SUMOylation Negatively Regulates Angiogenesis by Targeting Endothelial NOTCH SignalingNovelty and Significance
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.
Vascular network expansion is fundamental to embryonic development, tissue growth, and wound healing. New blood vessels are generated from the pre-existing ones by a series of successive steps, including endothelial sprouting and tube formation. Typically, these processes are regulated by angiogenic factors, such as vascular endothelial growth factors,1–3 fibroblast growth factors,4,5 and platelet-derived growth factors.6 In particular, VEGF-A (vascular endothelial growth factor-A) induces tip cell formation leading to vascular ingrowth into avascular areas. Endothelial sprouting is controlled by a feedback signaling loop during vascular network formation.7–9 Tip cells expressing DLL4 (Delta-like 4) activate NOTCH1 (neurogenic locus notch homolog protein-1) in adjoining cells to suppress the tip cell phenotype, thus committing them to stalk cell specification. Once bound to DLL4, activated NOTCH1 is cleaved by proteolytic enzymes within the membrane, thereby releasing its intracellular domain (N1ICD). The latter translocates into the nucleus to combine with coactivators and finally drives specific target genes. Activation of the endothelial NOTCH1 pathway represses expression of VEGF receptors and inhibits VEGF signaling, which leads to reduced endothelial cell (EC) angiogenic capability.10,11 The critical role of endothelial NOTCH1 in coordinating angiogenesis has been widely appreciated, but the regulatory and adaptive mechanisms of its signaling conduction are largely unclear.
Several studies have highlighted the importance of post-translational modifications on NOTCH functions. Glycosylation of NOTCH receptors, which is modulated by glycosyltransferases—Fringe family—regulates the tip–stalk cell selection by promoting the interaction between Delta ligands and the NOTCH receptor, thus decreasing Jagged-NOTCH affinity.10,12,13 On the contrary, the C-terminal region of N1ICD protein is the target of ubiquitin E3 ligase, FBXW7. It is strongly modified by ubiquitination, which disables NICD protein stability through hydrolysis.14–16 A recent study showed that SIRT1-mediated N1ICD acetylation significantly promotes its stabilization and enhances the activation of NOTCH1 signaling; this results in reduced vascular branching and density.17
Post-translational SUMOylation, like ubiquitination, is covalently attached to substrate proteins via an isopeptide bond between its C-terminal glycine and a lysine residue in the substrate proteins.18 A consensus SUMO (small ubiquitin-like modifier) acceptor site, consisting of the sequence ψKXE (ψ represents the hydrophobic amino acid and K is the SUMO conjugation site), has been identified.19,20 SUMOylation is considered to be an essential process that controls gene expression, protein stability, chromatin structure, signal transduction, and maintenance of the genome.17–22 Particularly, SUMOylation is a highly dynamic process attributed to a SUMO-specific proteases family, SENP (sentrin-specific protease), which reversely detaches SUMO molecules from the substrate proteins. Six members of the SENP family (SENP1, 2, 3, 5, 6, and 7) have been identified in mammals. Different members of these SUMO-specific proteases seem to localize in different cellular compartments where they regulate protein functions by altering protein stability, cellular localization, and protein–protein interactions.23–26
To date, most SUMOylation substrates are categorized as nuclear and perinuclear proteins. SUMOylation targeting of membrane proteins is indicated; however, a limited number of cell membrane substrates have been identified. On the contrary, recent studies suggest a potential involvement of SUMOylation in the pathogenesis of cardiovascular disease in which vascular cells could be the objects of modulation. Our previous study implies engagement of SENP1-regulated SUMOylation in vasculature formation during embryonic development (Figure 2A in the study by Yu et al26) and severe anemia as the major cause of death in SENP1 global knockout mice. Therefore, we considered the possibility that SUMO regulates endothelial receptors, the pivotal effectors of vascular extension.
In the present study, we identified SENP1-regulated SUMOylation as a novel post-translational modification to NOTCH1, which plays a critical role in coordinating developmental angiogenesis. Mechanistically, NOTCH1 SUMOylation facilitates NOTCH1 cleavage and stabilizes N1ICD to function as a cotranscriptional factor in EC, which consequently promotes NOTCH signaling.
