Glycosaminoglycan modification of neuropilin-1 modulates VEGFR2 signaling
2006; Springer Nature; Volume: 25; Issue: 13 Linguagem: Inglês
10.1038/sj.emboj.7601188
ISSN1460-2075
AutoresYasunori Shintani, Seiji Takashima, Yoshihiro Asano, Hisakazu Kato, Yulin Liao, Satoru Yamazaki, Osamu Tsukamoto, Osamu Seguchi, Hiroyuki Yamamoto, Tomi Fukushima, Kazuyuki Sugahara, Masafumi Kitakaze, Masatsugu Hori,
Tópico(s)Protein Tyrosine Phosphatases
ResumoArticle8 June 2006free access Glycosaminoglycan modification of neuropilin-1 modulates VEGFR2 signaling Yasunori Shintani Yasunori Shintani Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Suita, Osaka, Japan Search for more papers by this author Seiji Takashima Corresponding Author Seiji Takashima Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Suita, Osaka, Japan Search for more papers by this author Yoshihiro Asano Yoshihiro Asano Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Suita, Osaka, Japan Search for more papers by this author Hisakazu Kato Hisakazu Kato Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Suita, Osaka, Japan Search for more papers by this author Yulin Liao Yulin Liao Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Suita, Osaka, Japan Search for more papers by this author Satoru Yamazaki Satoru Yamazaki Cardiovascular Division of Medicine, National Cardiovascular Center, Suita, Japan Search for more papers by this author Osamu Tsukamoto Osamu Tsukamoto Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Suita, Osaka, Japan Search for more papers by this author Osamu Seguchi Osamu Seguchi Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Suita, Osaka, Japan Cardiovascular Division of Medicine, National Cardiovascular Center, Suita, Japan Search for more papers by this author Hiroyuki Yamamoto Hiroyuki Yamamoto Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Suita, Osaka, Japan Cardiovascular Division of Medicine, National Cardiovascular Center, Suita, Japan Search for more papers by this author Tomi Fukushima Tomi Fukushima Cardiovascular Division of Medicine, National Cardiovascular Center, Suita, Japan Search for more papers by this author Kazuyuki Sugahara Kazuyuki Sugahara Department of Biochemistry, Kobe Pharmaceutical University, Higashinada-ku, Kobe, Japan Present address: Laboratory of Proteoglycan Signaling and Therapeutics, Graduate School of Life Science, Hokkaido University, Frontier Research Center for Post-genomic Science and Technology, Sapporo, Japan Search for more papers by this author Masafumi Kitakaze Corresponding Author Masafumi Kitakaze Cardiovascular Division of Medicine, National Cardiovascular Center, Suita, Japan Search for more papers by this author Masatsugu Hori Masatsugu Hori Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Suita, Osaka, Japan Search for more papers by this author Yasunori Shintani Yasunori Shintani Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Suita, Osaka, Japan Search for more papers by this author Seiji Takashima Corresponding Author Seiji Takashima Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Suita, Osaka, Japan Search for more papers by this author Yoshihiro Asano Yoshihiro Asano Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Suita, Osaka, Japan Search for more papers by this author Hisakazu Kato Hisakazu Kato Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Suita, Osaka, Japan Search for more papers by this author Yulin Liao Yulin Liao Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Suita, Osaka, Japan Search for more papers by this author Satoru Yamazaki Satoru Yamazaki Cardiovascular Division of Medicine, National Cardiovascular Center, Suita, Japan Search for more papers by this author Osamu Tsukamoto Osamu Tsukamoto Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Suita, Osaka, Japan Search for more papers by this author Osamu Seguchi Osamu Seguchi Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Suita, Osaka, Japan Cardiovascular Division of Medicine, National Cardiovascular Center, Suita, Japan Search for more papers by this author Hiroyuki Yamamoto Hiroyuki Yamamoto Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Suita, Osaka, Japan Cardiovascular