SUMOylation Negatively Regulates Angiogenesis by Targeting Endothelial NOTCH Signaling

RATIONALE:The highly conserved NOTCH signaling pathway functions as a key cell-cell interaction mechanism controlling cell-fate and tissue patterning, while its dysregulation is implicated in a variety of developmental disorders and cancers. The pivotal role of endothelial NOTCH in regulation of angiogenesis is widely appreciated; however, little is known about what controls it signal transduction. Our previous study indicated the potential role of post-translational small ubiquitin-like modifier (SUMO) modification (SUMOylation) in vascular disorders. OBJECTIVE:To investigate the role of SUMOylation in endothelial NOTCH signaling and angiogenesis. METHODS AND RESULTS:Endothelial SENP1 deletion, in newly generated endothelial SENP1 (the major protease of the SUMO system) deficient mice, significantly delayed retinal vascularization by maintaining prolonged NOTCH1 signaling, as confirmed in cultured endothelial cells. An in vitro SUMOylation assay and immunoprecipitation revealed that when SENP1 associated with NOTCH1 intracellular domain (N1ICD) it functions as a deSUMOylase of N1ICD SUMOylation on conserved lysines. Immunoblot and immunoprecipitation analyses and dual luciferase assays of natural and SUMO-conjugated/nonconjugated NOTCH1 forms demonstrated that SUMO conjugation facilitated NOTCH1 cleavage. This released N1ICD from the membrane and stabilized it for translocation to the nucleus where it functions as a co-transcriptional factor. Functionally, SENP1-mediated NOTCH1 deSUMOylation was required for NOTCH signal activation in response to DLL4 stimulation. This in turn suppressed VEGF receptor signaling and angiogenesis, as evidenced by immunoblotted signaling molecules and in vitro angiogenesis assays. CONCLUSIONS:These results establish reversible NOTCH1 SUMOylation as a regulatory mechanism in coordinating endothelial angiogenic signaling; SENP1 acts as a critical intrinsic mediator of this process. These findings may apply to NOTCH-regulated biological events in non-vascular tissues and provide a novel therapeutic strategy for vascular diseases and tumors.