A detailed description of methods is provided in the Online Data Supplement and includes information on generating endothelial SENP1-deficient mouse, retina dissection and whole-mount staining, EC culture, transfection and viral infection, immunoprecipitation and immunoblotting, in vitro SUMOylation assay, protein stability assay, quantitative real-time polymerase chain reaction, dual-luciferase assay, in vitro EC migration and capillary-like structure formation assays, and statistical analyses.
Endothelial SENP1 Deficiency Compromises Neonatal Retina Angiogenesis
To investigate the involvement of SENP1 in regulation of angiogenesis, we generated mice with an EC-specific SENP1 deficiency (SENP1-ecKO) as described in the Online Data Supplement. These mice were found to be viable and fertile. Postnatal retina angiogenesis was assessed in P4 and P7 pups. SENP1 expression in control mice was readily evident throughout the retinal endothelium but was highly expressed in the angiogenic front of the vasculature (Figure 1A). A similar intracellular expression pattern of SENP1 was also observed in human microvascular endothelial cell (HMVEC; Figure 1B); this suggested a role for endothelial SENP1 in vascular sprouting. In contrast, SENP1 expression was profoundly reduced in the SENP1-ecKO retina endothelium (Online Figure IA and IB). However, SENP1-ecKO mice demonstrated a delayed expansion of the vascular plexus to the periphery, compared with controls; this was evidenced by a decrease in vascular branching and reduced vessel coverage at P4 (Online Figure IC through IL, IM, and IN) and P7 (Figure 1C through 1J, 1M, and 1N). Further analysis revealed that the tip cell number, tip sprouts, and vascular length were significantly reduced in the sprouting region in SENP1-ecKO (Figure 1O through 1Q; Online Figure IO and IP). Pulse BrdU (bromodeoxyuridine) labeling did not show any difference in the proliferation rate of retinal ECs between control and SENP1-ecKO mice (Figure 1K, 1L, and 1S). The inhibitory effect of SENP1 deficiency on ECs was further confirmed in cultured primary human umbilical vein endothelial cell (HUVEC) or HMVEC; SENP1 knockdown resulted in diminished spheroid sprouting (Online Figure IIA, left, and IIB, bars 1 and 3), migration (Online Figure IIC through IIF), and capillary-like structure formation (Online Figure IIH and III).
Inactivation of SENP1 Enhances Endothelial NOTCH1 Responses
The above findings suggested activation of endothelial Notch1 signaling. Control and SENP1-ecKO pups were treated with DAPT (N-[N-(3,5-difluorophenacetyl)-1-alanyl]-S-phenylglycine t-butyl ester), a γ-secretase inhibitor, to test whether increased Notch activity was responsible for the observed phenotype. DAPT treatment resulted in a large increase in vessel density and expansion of the vascular plexus in the postnatal retina of control mice (Figure 2A, 2C, 2E, and 2G). DAPT completely rescued the defect in SENP1-ecKO pups (Figure 2B, 2D, 2F, and 2H), which resulted in vascular length, vascular coverage, and tip sprouts growth comparable to the control group (Figure 2I through 2K). The rescue effect of DAPT on SENP1 deficiency–induced angiogenesis defect was further confirmed in an in vitro spheroid-based angiogenesis assay (Online Figure IIA, bottom, and IIB, bars 3 and 4).
The potential link between SENP1 and NOTCH1 signaling in ECs was further explored by examining the protein level of cleaved NOTCH1 and its main downstream effectors, HES1 and HEY1, in mouse lung endothelial cell isolated from wild-type and SENP1-ecKO mice. There was a pronounced increase in the amount of cleaved NOTCH1 N1ICD and expression of HES1 and HEY1 in SENP1-deficient mouse lung endothelial cell compared with control cells (Figure 2L).