Division of Medicine, National Cardiovascular Center, Suita, Japan Search for more papers by this author Tomi Fukushima Tomi Fukushima Cardiovascular Division of Medicine, National Cardiovascular Center, Suita, Japan Search for more papers by this author Kazuyuki Sugahara Kazuyuki Sugahara Department of Biochemistry, Kobe Pharmaceutical University, Higashinada-ku, Kobe, Japan Present address: Laboratory of Proteoglycan Signaling and Therapeutics, Graduate School of Life Science, Hokkaido University, Frontier Research Center for Post-genomic Science and Technology, Sapporo, Japan Search for more papers by this author Masafumi Kitakaze Corresponding Author Masafumi Kitakaze Cardiovascular Division of Medicine, National Cardiovascular Center, Suita, Japan Search for more papers by this author Masatsugu Hori Masatsugu Hori Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Suita, Osaka, Japan Search for more papers by this author Author Information Yasunori Shintani1, Seiji Takashima 1, Yoshihiro Asano1, Hisakazu Kato1, Yulin Liao1, Satoru Yamazaki2, Osamu Tsukamoto1, Osamu Seguchi1,2, Hiroyuki Yamamoto1,2, Tomi Fukushima2, Kazuyuki Sugahara3,4, Masafumi Kitakaze 2 and Masatsugu Hori1 1Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Suita, Osaka, Japan 2Cardiovascular Division of Medicine, National Cardiovascular Center, Suita, Japan 3Department of Biochemistry, Kobe Pharmaceutical University, Higashinada-ku, Kobe, Japan 4Present address: Laboratory of Proteoglycan Signaling and Therapeutics, Graduate School of Life Science, Hokkaido University, Frontier Research Center for Post-genomic Science and Technology, Sapporo, Japan ‡These authors contributed equally to this work *Corresponding authors: Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan. Tel.: +816 6879 3472; Fax: +816 6879 3473; E-mail: [email protected] Division of Medicine, National Cardiovascular Center, Suita, Japan. E-mail: [email protected] The EMBO Journal (2006)25:3045-3055https://doi.org/10.1038/sj.emboj.7601188 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Neuropilin-1 (NRP1) is a co-receptor for vascular endothelial growth factor (VEGF) that enhances the angiogenic signals cooperatively with VEGFR2. VEGF signaling is essential for physiological and pathological angiogenesis through its effects on vascular endothelial cells (ECs) and smooth muscle cells (SMCs), but the mechanisms coordinating this response are not well understood. Here we show that a substantial fraction of NRP1 is proteoglycan modified with either heparan sulfate or chondroitin sulfate on a single conserved Ser. The composition of the NRP1 glycosaminoglycan (GAG) chains differs between ECs and SMCs. Glycosylation increased VEGF binding in both cell types, but the differential GAG composition of NRP1 mediates opposite responsiveness to VEGF in ECs and SMCs. Finally, NRP1 expression and its GAG modification post-transcriptionally regulate VEGFR2 protein expression. These findings indicate that GAG modification of NRP1 plays a critical role in modulating VEGF signaling, and may provide new insights into physiological and pathological angiogenesis. Introduction Neuropilin-1 (NRP1) was originally discovered as a co-receptor for semaphorin-3A (Sema3A), an axon repellent factor (Kolodkin et al, 1997). However, NRP1 also acts as a co-receptor for vascular endothelial growth factor (VEGF), a molecule with no sequence or structural homology to Sema3A (Soker et al, 1998). VEGF (also referred as VEGF-A) is an essential factor promoting both embryonic angiogenesis and postnatal neovascularization. Additionally, VEGF plays a significant role in causing pathological angiogenesis associated with tumor growth, age-related macular degeneration, diabetic retinopathy, and other conditions (Ferrara et al, 2003). Indeed, a blocking anti-VEGF antibody that disrupts VEGF signaling is a promising anticancer therapy currently in development (Hurwitz et al, 2004). VEGF has three receptors, VEGF receptor 1 and 2 (VEGFR1, VEGFR2), and neuropilin (Veikkola and Alitalo, 1999; Ferrara et al, 2003). VEGFR2 is the primary receptor mediating the angiogenic activity of VEGF (Shalaby et al, 1995; Ferrara et