Shintani, Y. et al. Glycosaminoglycan modification of neuropilin-1 modulates VEGFR2 signaling. EMBO J. 25, 3045-3055

Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.
The EMBO Journal (Impact Factor: 10.43). 08/2006; 25(13):3045-55. DOI: 10.1038/sj.emboj.7601188
Source: PubMed


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.


Available from: Yasunori Shintani
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    • "Also, HS is thought to prevent diffusion of growth factors, such as the BMPs, away from the regions where they are likely to be required [17], and the concept of HSPG as a bioactive vehicle for GF delivery has been substantiated [12,13,18,19]. Highly sulfated forms of the proteoglycan GAG chondroitin sulfate (CS) have also been implicated in GF co-activation [20-23], though these interactions are less well characterized. "
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    ABSTRACT: Background Many growth factors, such as bone morphogenetic protein (BMP)-2, have been shown to interact with polymers of sulfated disacharrides known as heparan sulfate (HS) glycosaminoglycans (GAGs), which are found on matrix and cell-surface proteoglycans throughout the body. HS GAGs, and some more highly sulfated forms of chondroitin sulfate (CS), regulate cell function by serving as co-factors, or co-receptors, in GF interactions with their receptors, and HS or CS GAGs have been shown to be necessary for inducing signaling and GF activity, even in the osteogenic lineage. Unlike recombinant proteins, however, HS and CS GAGs are quite heterogenous due, in large part, to post-translational addition, then removal, of sulfate groups to various positions along the GAG polymer. We have, therefore, investigated whether it would be feasible to deliver a DNA pro-drug to generate a soluble HS/CS proteoglycan in situ that would augment the activity of growth-factors, including BMP-2, in vivo. Results Utilizing a purified recombinant human perlecan domain 1 (rhPln.D1) expressed from HEK 293 cells with HS and CS GAGs, tight binding and dose-enhancement of rhBMP-2 activity was demonstrated in vitro. In vitro, the expressed rhPln.D1 was characterized by modification with sulfated HS and CS GAGs. Dose-enhancement of rhBMP-2 by a pln.D1 expression plasmid delivered together as a lyophilized single-phase on a particulate tricalcium phosphate scaffold for 6 or more weeks generated up to 9 fold more bone volume de novo on the maxillary ridge in a rat model than in control sites without the pln.D1 plasmid. Using a significantly lower BMP-2 dose, this combination provided more than 5 times as much maxillary ridge augmentation and greater density than rhBMP-2 delivered on a collagen sponge (InFuse™). Conclusions A recombinant HS/CS PG interacted strongly and functionally with BMP-2 in binding and cell-based assays, and, in vivo, the pln.247 expression plasmid significantly improved the dose-effectiveness of BMP-2 osteogenic activity for in vivo de novo bone generation when delivered together on a scaffold as a single-phase. The use of HS/CS PGs may be useful to augment GF therapeutics, and a plasmid-based approach has been shown here to be highly effective.
    BMC Biotechnology 09/2012; 12(1):60. DOI:10.1186/1472-6750-12-60 · 2.03 Impact Factor
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    • "These findings indicate that NRP-1 plays a pivotal role in HTLV-1 binding. Because NRP-1 is modified by HS (Shintani et al., 2006), HTLV-1 SU might interact directly with HS conjugated to NRP-1. Thus, HTLV-1 SU will bind to NRP-1 in two ways: an HSPG-mediated indirect interaction and a KKPNR sequence-mediated direct interaction. "
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    ABSTRACT: Human T cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T cell leukemia (ATL) and HTLV-1 - associated myelopathy and tropical spastic paraparesis (HAM/TSP). HTLV-1 has a preferential tropism for CD4 T cells in healthy carriers and ATL patients, while both CD4 and CD8 T cells serve as viral reservoirs in HAM/TSP patients. HTLV-1 has also been detected other cell types, including monocytes, endothelial cells, and dendritic cells. In contrast to the limited cell tropism of HTLV-1 in vivo, the HTLV receptor appears to be expressed in almost all human or animal cell lines. It remains to be examined whether this cell tropism is determined by host factors or by HTLV-1 heterogeneity. Unlike most retroviruses, cell-free virions of HTLV-1 are very poorly