The role of SENP1 in NOTCH1 signaling was also examined in HMVEC. siRNA interference of SENP1 in HMVEC resulted in upregulation of NOTCH1 target genes, whereas the level of NOTCH1 itself was not affected (Figure 2M). This was confirmed by immunoblot analysis of protein levels of all the NOTCH1 forms (full-length, NTM [Notch transmembrane subunit], and N1ICD) and quantification of total NOTCH1 (Figure 2N). Furthermore, upregulation of N1ICD in HUVEC was observed in the presence of a SENP1 catalytic inactive mutant (SENP1 mutant; Figure 2O). Taken together, these results pointed to a strong link between SENP1 and NOTCH1 during angiogenesis (Figure 2P).
SENP1 Regulates NOTCH1 SUMOylation
The hyperactivation of NOTCH signaling was induced by either deleting endothelial SENP1 or altering its catalytic function in vivo and in vitro. SENP1 is an endopeptidase that deconjugates SUMOs from substrate proteins. Therefore, we reasoned that SUMOylation may directly regulate NOTCH1 and its signaling activation in ECs. This was tested by analyzing the amino acid sequence of NOTCH1 protein using computational system–based software. Several classic SUMO-binding motifs were predicted in the C-terminal N1ICD region (Online Figure IIIA). A truncated N1ICD fragment, containing putative SUMO-binding motifs, was used as a potential substrate in an in vitro SUMOylation assay in the presence of SUMO E1 (AOS1/UBA2), SUMO E2 (UBC9), and ATP. SUMOylated bands were observed, which indicated a SUMO modification of NOTCH1 (Online Figure IIIC through IIIE).
Next, we examined whether NOTCH1 is indeed SUMOylated in ECs. N1ICD was the predominant SUMOylated form of NOTCH1 in HMVEC (Figure 3A), although some amount of SUMOylation was also detected on full-length NOTCH1 (Online Figure IVA). The occurrence of the endogenous modification was further confirmed by overexpressing SUMO1 and N1ICD constructs in 293 cells (Figure 3B). These results corresponded to the computational prediction, which indicated the N1ICD region as the acceptor of SUMO modification. The predicted bioinformatical analysis (Online Figure IIIB) prompted our further investigation of putative SUMO-binding sites in N1ICD, which are evolutionarily conserved among vertebrates. Accordingly, we generated single mutants bearing lysine (K) to arginine (R) substitutions at 3 putative N1ICD SUMOylation sites K2049, K2150, and K2252, which we refer to as N1ICD K2049R, K2150R, and K2252R. Additionally, we generated a construct containing the triple mutation N1ICD-3KR. The K2049R, K2150R, and K2252R substitutions significantly diminished SUMOylation of N1ICD when coexpressed with SUMO1. This was further reduced in cells expressing N1ICD-3KR, thus indicating that K2049, K2150, and K2252 are the major SUMO-biding sites on NOTCH1 (Figure 3C).
N1ICD SUMOylation in mouse lung endothelial cell from control and SENP1-ecKO mice was examined to determine whether SENP1 functions as a NOTCH1 deSUMOylase in ECs. Intriguingly, the amount of SUMOylated N1ICD was significantly increased in SENP1-deficient ECs (Figure 3F). Also, N1ICD was coexpressed with SENP1 wild-type or a catalytic inactive mutant (SENP1 mutant) in an exogenous system; N1ICD SUMOylation was clearly detected in the presence of SUMO1. Importantly, SUMOylated N1ICD was robustly attenuated by SENP1 wild type, whereas SENP1 mutant increased the amount of SUMOylated N1ICD (Figure 3D). Moreover, we observed that N-ethylmaleimide, a SENP inhibitor that blocks SENP activity by modifying the active-site cysteine, intensely promoted N1ICD SUMOylation (Figure 3E). SENP1, SENP2, and SENP5 were reported as major functional deSUMOylases in mammalian cells.24,25 Coimmunoprecipitation experiments revealed a complex formed between N1ICD and SENP1; furthermore, N1ICD predominantly associated with SENP1, but only weakly with other SENPs (Online Figure IVB and IVC). These results suggested that SENP1 specifically mediates NOTCH1 deSUMOylation in ECs by deconjugating SUMO modification of N1ICD (Figure 3G).
