC, human umbilical vein endothelial cellNRP-1neuropilin-1EGNP-1the fusion protein with the extracellular domain of the EGF receptor and the transmembrane/intracellular domains of NRP-1EGDRthe fusion protein with the extracellular domain of the EGF receptor and the transmembrane/intracellular domain of VEGFR-2EGLTthe fusion protein with the extracellular domain of the EGF receptor and the transmembrane/intracellular domains of VEGFR-1EGNP-1ΔSEAthe mutant of EGNP-1 by deleting the C-terminal three amino acids of NRP-1 (SEA-COOH)PI3Kphosphatidylinositol 3-kinaseFACSfluorescence-activated cell sortingNIPNRP-1-interacting proteinEBMEC basic mediumPBSphosphate-buffered salineGSTglutathione S-transferaseTRBDGST-Rhotekin Rho binding domainDNdominant negative. sprouting from the vascular bed, occurs in many physiological or pathological processes (1.Folkman J. Nat. Med. 1995; 1: 27-31Crossref PubMed Scopus (7189) Google Scholar). Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) plays a major role in the regulation of angiogenesis; it is regarded as a key contributor to the growth of cancer and vascular disease (2.Neufeld G. Cohen T. Gengrinovitc S. Poltorak Z. FASEB J. 1999; 13: 9-22Crossref PubMed Scopus (3135) Google Scholar). VPF/VEGF activities are mediated by high affinity receptor tyrosine kinases that are associated primarily with ECs (2.Neufeld G. Cohen T. Gengrinovitc S. Poltorak Z. FASEB J. 1999; 13: 9-22Crossref PubMed Scopus (3135) Google Scholar). Two important VPF/VEGF binding receptor tyrosine kinases, VEGFR-1 (Flt-1) and VEGFR-2 (KDR/Flk-1), have been identified, both of which are functionally active during angiogenesis. Recent studies have found a third VPF/VEGF receptor, neuropilin-1 (NRP-1), that is expressed by ECs and tumor cells (3.Soker S. Fidder H. Neufeld G. Klagsbrun M. J. Boil. Chem. 1996; 271: 5761-5767Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar, 4.Soker S. Takashima S. Miao H.Q. Neufeld G. Klagsbrun M. Cell. 1998; 92: 735-745Abstract Full Text Full Text PDF PubMed Scopus (2067) Google Scholar). endothelial cell vascular permeability factor vascular endothelial growth factor epidermal growth factor EGF receptor HUVEC, human umbilical vein endothelial cell neuropilin-1 the fusion protein with the extracellular domain of the EGF receptor and the transmembrane/intracellular domains of NRP-1 the fusion protein with the extracellular domain of the EGF receptor and the transmembrane/intracellular domain of VEGFR-2 the fusion protein with the extracellular domain of the EGF receptor and the transmembrane/intracellular domains of VEGFR-1 the mutant of EGNP-1 by deleting the C-terminal three amino acids of NRP-1 (SEA-COOH) phosphatidylinositol 3-kinase fluorescence-activated cell sorting NRP-1-interacting protein EC basic medium phosphate-buffered saline glutathione S-transferase GST-Rhotekin Rho binding domain dominant negative. endothelial cell vascular permeability factor vascular endothelial growth factor epidermal growth factor EGF receptor HUVEC, human umbilical vein endothelial cell neuropilin-1 the fusion protein with the extracellular domain of the EGF receptor and the transmembrane/intracellular domains of NRP-1 the fusion protein with the extracellular domain of the EGF receptor and the transmembrane/intracellular domain of VEGFR-2 the fusion protein with the extracellular domain of the EGF receptor and the transmembrane/intracellular domains of VEGFR-1 the mutant of EGNP-1 by deleting the C-terminal three amino acids of NRP-1 (SEA-COOH) phosphatidylinositol 3-kinase fluorescence-activated cell sorting NRP-1-interacting protein EC basic medium phosphate-buffered saline glutathione S-transferase GST-Rhotekin Rho binding domain dominant negative. NRP-1 is a 130–135-kDa cell surface glycoprotein. It was originally characterized as a semaphorin III receptor that is important for guiding neural development (5.He Z. Tessier-Lavigne M. Cell. 1997; 90: 739-751Abstract Full Text Full Text PDF PubMed Scopus (966) Google Scholar, 6.Kolodkin A.L. Levengood D.V. Rowe E.G. Tai Y.T. Giger R.J. Ginty D.D. Cell. 1997; 90: 753-762Abstract Full Text Full Text PDF PubMed Scopus (997) Google Scholar). There is also evidence that NRP-1 mediates angiogenesis. NRP-1-null mice were found to be embryonic lethal and exhibit cardiovascular defects (7.Kawasaki T. Kitsukawa T. Bekku Y. Matsuda Y. Sanbo M. Yagi T. Fujisawa H.A. Development. 