al, 2003), and NRP1 functions as a co-receptor to enhance VEGFR2 signaling (Soker et al, 1998). Indeed, genetic ablation of NRP1 leads to severely impaired vascular development (Kawasaki et al, 1999; Takashima et al, 2002; Gu et al, 2003), indicating that NRP1 is essential for VEGF-mediated angiogenesis. In addition to promoting angiogenesis, VEGF is now thought to be required for the maintenance and stabilization of mature blood vessels (Zachary, 2001; Saint-Geniez and D'Amore, 2004). Signaling through VEGFR2, VEGF induces not only endothelial cell (EC) proliferation but also cell survival (Gerber et al, 1998), and the loss of VEGF signals in the choroidal endothelium is one factor promoting age-related macular degeneration (Blaauwgeers et al, 1999). Smooth muscle cells (SMCs), another important component of the vessel wall, also express both NRP1 (Kitsukawa et al, 1995; Kawasaki et al, 1999) and VEGFR2 (Grosskreutz et al, 1999; Ishida et al, 2001). However, SMCs in mature vessels typically do not respond to VEGF signals except in certain conditions such as atherosclerosis (Carmeliet, 2003; Jain, 2003; Khurana et al, 2004). Therefore, we wished to identify the mechanism(s) responsible for different cellular responses to VEGF in ECs and SMCs. In this study, we demonstrate that a substantial fraction of NRP1 is proteoglycan modified with either heparan sulfate (HS) or chondroitin sulfate (CS) attached to a single conserved Ser. The type of NRP1 glycosaminoglycan (GAG) chain modification differs between ECs and SMCs. Finally, we show that the type of NRP1 GAG modification critically and differentially modulates VEGFR2 signals in SMCs and ECs. Results A substantial fraction of NRP1 is proteoglycan modified with HS or CS The differential responsiveness of ECs and SMCs to VEGF could be explained by a number of factors, and we initially investigated the ability of VEGF to bind to these cells. When human coronary artery smooth muscle cells (CASMCs) were incubated with 125I-labeled VEGF, we detected two distinct binding proteins after cell-surface crosslinking (Figure 1A). The lower band was also detected in human umbilical vein endothelial cells (HUVECs) and was identified as NRP1. However, the upper band was not found in HUVECs, and this band did not correspond to VEGFR2. In contrast, the upper band was also seen in bronchial smooth muscle cells (BSMCs) (Figure 1A), a non-vascular SMC, and, because NRP1 alone cannot transduce VEGF signals, we initially thought that this binding protein represented a new VEGF receptor. However, after transfection of CASMCs, but not HUVECs, with FLAG-tagged NRP1, we observed an identical upper molecular weight band when blotted with an anti-FLAG antibody (Figure 1B). The high molecular weight band was not simply a covalently linked homodimer of NRP1 (Figure 1C), and we reasoned NRP1 could undergo post-translational modification. Although NRP1 itself undergoes N-glycosylation, we found that the high molecular weight NRP1 was not a form of N-glycosylation or O-glycosylation by enzyme treatment and lectin blot (data not shown), but it did contain GAG chains. Indeed, treatment of NRP1 immunoprecipitates with both heparitinase and chondroitinase, which digest HS and CS, respectively, led to the disappearance of the upper band, whereas the lower band was not affected. Next, we investigated the composition of GAG-modified endogenous NRP1 in both CASMCs and HUVECs. Heparitinase slightly decreased the modified NRP1 band in CASMCs, whereas chondroitinase digested the majority of the GAG present on NRP1, indicating that CS was the dominant GAG modification of NRP1 (Figure 1D). In contrast, NRP1 in HUVECs was also modified, but to a much lesser extent than that seen in CASMCs, and HUVEC NRP1 contained almost equivalent amounts of CS and HS (Figure 1D). By analyzing the band intensity, we determined the degree of each modification relative to untreated samples (non-modified/HS-modified/CS-modified NRP1—CASMCs: 100%/79%/174%, HUVECs: 100%/27.3%/24.6%, respectively) (Figure 1D, right panel). Figure 1.A substantial fraction of cellular NRP1 is proteoglycan, composed of either HS or CS. (A) 125I-labeled VEGF is