Furthermore, we investigated the status of NOTCH1 SUMOylation under hypoxia, a circumstance that is typically a critical inductive factor for both physiological and pathological angiogenesis. To this end, HUVEC cells were cultured in regular normoxia or hypoxia condition for 24 hours. Cell lysates were then subjected to immunoprecipitation with anti-N1ICD followed by immunoblotting with anti-N1ICD or anti-SUMO1. Hypoxia treatment significantly increased HIF1α expression level and nuclear accumulation but decreased the SUMO conjugation on N1ICD (Online Figure VA), as well as the expression of HES1 and HEY1 (Online Figure VA). These data indicated a suppressive effect of hypoxia on NOTCH1 SUMOylation and signaling.
SENP1-Regulated NOTCH1 SUMOylation Facilitates Its Cleavage
The interaction between SENP1 and NOTCH1 suggested that manipulation of SENP1 expression level or activity can affect NOTCH1 activity. Notably, SENP1 directly interacted with full-length NOTCH1, the cell membrane receptor form, in EC (Online Figure IVD). Proteolytic cleavage of NOTCH1 is the key event involved in activation of its signaling cascade that leads to release of N1ICD. The DLL4-NOTCH1 binding leads to proteolytic cleavage of full-length NOTCH1 into the NOTCH1 transmembrane subunit form (NTM, the intermediate cleaved form composed of transmembrane and intracellular domains) and NOTCH1 extracellular domain form (N1ECD); NTM is further cleaved into N1ICD, whereas N1ECD is removed by bound DLL4 (Figure 4A). Therefore, we tested whether SENP1-regulated NOTCH1 SUMOylation affects its cleavage. To this end, NOTCH1 and its cleaved forms were examined in HMVEC after SENP1 knockdown. In control ECs, NOTCH1 was mostly detected as full-length and the NTM subunit; N1ICD was detected to a lesser extent (Figure 4B, lane 1). The amounts of full-length and NTM were somewhat reduced in the absence of SENP1, whereas N1ICD was increased (Figure 4B, lane 2). This was accompanied by elevated HES1 expression, which demonstrated increased NOTCH signaling output (Figure 4B, lane 2); these data were consistent with the results in mouse retinal endothelium and mouse lung endothelial cell. A similar role for SENP1 was observed in Dll4-mediated NOTCH1 cleavage, which resulted in an even higher level of N1ICD (Figure 4B, lanes 3 and 4). Moreover, SENP1 wild type, but not high levels of SENP1 mutant (a catalytic inactive form that smothers the deSUMOylase activity), reversed the extensive N1ICD cleavage induced by SENP1 deficiency (Figure 4C). This suggested that SENP1-regulated SUMOylation may facilitate NOTCH1 cleavage. Indeed, SUMO1 knockdown significantly inhibited NOTCH1 cleavage and activation, which was completely opposite of the inactive SENP1 effect (Figure 4D). These data strongly support the hypothesis that SENP1-regulated SUMOylation facilitates NOTCH1 cleavage (Figure 4G). Furthermore, we examined the regulatory role of DLL4 in NOTCH1 SUMOylation in DLL4-treated HMVECs. Immunoprecipitation assays revealed that DLL4 induced increased NTM SUMOylation, which consequently promoted cleavage into N1ICD (Figure 4E). A similar enhancement of N1ICD SUMOylation was also detected in the assay using a comparable level of N1ICD input adjusted for better discernment (Figure 4F). Conversely, decreased N1ICD SUMOylation was exhibited by immunoprecipitation in the presence of soluble DLL4 (sDLL4), thus blocking the binding of NOTCH receptor with DLL4 (Online Figure VB). Taken together, we conclude that DLL4 positively regulates N1ICD SUMOylation for NOTCH1 cleavage and signaling in ECs.
