Endostatin-induced tyrosine kinase signaling through the Shb adaptor protein regulates endothelial cell apoptosis

• Published in: Blood 2000-06, Vol.95 (11), p.3403-3411
• Main Authors: Dixelius, Johan, Larsson, Helena, Sasaki, Takako, Holmqvist, Kristina, Lu, Lingge, Engstroüm, AÅke, Timpl, Rupert, Welsh, Michael, Claesson-Welsh, Lena
• Format: Article
• Language: English
• Subjects: Adaptor Proteins, Signal Transducing; Allantois - blood supply; Amino Acid Substitution; Animals; Antineoplastic Agents - pharmacology; Apoptosis; Cells, Cultured; Cerebrovascular Circulation; Chick Embryo; Chorion - blood supply; Collagen - pharmacology; Collagen Type XVIII; Endostatins; Endothelium, Vascular - cytology; Endothelium, Vascular - physiology; Fibroblast Growth Factor 2 - pharmacology; Humans; Mice; Mutagenesis, Site-Directed; Neovascularization, Physiologic - drug effects; Peptide Fragments - pharmacology; Protein-Tyrosine Kinases - metabolism; Proto-Oncogene Proteins - drug effects; Proto-Oncogene Proteins - physiology; Recombinant Proteins - metabolism; Recombinant Proteins - pharmacology; Signal Transduction - drug effects; Signal Transduction - physiology; src Homology Domains; Transfection
• ISSN: 0006-4971; 1528-0020; 1528-0020
• DOI: 10.1182/blood.V95.11.3403

Endostatin, which corresponds to the C-terminal fragment of collagen XVIII, is a potent inhibitor of angiogenesis. Fibroblast growth factor-2 (FGF-2)–induced angiogenesis in the chicken chorioallantoic membrane was inhibited by endostatin, but not by an endostatin mutant R158/270A, lacking heparin-binding ability. Endostatin was internalized by endothelial cells, but not by mouse fibroblasts. Treatment of murine brain endothelial (IBE) cells with endostatin reduced the proportion of cells in S phase, whereas growth-arrested IBE cells in collagen gels treated with endostatin displayed enhanced tubular morphogenesis. IBE cells overexpressing Shb, an adaptor protein implicated in angiostatin-induced apoptosis, displayed elevated apoptosis and decreased tubular morphogenesis in collagen gels in response to endostatin when added together with FGF-2. Induction of apoptosis was dependent on the heparin-binding ability of endostatin and the expression of Shb with a functional Src homology 2 (SH2)-domain. Endostatin treatment for 10 minutes or 24 hours induced tyrosine phosphorylation of Shb and formation of multiprotein complexes. An Shb SH2 domain fusion protein precipitated a 125-kd phosphotyrosyl protein in endostatin-treated cells. The 125-kd component either contained intrinsic tyrosine kinase activity or occurred in complex with a tyrosine kinase. In conclusion, our data show that endostatin induces tyrosine kinase activity and enhanced apoptosis in FGF-treated endothelial cells.