crosslinked to different proteins in ECs and SMCs. Arrow indicates VEGF-binding protein specifically seen in SMCs. CASMC: coronary artery smooth muscle cell; BSMC: bronchial smooth muscle cell; HUVEC: human umbilical vein endothelial cell. (B) Western blots of exogenously expressed NRP1 in either CASMCs or HUVECs. Adenovirus encoding FLAG-tagged NRP1 was transfected 2 days before analysis at the indicated MOI. LacZ-encoding adenovirus was used as a control. (C) The high molecular weight band was not simply a covalently linked homodimer of NRP1. Only FLAG-tagged NRP1 or both FLAG-tagged and V5-tagged NRP1 were transfected in CASMCs, and the cell lysates were immunoprecipitated and detected by the indicated antibody. (D) Endogenous NRP1 was modified by GAG chain addition in both SMCs and ECs. The upper band in CASMC immunoprecipitates disappeared following treatment with both HSase and CSase. HUVEC-expressed NRP1 is also GAG modified. The band intensity was analyzed and the proportion of each glycanated form of NRP1 was determined. Data are from three separate experiments. HSase: heparitinase; CSase: chondroitinase. Download figure Download PowerPoint GAG chains are covalently added to Ser residues of the core protein contained within a Ser-Gly consensus sequence (Esko and Zhang, 1996). We mutated the nine consensus sequences present in NRP1, and the GAG chains are attached only to Ser612 (Figure 2A). As a single GAG chain cannot contain both HS and CS simultaneously, endogenous NRP1 exists as either an HS proteoglycan or CS proteoglycan but not as a hybrid proteoglycan. Moreover, as shown in Figure 1D, non-modified NRP1 (130 kDa, the core protein) was always detected in both CASMCs and HUVECs. Ser612 is located in the bridge region between the b1b2 and MAM domains of NRP1 (Figure 2D), and multiple sequence alignments suggested that Ser612 was remarkably conserved among vertebrates (Figure 2B), and the peptide sequence around Ser612 was also well conserved, especially the acidic amino acids that are important for HS attachment (Esko and Zhang, 1996). NRP2, a mammalian homolog of NRP1, does not have this conserved Ser residue, and adenovirus-mediated expression of NRP2 in CASMCs demonstrated that NRP2 is not GAG modified (Figure 2C). Figure 2.(A) NRP1 is GAG modified on a single Ser612 residue. CASMCs were transfected with adenoviral vectors encoding WT or S612A mutant NRP1. NRP1 S612A is not GAG modified. (B) Multiple alignments of NRP1 from different species. Ser612 is highly conserved among vertebrates. (C) NRP2, an NRP family member, is not GAG modified. (D) Design of siRNA and adenovirus constructs. Ser612 exists in the bridge region between the b1b2 and MAM domains. (E) Replacement of Ser612 by Ala612 of NRP1 did not change binding to VEGF. Cos7 cells were transfected with either NRP1 WT′ or S612A expression vector and preincubated with heparitinase (1.5 mU/ml), heparinase (1.5 mU/ml), and chondroitinase (20 mU/ml) in the culture medium at 37°C for 2 h to make NRP1 non-GAG form. After incubation with 125I-labeled VEGF for 30 min at room temperature, cell lysates were immunoprecipitated by anti-NRP1 antibody, and the bound radioactivity was quantitated using a gamma counter. Data are from three independent experiments. For panel E, error bars represent s.e. Download figure Download PowerPoint GAG modification of NRP1 enhances VEGF binding Addition of both chondroitinase and heparitinase to culture medium completely digests all GAGs attached to other core proteins on the cell surface, and this would dramatically complicate the interpretation of any experiments using this technique. Therefore, to further investigate the function of the GAG of only NRP1, we used RNAi to knock down endogenous NRP1 while expressing mutant NRP1. We designed an siRNA, named N-G, targeting the GAG attachment site of NRP1 (Figure 2D). We further generated two NRP1-encoding adenovirus constructs: NRP1 S612A, in which Ser612 was replaced by Ala612 and there was a three-base mismatch with N-G siRNA; and NRP1 WT′, which contains the glycan accepting residue but had a four-base mismatch with N-G siRNA (Figure 2D). In cells transfected with N-G siRNA, transfection