SENP1-Regulated SUMOylation Enhances Transcriptional Activity and Stability of N1ICD
Next, we considered that enhanced NOTCH1 signaling in SENP1-deficient EC may also be because of SUMO modification on N1ICD in the cytoplasm. SUMOylation has multiple effects on its substrates, including intracellular localization, protein stability, and protein activity; this is unlike ubiquitination, which usually induces degradation of target proteins. The biological effect of SUMOylation on N1ICD was examined by generating a SUMO1–N1ICD fusion protein. We used a SUMO fusion strategy described in our previous study,26 which mimics constant N1ICD SUMOylation (Online Figure VIA and VIB). N1ICD, with or without SUMO fusion, and SUMOylation defective mutant N1ICD-3KR showed a similar pattern of specific localization to the nucleus, as detected by immunofluorescence (Online Figure VIC). On the contrary, N1ICD entered the nucleus and activated downstream signaling by forming a complex with the transcriptional factor RBP-J (recombination signal binding protein J). SUMO conjugation had little effect on intracellular localization of N1ICD, but it significantly promoted the binding of N1ICD to RBP-J, as demonstrated by coimmunoprecipitation (Figure 5A and 5B). In contrast, N1ICD-3KR had a weaker involvement in the RBP-J complex (Figure 5C and 5D).
The effect of SUMOylation on NOTCH1 cotranscriptional activity was further examined in a dual-luciferase assay. As expected, SUMO-N1ICD strengthened while N1ICD-3KR diminished RBP-J binding to the HES1 promoter, the classic downstream gene driven by NOTCH1 signaling in EC (Figure 5E and 5F). A trend was confirmed by a similar finding, which showed that SUMOylated N1ICD promoted transcriptional binding of RBP-J to its general binding motif (Figure 5G). Furthermore, SUMO conjugation to N1ICD substantially enhanced the amplitude and duration of the N1ICD half-life in the presence of protein synthesis inhibitor cycloheximide (Figure 5H and 5I). Importantly, treatment with the SENP inhibitor, N-ethylmaleimide, significantly improved the stability of endogenous N1ICD in HMVEC (Figure 5J and 5K) and overexpressed exogenous N1ICD (Figure 5L and 5M).
Concomitantly, expression of NOTCH1 target genes was extensively elevated in Ad-SUMO1-N1ICD–infected HUVECs compared with the moderate increase induced by N1ICD overexpression (Figure 5N). Together, these data revealed that SENP1-regulated N1ICD SUMOylation positively modulates its cotranscriptional activity and protein stability but not intracellular localization.
NOTCH1 SUMOylation Negatively Regulates Angiogenic Signaling in ECs
VEGFR signaling is the dominant driver of endothelial sprouting. Its impact on tip/stalk cell balance is mediated via activation of NOTCH signaling. We examined angiogenic signaling in ECs to define the biological function of SENP1-regulated NOTCH1 SUMOylation in angiogenesis. SENP1 knockdown in HUVEC resulted in a significant decrease in VEGFR2 phosphorylation and an increase in VEGFR1 expression level; both were further promoted by Dll4 treatment (Figure 6A). The SENP1 deficiency–induced VEGFR2 phosphorylation blockage was confirmed in SENP1-ecKO retinal endothelium compared with control mice (Figure 6B). On the contrary, SUMO1 knockdown reversed the effect of SENP1 deficiency on VEGFR2 activation and VEGFR1 expression (Figure 6C). We further observed that SUMO1 augmented N1ICD-regulated VEGFR1 expression (Figure 6D and 6E), which was attributed to N1ICD SUMO conjugation (Figure 6F and 6G).
We further examined the role of SUMOylated N1ICD in VEGFR signaling by transducing HUVECs and HMVEC with adenoviral vectors containing N1ICD (Ad-Flag-N1ICD) or Flag-SUMO1-N1ICD (Ad-Flag-SUMO1-N1ICD). VEGF-A treatment resulted in significant inhibition of VEGFR2 phosphorylation and its downstream signaling molecules in ECs overexpressing N1ICD. However, inhibition of VEGFR2 signaling was augmented in the SUMO1-N1ICD transgene group (Figure 6H and 6I). A similar trend was observed in VEGF-C–induced VEGFR3 activation (Online Figure VIIA), with a slight decrease of VEGFR3 expression in the presence of SUMO1-N1ICD in EC (Online Figure VIIA) or SENP1 deficiency in retina endothelium (Online Figure VIIB and VIIC). Thus, SENP1-regulated NOTCH1 SUMOylation negatively modulates angiogenesis by upregulating VEGFR1 expression, thereby suppressing VEGFR2 and VEGFR3 angiogenic signaling in ECs.
