Xiaolong Zhu1, Sha Ding1, Cong Qiu1, Yanna Shi1, Lin Song1, Yueyue Wang1, Yuewen Wang1, Jinying Li1, Yiran Wang1, Yi Sun1, Lingfeng Qin1, Jun Chen1, Michael Simons1, Wang Min1, Luyang Yu2. 1. From the Institute of Genetics and Regenerative Biology, College of Life Sciences (X.Z., S.D., C.Q., Y. Shi, L.S., Yueyue Wang, Yuewen Wang, J.L., Yiran Wang, Y. Sun, L.Y.), Research Center for Air Pollution and Health (X.Z., S.D., C.Q., Y. Shi, L.S., Yuewen Wang, J.L., Yiran Wang, L.Y.), and Key Laboratory for Molecular Animal Nutrition, Ministry of Education, Innovation Center for Cell Signaling Network, College of Life Sciences (J.C.), Zhejiang University, Hangzhou, China; Yale Cardiovascular Research Center, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT (L.Q., M.S.); Interdepartmental Program in Vascular Biology and Therapeutics, Department of Pathology, Yale University School of Medicine, New Haven, CT (L.Q., W.M.); and The First Affiliated Hospital, Center for Translational Medicine, Sun Yat-sen University, Guangzhou, China (W.M.). 2. From the Institute of Genetics and Regenerative Biology, College of Life Sciences (X.Z., S.D., C.Q., Y. Shi, L.S., Yueyue Wang, Yuewen Wang, J.L., Yiran Wang, Y. Sun, L.Y.), Research Center for Air Pollution and Health (X.Z., S.D., C.Q., Y. Shi, L.S., Yuewen Wang, J.L., Yiran Wang, L.Y.), and Key Laboratory for Molecular Animal Nutrition, Ministry of Education, Innovation Center for Cell Signaling Network, College of Life Sciences (J.C.), Zhejiang University, Hangzhou, China; Yale Cardiovascular Research Center, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT (L.Q., M.S.); Interdepartmental Program in Vascular Biology and Therapeutics, Department of Pathology, Yale University School of Medicine, New Haven, CT (L.Q., W.M.); and The First Affiliated Hospital, Center for Translational Medicine, Sun Yat-sen University, Guangzhou, China (W.M.). luyangyu@zju.edu.cn.
Abstract
RATIONALE: The highly conserved NOTCH (neurogenic locus notch homolog protein) signaling pathway functions as a key cell-cell interaction mechanism controlling cell fate and tissue patterning, whereas 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 its signal transduction. Our previous study indicated the potential role of post-translational SUMO (small ubiquitin-like modifier) modification (SUMOylation) in vascular disorders. OBJECTIVE: The aim of this study was to investigate the role of SUMOylation in endothelial NOTCH signaling and angiogenesis. METHODS AND RESULTS: Endothelial SENP1 (sentrin-specific protease 1) 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 N1ICD (NOTCH1 intracellular domain), 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 cotranscriptional factor. Functionally, SENP1-mediated NOTCH1 deSUMOylation was required for NOTCH signal activation in response to DLL4 (Delta-like 4) stimulation. This in turn suppressed VEGF (vascular endothelial growth factor) 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 nonvascular tissues and provide a novel therapeutic strategy for vascular diseases and tumors.
RATIONALE: The highly conserved NOTCH (neurogenic locus notch homolog protein) signaling pathway functions as a key cell-cell interaction mechanism controlling cell fate and tissue patterning, whereas 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 its signal transduction. Our previous study indicated the potential role of post-translational SUMO (small ubiquitin-like modifier) modification (SUMOylation) in vascular disorders. OBJECTIVE: The aim of this study was to investigate the role of SUMOylation in endothelial NOTCH signaling and angiogenesis. METHODS AND RESULTS: Endothelial SENP1 (sentrin-specific protease 1) 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 N1ICD (NOTCH1 intracellular domain), 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 cotranscriptional factor. Functionally, SENP1-mediated NOTCH1 deSUMOylation was required for NOTCH signal activation in response to DLL4 (Delta-like 4) stimulation. This in turn suppressed VEGF (vascular endothelial growth factor) 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 nonvascular tissues and provide a novel therapeutic strategy for vascular diseases and tumors.
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