of both adenovirus constructs led to the expression of the appropriate NRP1 molecules. We confirmed that addition of FLAG tag to NRP1 does not affect VEGF binding (data not shown) and that the mutation itself (NRP1 S612A) did not change VEGF binding to the core protein of NRP1 (Figure 2E). We next examined the ability of VEGF to bind to experimentally replaced NRP1 in both SMCs and ECs. Transfection of both N-G siRNA and equal multiplicity of infection (MOI) adenoviral constructs successfully replaced endogenous NRP1 with either the GAG-acceptor (NRP1 WT′) or mutated (NRP1 S612A) NRP1 (Figure 3A). Throughout these experiments, MOIs were used to generate NRP1 WT′ or S612A expression levels comparable to endogenous NRP1. To determine whether GAG modifications affect the ability of NRP1 to bind VEGF, we measured the binding of 125I-labeled VEGF to FLAG-tagged NRP1 in these cells. After incubation with 125I-labeled VEGF, cell lysates were immunoprecipitated with an anti-FLAG antibody, and bound radioactivity was counted. NRP1 WT′ bound VEGF with 3.87- and 2.27-fold higher than NRP1 S612A in SMCs and ECs, respectively (Figure 3B). We found that heparitinase and chondroitinase treatment with these immunoprecipitates could not entirely eliminate the enhancement of VEGF binding (Figure 3B), different from the results of Figure 2E in which we showed that VEGF equally binds NRP1 WT′ and S612A pretreated with heparitinase and chondroitinase before the exposure to VEGF. These results suggest that GAG modifications of NRP1 in SMCs and ECs enhance VEGF binding mainly to NRP1 core protein and not to only GAG chain of NRP1. Figure 3.GAG modifications differentially affect NRP1 function in SMCs and ECs. (A) Experimental replacement of NRP1 in SMCs and ECs. After transfection with both N-G siRNA and adenoviral constructs, endogenous NRP1 was successfully replaced with either the glycanated form (NRP1 WT′) or non-glycanated form (NRP1 S612A) of NRP1. Tubulin was used as a loading control. (B) Addition of GAG to NRP1 enhances binding to VEGF in both types of cells. Two days after NRP1 replacement, cell lysates were immunoprecipitated with anti-FLAG antibody after incubation with 125I-labeled VEGF (25 ng/ml) for 40 min at room temperature, and bound radioactivity was quantitated using a gamma counter. Heparitinase and chondroitinase treatment with these immunoprecipitates could not entirely eliminate the enhancement of VEGF binding. Data are from three independent experiments. (C) VEGF (50 ng/ml) induced greater cell migration in SMCs expressing non-modified NRP1 S612A than those expressing NRP1 WT′. Migrated cells were quantified by counting cells in three random high-power fields (HPF, × 200). Similar results were obtained from additional two independent experiments. (D) VEGF (50 ng/ml) increased cell viability in ECs expressing NRP1 WT′ to a greater extent than in ECs expressing NRP1 S612A. Data are from three independent experiments. For panels B–D, error bars represent s.e. *P<0.05, versus adeno-NRP1 WT′ in panel B. Download figure Download PowerPoint NRP1 GAG modifications lead to differential VEGF responsiveness in SMCs and ECs We next investigated whether GAG modifications of NRP1 in both SMCs and ECs affected cellular responsiveness to VEGF. VEGF increases the motility of vascular SMCs (Grosskreutz et al, 1999; Ishida et al, 2001), and induces proliferation, migration, and cell survival in ECs (Ferrara et al, 2003). These actions are primarily mediated through the VEGFR2 signaling pathway likely in conjunction with NRP1. Notably, VEGF induced the migration of SMCs expressing NRP1 S612A (non-modified) stronger than those expressing NRP1 WT′ (GAG modified) (Figure 3C). In contrast, VEGF increased the viability of ECs expressing NRP1 WT′ to a greater extent than those expressing NRP1 S612A (Figure 3D). The observed increased viability seen in ECs expressing NRP1 WT′ in response to VEGF is consistent with the increased VEGF binding shown in Figure 3B. However, the decreased motility seen in SMCs expressing NRP1 WT′ was unexpected. To further explore this discrepancy, we examined the influence of different GAG chains on the expression