N1ICD and SUMO1-N1ICD were each expressed in HUVEC or HMVEC to verify the role of NOTCH1 SUMOylation in EC angiogenesis. Vessel sprouting (Online Figure VID and VIE), migration (Online Figure VIF and VIG), and capillary-like structure formation capabilities (Online Figure VIH and VI I) were significantly diminished in EC expressing SUMO1-N1ICD compared with the N1ICD-infected ECs. This further proved the negative regulation of NOTCH1 SUMOylation in endothelial angiogenic activity.
Endothelial tip cell–derived Dll4 binds to NOTCH receptor on adjacent stalk EC during sprouting angiogenesis, which tightly coordinates the growth and patterning of the vessel network.10,11,27 However, the complete regulatory mechanism of endothelial NOTCH activation remains unknown. This study revealed that SENP1, the major protease for post-translational SUMO modification, acts as an intrinsic positive modulator of endothelial NOTCH1 signaling in developmental angiogenesis. The diminished NOTCH1 activity and cellular growth of EC deficient in SENP1 or bearing SENP1 deSUMOylase inactive mutant confirmed the in vivo retina phenotype, thus indicating the regulatory effect of SUMOylation on the NOTCH response in EC. Indeed, constitutive SUMOylation of endothelial NOTCH1 was identified both endogenously and exogenously, which was reversibly regulated by SENP1. Functionally, SENP1-regulated endothelial NOTCH1 SUMOyaltion is required for NOTCH1 cleavage at the cell membrane, maintenance of N1ICD protein stability, and cotranscriptional activity in the nucleus. Augmented NOTCH1 signaling, in turn, counteracts VEGF/VEGFR signaling in EC and consequent angiogenic growth (Figure 7).
Post-Translational SUMOylation Tightly Regulates Molecular Properties of NOTCH1 in EC
NOTCH1 undergoes many post-translational modifications, including glycosylation,12,13,28 ubiquitination,16,29 and acetylation.17 In the present study, we discovered that SUMOylation is a novel post-translational modification of the NOTCH receptor. NOTCH1 is normally modified in a SUMO1 conjugation preferred manner throughout its cellular functions. Dll4 binding promotes SUMOylation upon which SENP1 selectively acts as the key deSUMOylation regulator. SUMOyaltion is not a typical post-translational modification. The biochemical effect of SUMO modification is still controversial, because SUMOyaltion of specific targets correlates with a plethora of altered protein properties. Our investigation reveals that SUMOylation does not disturb N1ICD localization in the nucleus. However, intranuclear SUMOylation directly participates in the binding of N1ICD to RBP-J, the dominant transcriptional factor for driving downstream molecules in the NOTCH signaling cascade and formation of a NOTCH active transcriptional complex. The SUMO modification-induced exposure or creation of a relevant interaction surface may provide an elegant explanation for the biochemical change. Accordingly, the SUMOylation-promoted N1ICD interaction may result in a complex choreography by facilitating the recruitment of other components in the same transcriptional complex in a context-dependent pattern.
SUMOylated N1ICD was found to have greater stability. On the contrary, the SUMOylation-deficient mutant displayed attenuated protein stability. SUMO1 shares ≈18% homology and a similar 3-dimensional structure with ubiquitin. However, SUMOylation usually does not trigger proteolysis of a conjugated protein but mediates an abundance of substrate via interplay with the ubiquitin–proteasome system.30 It is well established that ubiquitin-dependent proteasomal degradation mediates NOTCH processing and NICD accumulation,28 but evidence for an exact antagonism between SUMOylation and ubiquitination on NOTCH levels remains under investigation. Additionally, SUMOylation shares at least one common lysine residue (K2150) with acetylated NICD, which is based on the 3 major SUMOylation sites identified in our study.17 NICD acetylation and SUMOylation play similar roles in the maintenance of N1ICD stability and promotion of NOTCH signature signaling in spite of the fact that they are within different molecular frameworks. Therefore, it is worthwhile to determine the crosstalk between SUMOylation and acetylation of NOTCH in future studies.