1999; 126: 4895-4902Crossref PubMed Google Scholar). Furthermore, overexpression of NRP-1 in mice resulted in excessive capillary and blood vessel formation and hemorrhaging in embryos (8.Kitsukawa T. Shimono A. Kawakami A. Kondoh H. Fujisawa H. Development. 1995; 121: 4309-4318Crossref PubMed Google Scholar). NRP-1 also contributes to tumor angiogenesis. Induction of NRP-1 expression in tumor cells in vivo resulted in larger and more vascular tumors (9.Miao H.Q. Lee P. Lin H. Soker S. Klagsbrun M. FASEB J. PubMed Scopus Google Scholar). evidence for the role of NRP-1 in angiogenesis is its expression on ECs in the G. PubMed Scopus Google and on in J. Full Text Full Text PDF PubMed Scopus Google Scholar). it has been that expression of NRP-1 on ECs the VEGF165 binding to VEGFR-2 and of VEGF165 S. Takashima S. Miao H.Q. Neufeld G. Klagsbrun M. Cell. 1998; 92: 735-745Abstract Full Text Full Text PDF PubMed Scopus (2067) Google Scholar). that NRP-1 is a co-receptor for which has to that NRP-1 is in angiogenesis in studies on NRP-1 have also evidence for its role in EC as a VEGFR-2 co-receptor that signaling through this tyrosine receptor J. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the study in cells which NRP-1 but not VEGFR-2 found that the expression of NRP-1 resulted in tumors associated with tumor angiogenesis (9.Miao H.Q. Lee P. Lin H. Soker S. Klagsbrun M. FASEB J. PubMed Scopus Google Scholar). study that NRP-1 with high affinity to VEGFR-1 and that this the binding of VEGF165 to NRP-1 G. J. Chem. Full Text Full Text PDF PubMed Google Scholar). a study by A. J. G. Google first that NRP-1 VPF/VEGF in cells VEGFR-2 this evidence the that NRP-1 alone or in with tyrosine to the VPF/VEGF signaling in However, of the signaling has been to NRP-1 a extracellular domain of amino a transmembrane domain of amino and an intracellular domain of amino Previous studies were mainly focused on the extracellular domain that of and domains of NRP-1 the for both VPF/VEGF and the of R.J. Levengood D.V. Ginty D.D. A.L. 1999; Scholar, M. T. 1999; Scholar, Z. M. J. Klagsbrun M. J. Chem. Full Text Full Text PDF PubMed Scopus Google that are the for NRP-1 as a VEGFR-2 co-receptor for VPF/VEGF from the that the intracellular domain of NRP-1 was not required for semaphorin signaling M. T. 1999; and it not of to be in to intracellular signaling the transmembrane and intracellular domains of NRP-1 amino S. Y. M. T. M. Kawakami A. Fujisawa H. 1995; PubMed Scopus Google Scholar, A. Kitsukawa T. S. Fujisawa H. J. 1996; PubMed Scopus Google which an important role for this domain in of the NRP-1 the C-terminal three amino acids of NRP-1 which are from to were for binding to a domain J. 1999; PubMed Google Scholar, G. S. A. 1998; PubMed Scopus Google Scholar). and NRP-1 are expressed on it is to the and signaling of in ECs by to the roles of in an the chimeric of of the by the extracellular domain of with the extracellular domain of epidermal growth factor receptor was in H. J. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In this a chimeric receptor EGNP-1 the extracellular domain of the and the transmembrane/intracellular domains of we the of the intracellular domain of NRP-1 and its signaling in vascular We found that NRP-1 alone can mediate HUVEC migration but not and the C-terminal three amino acids of NRP-1 (SEA-COOH) are required for NRP-1-mediated HUVEC migration. of RhoA can be mediated by NRP-1 through Gq family Gβγ and that are required for HUVEC migration. human and EC basic medium were from the domain, the domain, and RhoA were from was from were from was from and Ly294002 were from were as H. J. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). 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Full Text Full Text PDF PubMed Scopus Google and mutant the VEGFR-1 were transduced into HUVECs with and We found that the migration of HUVECs with EGNP-1 and or EGNP-1 and from that of cells transduced with EGNP-1 alone In the expression of EGNP-1 was we the VEGFR-2 by on or results that VEGFR-2 HUVEC migration by VPF/VEGF but not HUVEC migration by EGF demonstrate that the of HUVEC migration can be or of NRP-1-mediated the role of in NRP-1-mediated HUVEC a was of HUVECs with a of HUVEC migration in to EGF was observed a and an and in tyrosine receptor signaling 1999; PubMed Scopus Google Scholar). with the p85 the in an active We used the dominant negative of the p85 and the mutant of to is required for migration. results that the HUVECs expressed of EGNP-1 but the of HUVECs with EGNP-1 and reduced migration as with that of HUVECs with EGNP-1 and However, the migration by These results that migration of HUVECs is through the signaling Previous on the has that NRP-1 be in the regulation of by with the signaling J. 1999; PubMed Google Scholar). we proteins are in the signaling of HUVEC migration. It has been that migration and HUVEC by VPF/VEGF S. H.A. A. 1996; PubMed Google Scholar, H. J. Chem. Full Text Full Text PDF PubMed Scopus Google which that proteins in Recent studies from have that Gq family proteins are important for HUVEC and migration. and HUVECs were with for or fusion that of a an and can of Gq family proteins in Google Scholar, A. A. 1999; PubMed Scopus Google and for and with show that with not have on HUVEC but significantly the migration These results that Gq family proteins are in HUVEC migration. It is that proteins into and subunits that can signaling PubMed Scopus Google Scholar, J. Chem. 1998; Full Text Full Text PDF Scopus Google Scholar). Gβγ is also to be in and signaling H. J. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, H. J. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). we the role of Gβγ subunits in NRP-1/EGNP-1 mediated HUVEC migration. to the C-terminal domain of human that with Gβγ and, as a intracellular Gβγ by H.A. G. R.J. 1998; PubMed Scopus Google Scholar, J. R.J. J. Chem. Full Text PDF PubMed Google Scholar). Our results that overexpression of reduced the migration of HUVECs mediated by EGNP-1 the of EGNP-1 were expressed in the cells that Gβγ subunits are required for HUVEC migration. RhoA family of the has been to an important role in cell growth and migration P. 1999; PubMed Scopus Google Scholar). we that and are required for HUVEC H. J. Chem. Full Text Full Text PDF PubMed Scopus Google RhoA and are important for HUVEC migration H. J. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the the of the dominant negative of and RhoA that were to a retroviral in H. J. Chem. 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HUVECs were with EGNP-1 and or the cells expressed EGNP-1 that with the or cells not HUVEC but the HUVECs of migration. results that RhoA is in migration of HUVECs mediated by NRP-1/EGNP-1 and of the dominant negative mutant of RhoA HUVEC migration mediated by EGNP-1, we EGNP-1 can mediate RhoA of RhoA was by a a fusion protein that to the of HUVECs transduced with EGNP-1 or were with EGF for as were with to the were to the results show that RhoA was not by EGF in transduced HUVECs but was by EGF in HUVECs as as and high EGF the signaling that mediate RhoA HUVECs were with a for and with EGF for were used to the of show that Ly294002 RhoA in HUVECs EGF Furthermore, the dominant negative mutant of RhoA These results that is