of VEGR2 and the formation of the VEGF–VEGFR2–NRP1 ternary complex, both important determinants of VEGF signaling (Soker et al, 2002). We first analyzed VEGFR2 protein expression in cells expressing either NRP1 WT′ or S612A. In SMCs expressing S612A mutant, VEGFR2 expression was two-fold higher than in cells expressing NRP1 WT′ (Figure 4A (left) and B), but expression of either NRP1 WT′ or S612A did not affect VEGFR2 expression in ECs (Figure 4A (right) and B). The increased VEGFR2 protein expression in SMCs was not accompanied by changes in mRNA levels (Figure 4C), indicating that post-transcriptional mechanisms regulate VEGFR2 expression. Figure 4.Different roles of the GAG of NRP1 on VEGFR2 in SMCs. (A) Experimental replacement with NRP1 S612A increased VEGFR2 expression in SMCs, but replacement did not affect VEGFR2 expression in ECs. Two days after transfection with siRNA and adeno-NRP1, cells were analyzed by Western blotting. Data are representative of at least three independent experiments. (B) Quantitative results of Western blot. (C) Experimental replacement with NRP1 S612A increased VEGFR2 protein expression without any transcriptional change in SMCs. Each sample was analyzed in duplicate and the experiments were performed in triplicate for the full set of genes. (D) CS-modified NRP1 had the same affinity for VEGF as HS-modified NRP1 and non-modified NRP1. Note that 125I-labeled VEGF bound CS-modified NRP1 with a similar ratio before crosslink (upper band in lanes 4 and 8, CS-modified NRP1:HS-modified NRP1=2:1). Increasing amounts of cold VEGF equally inhibited 125I-labeled VEGF binding to all forms of NRP1. (E) Co-immunoprecipitation of NRP1 with VEGFR2. CS-modified NRP1 (left panel, about 250 kDa in lane 4) minimally associated with VEGFR2 compared to non-modified (130 kDa, in lanes 2, 4, 6, 8) and HS-modified NRP1 (about 250 kDa in lane 6), although there was a two-fold excess of CS-modified NRP1 compared to HS-modified NRP1 at input (right panel). The membrane was stripped and re-probed with anti-V5 as a loading control. Data are representative of at least three independent experiments. (F) The rate of VEGFR2 degradation was decreased in NRP1 WT′ ECs compared to NRP1 S612A ECs. Phosphorylated VEGFR2 was also much higher in NRP1 WT′ ECs than in NRP1 S612A ECs at any time point after VEGF. Data are representative of at least three independent experiments. For panels B, C, F, error bars represent s.e. *P<0.05, versus NG/ NRP1 S612A at the same period as in panel F. HSase: heparitinase; CSase: chondroitinase. Download figure Download PowerPoint Both the extent of GAG modification and the predominant GAG chain added (i.e. HS or CS) differ between ECs and SMCs. Therefore, we examined whether the type of GAG modification affected ternary complex formation. We used enzymatic digestions and 125I-labeled VEGF binding to assess the contribution of CS- and HS-modified NRP1 to VEGF binding, and found that all forms of NRP1 bound VEGF equally well (Figure 4D). We next examined the ability of GAG-modified NRP1 to associate with VEGFR2 in the presence of VEGF by co-immunoprecipitation. After pretreatment with heparitinase and/or chondroitinase, V5-tagged VEGFR2 was precipitated from SMC lysates in the presence of VEGF. As shown in Figure 4E, CS-modified NRP1 minimally associated with VEGFR2 compared to non-modified or HS-modified NRP1. Thus, in SMCs, GAG-modified NRP1 post-transcriptionally downregulates VEGFR2 expression, and CS-modified NRP1 may act as a decoy receptor, rather than a co-receptor. It is likely that a combination of these factors explains the differences in VEGF activity seen in SMCs expressing NRP1 WT′ or S612A (Figure 3C). Based on the results of receptor complex formation in the presence of VEGF in Figure 4E, we hypothesized that NRP1 might affect VEGFR2 internalization/degradation after ligand binding, because degradation of the receptor tyrosine kinase is an important regulator of signaling intensity (Duval et al, 2003; Rubin et al, 2005). Before exposure to VEGF, VEGFR2 expression was not different between ECs expressing NRP1 WT′ and S612A (Figure 4A). However, the rate of VEGFR2 degradation was decreased