infectious. The lack of completely HTLV-1-resistant cells and the low infectivity of HTLV-1 have hampered research on the HTLV entry receptor. Entry of HTLV-1 into target cells is thought to involve interactions between the env (Env) glycoproteins, a surface glycoprotein (surface unit), and a transmembrane glycoprotein. Recent studies have shown that glucose transporter GLUT1, heparan sulfate proteoglycans (HSPGs), and neuropilin-1 (NRP-1) are the three proteins important for the entry of HTLV-1. Studies using adherent cell lines have shown that GLUT1 can function as a receptor for HTLV. HSPGs are required for efficient entry of HTLV-1 into primary CD4 T cells. NRP-1 is expressed in most established cell lines. Further studies have shown that these three molecules work together to promote HTLV-1 binding to cells and fusion of viral and cell membranes. The virus could first contact with HSPGs and then form complexes with NRP-1, followed by association with GLUT1. It remains to be determined whether these three molecules can explain HTLV-1 cell tropism. It also remains to be more definitively proven that these molecules are sufficient to permit HTLV-1 entry into completely HTLV-1-resistant cells.
    Frontiers in Microbiology 06/2012; 3:222. DOI:10.3389/fmicb.2012.00222 · 3.99 Impact Factor
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    • "Such studies should help us understand the functional consequences of CS/DS-GAG, PG and P-selectin interactions and provide data that may, in future studies, be used to manipulate the expression of the polysaccharide by targeting the core protein(s). Several membrane PGs, including SDC-1, SDC-4, NRP-1, and CSPG4 can potentially present GAG chains on the surface of tumor cells [53-55]. CSPG4 is the only cell surface PG that is exclusively decorated with CS-GAGs [41] and therefore, it may play a major role in forming cell surface CS-GAGs. "
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    ABSTRACT: We have previously demonstrated that chondroitin sulfate glycosaminoglycans (CS-GAGs) on breast cancer cells function as P-selectin ligands. This study was performed to identify the carrier proteoglycan (PG) and the sulfotransferase gene involved in synthesis of the surface P-selectin-reactive CS-GAGs in human breast cancer cells with high metastatic capacity, as well as to determine a direct role for CS-GAGs in metastatic spread. Quantitative real-time PCR (qRT-PCR) and flow cytometry assays were used to detect the expression of genes involved in the sulfation and presentation of chondroitin in several human breast cancer cell lines. Transient transfection of the human breast cancer cell line MDA-MB-231 with the siRNAs for carbohydrate (chondroitin 4) sulfotransferase-11 (CHST11) and chondroitin sulfate proteoglycan 4 (CSPG4 ) was used to investigate the involvement of these genes in expression of surface P-selectin ligands. The expression of CSPG4 and CHST11 in 15 primary invasive breast cancer clinical specimens was assessed by qRT-PCR. The role of CS-GAGs in metastasis was tested using the 4T1 murine mammary cell line (10 mice per group). The CHST11 gene was highly expressed in aggressive breast cancer cells but significantly less so in less aggressive breast cancer cell lines. A positive correlation was observed between the expression levels of CHST11 and P-selectin binding to cells (P < 0.0001). Blocking the expression of CHST11 with siRNA inhibited CS-A expression and P-selectin binding to MDA-MB-231 cells. The carrier proteoglycan CSPG4 was highly expressed on the aggressive breast cancer cell lines and contributed to the P-selectin binding and CS-A expression. In addition, CSPG4 and CHST11 were over-expressed in tumor-containing clinical tissue specimens compared with normal tissues. Enzymatic removal of tumor-cell surface CS-GAGs significantly inhibited lung colonization of the 4T1 murine mammary cell line (P = 0.0002). Cell surface P-selectin binding depends on CHST11 gene expression. CSPG4 serves as a P-selectin ligand through its CS chain and participates in P-selectin binding to the highly metastatic breast cancer cells. Removal of CS-GAGs greatly reduces metastatic lung colonization by 4T1 cells. The data strongly indicate that CS-GAGs and their biosynthetic pathways are promising targets for the development of anti-metastatic therapies.
    Breast cancer research: BCR 06/2011; 13(3):R58. DOI:10.1186/bcr2895 · 5.49 Impact Factor
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