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Endostatin-induced tyrosine kinase signaling through the Shb adaptor protein regulates endothelial cell apoptosis
J Dixelius1, H Larsson, T Sasaki
1Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala, Sweden.
Abstract:
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.
Insights
Endostatin inhibits angiogenesis by inducing tyrosine kinase activity and apoptosis in endothelial cells, particularly when Shb is expressed. Its heparin-binding ability is crucial for these effects.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Endostatin, a collagen XVIII fragment, is a known inhibitor of angiogenesis.
- Fibroblast growth factor-2 (FGF-2) is a key inducer of angiogenesis.
- The role of endostatin's heparin-binding ability and intracellular signaling in its anti-angiogenic effects requires elucidation.
Purpose of the Study:
- To investigate the mechanism by which endostatin inhibits FGF-2-induced angiogenesis.
- To determine the role of endostatin's heparin-binding ability and Shb protein in endostatin's effects on endothelial cells.
- To explore endostatin's impact on endothelial cell cycle, morphogenesis, and apoptosis.
Main Methods:
- Utilized the chicken chorioallantoic membrane model to assess angiogenesis inhibition.
- Employed murine brain endothelial (IBE) cells, including those overexpressing Shb, to study cellular responses.
- Analyzed cell cycle progression, tubular morphogenesis in collagen gels, apoptosis, and protein tyrosine phosphorylation.
Main Results:
- Endostatin inhibited FGF-2-induced angiogenesis, an effect dependent on its heparin-binding ability.
- Endostatin induced apoptosis and altered tubular morphogenesis in IBE cells, particularly when Shb was overexpressed.
- Endostatin treatment led to tyrosine phosphorylation of Shb and the formation of multiprotein complexes involving a tyrosine kinase.
Conclusions:
- Endostatin's anti-angiogenic effects are mediated by inducing tyrosine kinase activity and apoptosis in endothelial cells.
- Heparin-binding ability of endostatin and Shb expression are critical for its pro-apoptotic and anti-angiogenic functions.
- Endostatin modulates endothelial cell behavior through intracellular signaling pathways involving tyrosine kinases.