Endostatin-induced tyrosine kinase signaling through the Shb adaptor protein regulates endothelial cell apoptosis.
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.
Dixelius
J
J
Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala, Sweden.
Larsson
H
H
Sasaki
T
T
Holmqvist
K
K
Lu
L
L
Engström
A
A
Timpl
R
R
Welsh
M
M
Claesson-Welsh
L
L
D048868:Adaptor Proteins, Signal Transducing; D000482:Allantois / Q000098:blood supply; D019943:Amino Acid Substitution; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis; D002478:Cells, Cultured; D002560:Cerebrovascular Circulation; D002642:Chick Embryo; D002823:Chorion / Q000098:blood supply; D003094:Collagen / Q000494:pharmacology*; D043170:Collagen Type XVIII; D043169:Endostatins; D004730:Endothelium, Vascular / Q000166:cytology* / Q000502:physiology*; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D006801:Humans; D051379:Mice; D016297:Mutagenesis, Site-Directed; D018919:Neovascularization, Physiologic / Q000187:drug effects; D010446:Peptide Fragments / Q000494:pharmacology*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000187:drug effects / Q000502:physiology*; D011994:Recombinant Proteins / Q000378:metabolism / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D014162:Transfection; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; D000970:Antineoplastic Agents; D043170:Collagen Type XVIII; D043169:Endostatins; D010446:Peptide Fragments; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; C490081:SHB protein, human; C085634:Shb protein, mouse; D016222:Fibroblast Growth Factor 2; D003094:Collagen; D011505:Protein-Tyrosine Kinases