Syndecan-4 contributes to endothelial tubulogenesis through interactions with two motifs inside the pro-angiogenic N-terminal domain of thrombospondin-1.
Thrombospondin-1 (TSP-1) is an extracellular matrix protein that modulates focal adhesion in mammalian cells and exhibits dual roles in angiogenesis. In a previous work, we showed that a recombinant 18 kDa protein encompassing the N-terminal residues 1-174 of human TSP-1 (TSP18) induced tubulogenesis of human umbilical vein endothelial cells and protected them from apoptosis. Our results indicated that these effects were possibly mediated by syndecan-4 proteoglycan, since binding of TSP18 to endothelial extracts was inhibited by anti-syndecan-4 antibody. Syndecan-4 is a heparan-sulfate proteoglycan that regulates cell-matrix interactions and is the only member of its family present in focal adhesions. In this report, we demonstrate that a monoclonal antibody against syndecan-4 blocks TSP18-induced tubulogenesis. Furthermore, through 2D adhesion and 3D angiogenic assays, we demonstrate that two sequences, TSP Hep I and II, retain the major pro-angiogenic activity of TSP18. These TSP-1 motifs also compete with the fibronectin Hep II domain for binding to syndecan-4 on endothelial cell surface, indicating that they may exert their effects by interfering with the recognition of fibronectin by syndecan-4. Additionally, TSP18 and its derived peptides activate the PKC-dependent Akt-PKB signaling pathway. Blockage of PKC activation prevented HUVEC spreading when seeded on TSP18 fragment, and on TSP Hep I and TSP Hep II peptides, but not on gelatin-coated substrates. Our results identify syndecan-4 as a novel receptor for the N-terminus of TSP-1 and suggest that TSP-1 N-terminal pro-angiogenic activity is linked to its capacity of interfering with syndecan-4 functions in the course of cell adhesion.
Nunes
Sara Santana
SS
Departamento de Biologia Celular e Genética, Laboratório de Biologia da Célula Endotelial e da Angiogênese (LabAngio), Instituto de Biologia Roberto Alcantara Gomes, Universidade do Estado do Rio de Janeiro, UERJ, Rio de Janeiro, RJ, Brazil.
Outeiro-Bernstein
Marianna A Ferrari do
MA
Juliano
Luiz
L
Vardiero
Francisco
F
Nader
Helena B
HB
Woods
Anne
A
Legrand
Chantal
C
Morandi
Verônica
V
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D042501:Angiogenic Proteins / Q000378:metabolism*; D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D002448:Cell Adhesion / Q000187:drug effects; D002465:Cell Movement / Q000187:drug effects; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects; D004727:Endothelium / Q000098:blood supply* / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects; D005353:Fibronectins / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D018919:Neovascularization, Physiologic / Q000187:drug effects; D010446:Peptide Fragments / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D013552:Swine; D053671:Syndecan-4 / Q000378:metabolism*; D019700:Thrombospondin 1 / Q000737:chemistry* / Q000378:metabolism*
D042501:Angiogenic Proteins; D000906:Antibodies; D005353:Fibronectins; D010446:Peptide Fragments; D047428:Protein Kinase Inhibitors; D053671:Syndecan-4; D019700:Thrombospondin 1; D051057:Proto-Oncogene Proteins c-akt; D011493:Protein Kinase C