in NRP1 WT′ ECs compared to NRP1 S612A ECs. Phosphorylated VEGFR2 was also much higher in ECs expressing NRP1 WT′ than those expressing NRP1 S612A at any time points after VEGF (Figure 4F). These results suggested that the GAG modification of NRP1 enhances VEGF signaling in ECs by delaying the degradation of VEGFR2 in the presence of VEGF, and not just by the enhancement of VEGF binding. NRP1 post-transcriptionally modulates VEGFR2 expression NRP1 knockout mice exhibit severely impaired vascular development and die around E13.5 (Kitsukawa et al, 1995; Kawasaki et al, 1999). VEGF has several splicing isoforms (its major forms in mice are VEGF120, 164, 188) and NRP1 does not bind VEGF120. In contrast, VEGFR2 can bind all of VEGF isoforms. Although NRP1 is a common receptor for both VEGF and Sema3A, impaired VEGF signaling is responsible for the observed vascular defects in these mice (Gu et al, 2003). However, the vascular defect in NRP1−/− mice is more severe than that seen in VEGF120/120 mice, in which only VEGF120 is expressed (Carmeliet et al, 1999; Stalmans et al, 2003). Thus, NRP1 appears to play a more prominent role in VEGF signaling than simply functioning as a co-receptor for some VEGF isoforms. Based on the results that CS-dominant GAG of NRP1 negatively affects VEGFR2 expression levels in SMCs, we hypothesized that NRP1 basically stabilizes VEGFR2 leading to increased expression. Thus, VEGFR2 expression should be lower in NRP1−/− mice, leading to a more pronounced vascular phenotype. To test this hypothesis in cells, we knocked down NRP1 in ECs using siRNA. Before the addition of VEGF, VEGFR2 expression was substantially decreased in cells transfected with NRP1 siRNAs (Figure 5A). Two siRNAs targeting NRP1 were used to exclude the possibility of an off-target effect of RNAi. VEGFR2 mRNA levels were unaffected by NRP1 knockdown, however (Figure 5B). Additionally, VEGFR1, another VEGF receptor, was not affected by either NRP1 siRNA, suggesting that NRP1 specifically regulates VEGFR2 expression (Figure 5A). As VEGFR2 protein level was not associated with transcription level, we conducted pulse–chase experiments in HUVECs treated with NRP1 siRNA to determine the rate of VEGFR2 degradation in the absence of VEGF. Notably, we found that the rate of VEGFR2 degradation was not changed by NRP1 knockdown (Figure 5C), which was different from the results in the presence of VEGF (Figure 4F). Figure 5.NRP1 post-transcriptionally regulates the expression of VEGFR2. (A) Both NRP1 siRNAs (N-G, N-1) decreased VEGFR2 expression. In contrast, VEGFR1 was not influenced by NRP1 knockdown. Tubulin was used as a loading control. Data are representative of at least three independent experiments. (B) Transcription levels of both VEGFR1 and VEGFR2 were not influenced by NRP1 knockdown. Each sample was analyzed in duplicate and experiments were performed in triplicate for the full set of genes. (C) Pulse–chase experiments in HUVECs. The rate of degradation of VEGFR2 was not changed by NRP1 knockdown. Data are from four independent experiments. (D) NRP1 significantly upregulated VEGFR2 protein levels in Flp293/VEGFR2 cells. Transfected NRP1 in Flp293/VEGFR2 cells was GAG modified similar to ECs. Data are representative of two independent experiments. (E) NRP1 regulates cell-surface VEGFR2 expression. Transient expression of FLAG-tagged NRP1 WT upregulated the cell membrane-associated VEGFR2 expression compared to adjacent non-transfected cells. Flp293/VEGFR2 cells were transfected with either NRP1 WT or mock and stained without permeabilization using anti-VEGFR2 (green) and anti-FLAG-Cy3 (red). Blue: DAPI nuclear staining. For panels A and C, numeric represents the mean of band intensity of three experiments. For panel B, error bars represent s.e. Download figure Download PowerPoint We next examined whether the ability of NRP1 to promote VEGFR2 expression was specific for ECs, and we generated Flp293/VEGFR2 cells stably expressing VEGFR2. These cells express much less NRP1 than either ECs or SMCs. When these cells were transfected with NRP1, NRP1 was GAG modified similar to ECs (Figure 5D
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