Biological Significance of SUMOylation on NOTCH Response in Angiogenesis
Transmembrane receptor proteins in ECs are a predominant part of the communication system for endothelial growth and homeostasis. Their molecular flexibility in response to angiogenic stimuli permits fast adaptation to the cellular microenvironment to promote angiogenesis. Protein SUMOylation predominantly occurs in nuclei. However, many cell membrane proteins were identified as SUMO modification substrates in a highly dynamic but specific pattern, which suggests that the SUMOylation system could be well suited to managing and fine-tuning the angiogenic ligand–receptor network.31,32 Thus far, little is known about SUMOylation of endothelial receptor proteins and how it impacts their function. Our current study reveals the significant involvement of SUMOylation in endothelial NOTCH functions during angiogenesis. The underlying biological consequences apply to both NOTCH cleavage and activation of NOTCH signaling.
The ligand-bound NOTCH receptors require successive proteolytic cleavage across the cell membrane to trigger NOTCH signaling in ECs during angiogenesis. Importantly, cleavage of S2 by ADAM (a disintegrin and a metalloprotease) metalloproteinases and γ-secretase cleavage of S3 result in NICD release from the cell membrane, which activates intracellular NOTCH signaling.28,33 The receptor processing delineates the framework for initiation of the NOTCH cascade, but the regulatory machinery for converting the molecular information into proper biological output is still unclear. Our study identified SENP1-regulated SUMOylation as an essential requirement for the NOTCH cleavage process in angiogenic ECs. The positive regulation of Dll4 in NOTCH SUMOylation indicates an intrinsic modulatory mechanism of Dll4-activated NOTCH cleavage and signaling. Ubiquitination, which targets N1ICD for degradation within recycling endosomes, contrasts with SUMO conjugation to the NOTCH C terminus that promotes N1ICD turnover and ensures its stability. We further demonstrate that intracellular N1ICD SUMOylation enhances the intensity and duration of NOTCH target gene expression, which is a result of augmented assembly of the cotranscriptional complex. Thus, SUMOylation of NOTCH functions positively on the spatiotemporal regulation of the NOTCH responses for EC behavior. Moreover, the suppressive effect of hypoxia on NOTCH signaling and its required N1ICD SUMOylation might be new contribution to the regulatory mechanism for hypoxia-induced endothelial sprouting and angiogenesis. Consequently, our study provides an appealing approach for tailoring the linear transmission of NOTCH signaling to adjust ongoing EC growth and communication during angiogenesis. In view of the current finding that SENP1 associates with full or cleaved forms of NOTCH, we propose that SENP1 functions as an autonomous rheostat to reconcile NOTCH signaling scenarios in different spatial and temporal contexts in ECs. To our knowledge, our finding is the first demonstration of the critical role of the SUMO system in angiogenesis.
An understanding of the details of SUMOylation regulation of the dynamic fluctuations of endothelial NOTCH responses should be of great importance given the highly dynamic characteristic of the SUMOylation procedure. The initial focus could be on the effect of NOTCH SUMOylation on the γ-secretase complex composition and its subcellular localization. This event influences the rapid-turnover of NICD in response to NOTCH activation. On the contrary, we also observed that SENP1 mediates HES1 deSUMOylation in ECs (Online Figure VIII), based on a previous report.34 N1ICD SUMOylation occurs in the cytosol or on the membrane, whereas HES1 SUMOylation may occur in the nucleus; therefore, the synergistic modulation of SENP1 in NOTCH signaling–controlled endothelial growth is worthy of investigation. Moreover, there is little knowledge about the cooperation of SUMOylation with other post-translational modifications, such as ubiquitination and phosphorylation, to manipulate the efficiency of NOTCH signaling.