a key of RhoA Gq family proteins and Gβγ subunits are for HUVEC we Gq family proteins have on RhoA mediated by HUVECs transduced with EGNP-1 were with for or for by with EGF for were to RhoA results that the RhoA mediated by EGNP-1, but not we Gβγ subunits are required for RhoA HUVECs with EGNP-1 and or were with were to the RhoA that overexpression of the of RhoA that Gβγ subunits are required for RhoA C-terminal of NRP-1 (SEA-COOH) for HUVEC the signaling by NRP-1 in a chimeric mutant receptor was by deleting the C-terminal three amino acids of NRP-1 (SEA-COOH) and HUVECs transduced with EGNP-1, and expressed of NRP-1 and, expressed of EGNP-1 and that the mutant protein is expressed on the cell surface as a receptor, the transduced cells were to with and as a results that of HUVECs with and of HUVECs with expressed the on the cell was on HUVECs transduced with we the of the on HUVEC migration. in in HUVECs transduced with EGNP-1, EGF migration by more HUVECs transduced with or not to that the transduced cells expressed of EGNP-1 or However, HUVECs transduced with a to VPF/VEGF that the of migration of is not to the of the These results that the C-terminal three amino acids of NRP-1 (SEA-COOH) are essential for HUVEC migration. We has on NRP-1-mediated RhoA in RhoA was in HUVECs transduced with in not mediate RhoA in to EGF that the C-terminal three amino acids of NRP-1 (SEA-COOH) are required for the signaling of HUVEC migration. on and is the it is that NRP-1 as an important of VPF/VEGF during and its exact functional roles and related signaling mechanisms in processes are S. Takashima S. Miao H.Q. Neufeld G. Klagsbrun M. Cell. 1998; 92: 735-745Abstract Full Text Full Text PDF PubMed Scopus (2067) Google Scholar, H.Q. Lee P. Lin H. Soker S. Klagsbrun M. FASEB J. PubMed Scopus Google Scholar, J. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, A. J. G. Google Scholar). study has of the functional roles of NRP-1 in EC migration and related signaling the of which are study evidence for the of NRP-1 in migration in the functional role of NRP-1 in we used an in H. J. Chem. Full Text Full Text PDF PubMed Scopus Google to a chimeric receptor EGNP-1 by fusing the extracellular domain of the EGF receptor with the transmembrane and intracellular domains of In this we for the first time that EGNP-1 mediates HUVEC migration but not proliferation. Furthermore, we that HUVEC migration is observed by and of with and a study by J. Full Text Full Text PDF PubMed Scopus Google it was that the migration of human endothelial cells to VEGF165 was in in the of and this was These that NRP-1 mediates EC migration. study NRP-1 alone can mediate EC migration. There are three of evidence in results that this functional role of we found that EGF a of of migration in HUVECs transduced with EGNP-1 or chimeric receptor to the of VEGFR-2 EGF H. J. Chem. Full Text Full Text PDF PubMed Scopus Google was on HUVEC migration HUVECs were with EGNP-1 and or is a mutant of in tyrosine of VEGFR-2 that the EGF to in HUVECs transduced with H. S. J. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). VEGFR-2 with an on HUVECs on HUVEC migration. results that NRP-1/EGNP-1 can mediate HUVEC migration as an is by a of binding with VPF/VEGF cell the expression of by the A. J. G. Google Scholar). However, the study to S. Takashima S. Miao H.Q. Neufeld G. Klagsbrun M. Cell. 1998; 92: 735-745Abstract Full Text Full Text PDF PubMed Scopus (2067) Google that migration to VEGF165 NRP-1 alone was expressed on is that cells not of or NRP-1 and not to VPF/VEGF S. Takashima S. Miao H.Q. Neufeld G. Klagsbrun M. Cell. 1998; 92: 735-745Abstract Full Text Full Text PDF PubMed Scopus (2067) Google Scholar, J. A. M. J. Chem. Full Text PDF PubMed Google Scholar). cells related signaling with the of a receptor and be of vascular the HUVECs used in this the VEGFR-2 to VEGF165 more in cells and this was into a to VEGF165 as with the of cells VEGFR-2 but not NRP-1 to as an of VEGFR-2 in the of is of a formation VEGFR-2 and NRP-1 J. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). study is the first that the C-terminal three amino acids of NRP-1 (SEA-COOH) are essential for NRP-1-mediated HUVEC migration. It is that the C-terminal three amino acids of NRP-1 (SEA-COOH) are for with the C-terminal of NRP-1-interacting protein J. 1999; PubMed Google Scholar, G. S. A. 1998; PubMed Scopus Google Scholar). of the important roles of proteins is to act as that proteins to or into associated protein J. 1996; Full Text PDF PubMed Scopus Google Scholar, M. A. M. 1995; PubMed Scopus Google Scholar, Lee A. J. M. Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). and of NRP-1 and in the that NRP-1 functionally with J. 1999; PubMed Google Scholar). has been independently as it was by of its with the of protein to the of and was to in the regulation of by with the signaling G. S. A. 1998; PubMed Scopus Google Scholar). In of as well as we that be a in HUVECs that in the regulation of NRP-1-mediated migration signaling by with the C-terminal three amino acids of NRP-1 signaling for HUVEC migration were in this We found that inhibitor Ly294002 and the dominant negative mutant of p85 HUVEC migration by but the mutant of expression results in more migration. These that is in the NRP-1-mediated migration signaling which is with the by A. J. G. Google that the by VPF/VEGF in cells which is important for the of cells A. J. G. Google Scholar). in proteins that a of Gq of HUVEC migration by and that significantly HUVEC migration. These demonstrate that Gq family proteins are in NRP-1-mediated HUVEC and Gβγ subunits are required for this signaling In with the study in that protein and Gβγ subunits are required for EC migration H. J. Chem. Full Text Full Text PDF PubMed Scopus Google this study that Gq family proteins and Gβγ subunits are required for EC migration. study found that the RhoA dominant negative significantly HUVEC migration. that the RhoA is in the NRP-1-mediated EC migration signaling and that the of VPF/VEGF on EC is mediated through the RhoA in HUVECs H. J. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). is not in with P. J. PubMed Scopus Google that overexpression of a dominant negative Rho not endothelial cell migration P. J. PubMed Scopus Google Scholar). for the are that ECs have physiological 1999; PubMed Scopus Google and that the HUVECs in have a of expression of VPF/VEGF from endothelial cells of used in Ly294002 the VEGFR-1 signaling and, migration of endothelial cells J. Chem. Full Text Full Text PDF PubMed Scopus Google it is that VEGFR-1 mediate migration of endothelial NRP-1 to HUVEC migration was by a RhoA in this the results of we found that EGNP-1 mediated RhoA in but the mutant of EGNP-1, not show the of Furthermore, we the inhibitor and the dominant negative mutant this with an of Gq family RhoA which the of Gq family proteins in NRP-1 that Gβγ is the of NRP-1 signaling to RhoA results demonstrate that NRP-1-mediated RhoA Gq family Gβγ and for to EC migration is VPF/VEGF NRP-1 protein Gq family proteins Gβγ subunits RhoA EC migration. Our studies be more mechanisms of NRP-1 functional and the functional activities in major of this study is the that NRP-1 alone can mediate EC that the intracellular domain of NRP-1 is in this and that the C-terminal three amino acids are essential for this function. and is as J. PubMed Scopus Google EC migration and tumor cell migration as well as have from in cancer that NRP-1 alone can mediate EC migration with the that NRP-1 is not expressed in EC but also in tumor cells S. Takashima S. Miao H.Q. Neufeld G. Klagsbrun M. Cell. 1998; 92: 735-745Abstract Full Text Full Text PDF PubMed Scopus (2067) Google that NRP-1 be an important of this study both in the mechanisms of tumor cell and in NRP-1 for