CRSBP-1/LYVE-1 ligands disrupt lymphatic intercellular adhesion by inducing tyrosine phosphorylation and internalization of VE-cadherin

Frank E. Johnson,Wei-Hsien Hou,Shuan Shian Huang,Jung San Huang,Cheng C. Tsai,I-Hua Liu

doi:10.1242/jcs.078154

Frank E. Johnson, Wei-Hsien Hou + Show 4 more

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https://doi.org/10.1242/jcs.078154

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Abstract

Cell-surface retention sequence (CRS) binding protein (CRSBP-1) is a membrane glycoprotein identified by its ability to bind PDGF-BB and VEGF-A via their CRS motifs (clusters of basic amino acid residues). CRSBP-1 is identical to LYVE-1 and exhibits dual ligand (CRS-containing proteins and hyaluronic acid) binding activity, suggesting the importance of CRSBP-1 ligands in lymphatic function. Here, we show that CRSBP-1 ligands induce disruption of VE-cadherin-mediated intercellular adhesion and opening of intercellular junctions in lymphatic endothelial cell (LEC) monolayers as determined by immunofluorescence microscopy and Transwell permeability assay. This occurs by interaction with CRSBP-1 in the CRSBP-1-PDGFβR-β-catenin complex, resulting in tyrosine phosphorylation of the complex, dissociation of β-catenin and p120-catenin from VE-cadherin, and internalization of VE-cadherin. Pretreatment of LECs with a PDGFβR kinase inhibitor abolishes ligand-stimulated tyrosine phosphorylation of VE-cadherin, halts the ligand-induced disruption of VE-cadherin intercellular adhesion and blocks the ligand-induced opening of intercellular junctions. These CRSBP-1 ligands also induce opening of lymphatic intercellular junctions that respond to PDGFβR kinase inhibitor in wild-type mice (but not in Crsbp1-null mice) as evidenced by increased transit of injected FITC-dextran and induced edema fluid from the interstitial space into lymphatic vessels. These results disclose a novel mechanism involved in the opening of lymphatic intercellular junctions.

Highlights

Cell-surface retention sequence (CRS) binding protein-1 (CRSBP1) is a membrane glycoprotein first identified by its ability to mediate cell-surface retention of the simian sarcoma virus (SSV) oncogene v-sis gene product (PDGFBB) in SSV-transformed fibroblasts (Boensch et al, 1995; Boensch et al, 1999; Lokeshwar et al, 1990)
To test the hypothesis that CRSBP-1 ligands cause opening of lymphatic intercellular junctions by compromising VE-cadherinand -catenin-mediated intercellular adhesion, we analyzed the subcellular localization of VE-cadherin and its associated protein, -catenin, in monolayers of SVEC4-10 cells treated with CRSBP1 ligands using immunofluorescence confocal microscopy
When compared with cells treated with vehicle only, gaps and slits appeared between endothelial cells treated with CRSBP-1 ligand, which we believe is due to loss of cell–cell adhesion (Fig. 1F,I)

Summary

Introduction

Cell-surface retention sequence (CRS) binding protein-1 (CRSBP1) is a membrane glycoprotein first identified by its ability to mediate cell-surface retention (after synthesis and secretion) of the simian sarcoma virus (SSV) oncogene v-sis gene product (PDGFBB) in SSV-transformed fibroblasts (Boensch et al, 1995; Boensch et al, 1999; Lokeshwar et al, 1990). All members of the PDGF superfamily, including PDGF-AA, PDGF-BB, placental growth factor (PlGF) and vascular endothelial cell growth factorA, -C and -D (VEGF-A, VEGF-C and VEGF-D), possess CRS motifs and exhibit cell-surface retention during secretion (Carmeliet et al, 1999; Houck et al, 1991; Joukov et al, 1996; Joukov et al, 1997; LaRochelle et al, 1991; Ostman et al, 1991). The primary localization of CRSBP-1/LYVE-1 to lymphatic endothelium has made it a suitable marker for studying lymphangiogenesis (Alitalo and Carmeliet, 2002; Jackson, 2003; Jussila and Alitalo, 2002) This implies the likely importance of the dual ligand binding activity of CRSBP-1 in regulating the function of lymphatic vessels

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