Role of cell-surface retention sequence binding protein-1 (CRSBP-1) regulation of interstitial-lymphatic flow

CRSBP-1 is a membrane glycoprotein and exhibits distinct dual ligand (CRS motif and hyaluronic acid) binding activity. CRSBP-1 specifically localizes to lymphatic endothelial cells (LECs). To determine the in vivo role of CRSBP-1, we generated CRSBP-1-null mice. These mice are overtly normal and fertile but exhibit identifiable morphological and functional alterations of lymphatic vessels, including distended lumens and constitutively increased interstitial-lymphatic flow. Putative CRSBP-1 ligands PDGF-BB and hyaluronic acid stimulate interstitial-lymphatic flow in wild-type mice but not in CRSBP-1-null mice as determined by FITC-dextran egress (in tails) assay. These preliminary results indicate that CRSBP-1 and its ligands may regulate interstitial-lymphatic flow. This thesis is to define the roles of CRSBP-1 and its ligands in the regulation of interstitial-lymphatic flow.;Using FITC-dextran egress and carrageenan-induced paw edema assays, this thesis demonstrates that CRSBP-1-null mice have constitutively increased interstitial-lymphatic flow. Specific CRSBP-1 ligands (PDGF peptide and VEGF peptide), which interact with CRSBP-1 but not PDGFbetaR and VEGFR3, stimulate interstitial-lymphatic flow. Immunofluorescent confocal microscopic analysis indicates that CRSBP-1 is localized to the plasma membrane as well as intracellularly to the ER network in SVEC4-10 cells (LEC-like cells) and primary human dermal LECs (HDLECs) and that specific CRSBP-1 ligands stimulate contraction of the CRSBP-1-associated ER network in these LECs. Stimulation by specific CRSBP-1 ligands also causes dissociation of p120- and beta-catenin from VE-cadherin, internalization of VE-cadherin and opening of VE-cadherin-mediated intercellular junctions, resulting in increased permeability in these LEC cell monolayers. Immunoprecipitation/immunoblot analysis in SVEC4-10 cells reveals that CRSBP-1 forms complexes with PDGFbetaR and beta-catenin and that specific CRSBP1 ligands stimulate tyrosine phosphorylation of PDGFbetaR, VE-cadherin and beta-catenin. Pretreatment of cells with a PDGFbetaR kinase inhibitor, Tyrphostin AG 1296, abolishes tyrosine phosphorylation of these proteins and specific CRSBP-1 ligand-induced disassembly and opening of intercellular junctions and increased permeability in SVEC4-10 cell monolayers. Co-injection of Tyrphostin AG 1296 abolishes CRSBP-1 ligand-stimulated interstitial-lymphatic flow in mice.;In conclusion, CRSBP-1 ligands stimulate permeability in LEC monolayers and interstitial-lymphatic flow in mice by sequentially inducing activation of PDGFbetaR in the CRSBP-1/PDGFbetaR/beta-catenin complex, tyrosine phosphorylation of beta-catenin and VE-cadherin, VE-cadherin internalization, and disassembly and opening of intercellular junctions.