Novel Modulatory Machinery of NOTCH-VEGFR Signaling Interaction
The NOTCH pathway has been identified as a prominent negative modulator of angiogenesis, which counteracts VEGFR signaling in ECs. However, the precise interacting mechanism is not completely understood. VEGF-stimulated VEGFR2 and VEGFR3 signaling potently promote angiogenesis and vascular development, whereas VEGFR1 negatively regulates signaling by trapping VEGF with high ligand affinity.2,8,10,35,36 Our study shows that over-SUMOylation of NOTCH leads to direct suppression of VEGFR2 and 3 activation in EC, which accounts for the dysfunction of EC growth and new vessel formation. A potential explanation is that it forms another link between endothelial VEGFR and NOTCH pathways, which would indicate the importance of crosstalk at a post-translational level. Comprehensively, the effect of NOTCH SUMOylation spans endothelial VEGFR1-3 by targeting VEGFR expression and its corresponding signal transduction. On the contrary, the positive regulation of Dll4 and negative regulation of hypoxia in NOTCH SUMOylation in ECs may indicate another interaction mode between Dll4-NOTCH signaling and hypoxia-VEGF/VEGFR signaling in coordination of angiogenesis. Therefore, we speculate a novel fine-tuned mechanism for the VEGFR-NOTCH balance in angiogenesis via SUMOylation or deSUMOylation of NOTCH. The direct effect of NOTCH signaling on VEGFR2 expression is via HEY1/HEY2/HEYL binding to the VEGFR2/VEGFR3 promoter.37 It will be interesting to investigate whether SENP1 synergistically regulates other NOTCH pathway components during different stages of angiogenesis.
We thank Dr Adolfo Ferrando (Columbia University) for providing Notch1 full-length construct, Dr R. Adams (Max Planck Institute, Munster) for Cdh5-CreERT2 mouse, and Dr Xuri Li (Sun Yat-sen University, China) for helpful discussion of the aritcle.
Sources of Funding
The present study was supported by the National Natural Science Foundation of China (grant numbers 81422005, 81270357, and 31470057), the Zhejiang Provincial Natural Science Foundation of China (grant number LR14H020002), the Fundamental Research Funds for the Central Universities to L. Yu, China Postdoctoral Science Foundation (grant number 508000-X91402) to X. Zhu, and National Natural Science Foundation of China (grant number 81600354) to C. Qiu. This work was partly supported by National Key Research and Development Program of China (2016YFC1300600), National Natural Science Foundation of China (number 91539110) to W. Min, R01 HL109420, R01 HL115148, and CT Stem Cell Innovation Award (Established Investigator Grant) 14-SC B-YALE-17 to W. Min.
In June 2017, the average time from submission to first decision for all original research papers submitted to Circulation Research was 12.45 days.
The online-only Data Supplement is available with this article at http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA.117.310696/-/DC1.
- Nonstandard Abbreviations and Acronyms
- Delta-like 4
- endothelial cell
- human microvascular endothelial cell
- human umbilical vein endothelial cell
- NOTCH1 intracellular domain
- sentrin-specific protease
- small ubiquitin-like modifier
- vascular endothelial growth factor
- Received January 29, 2017.
- Revision received July 22, 2017.
- Accepted July 28, 2017.
- © 2017 American Heart Association, Inc.
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- Heisig J,
- Weber D,
- Englberger E,
- Winkler A,
- Kneitz S,
- Sung WK,
- Wolf E,
- Eilers M,
- Wei CL,
- Gessler M
Novelty and Significance
What Is Known?
The NOTCH (neurogenic locus notch homolog protein) pathway is the prominent negative regulator of endothelial sprouting and vascular growth.
Activation of NOTCH leads to its proteolytic cleavage, which releases its intracellular domain and activates transcription of downstream genes.
Post-translational modification of proteins by SUMOylation regulates gene expression, protein stability, chromatin structure, and signal transduction. It is reversed by the SENP (sentrin-specific protease).
What New Information Does This Article Contribute?
Endothelial SENP1 deficiency delays developmental angiogenesis in mice and elevates NOTCH signaling.
NOTCH1 SUMOyaltion is required for NOTCH1 cleavage at the cell membrane, maintenance of N1ICD protein stability, and cotranscriptional activity in the nucleus.
SENP1 functions as a critical intrinsic mediator of NOTCH1 SUMOylation to reconcile NOTCH signaling scenarios in endothelial cells.
NOTCH1 SUMOylation coordinates hypoxia-induced angiogenesis by facilitating NOTCH signaling, which, in turn, suppresses VEGFR (vascular endothelial growth factor receptor) signaling.
Angiogenesis is essential for embryonic development and tissue growth. NOTCH signaling plays a pivotal role in angiogenesis; however, the mechanisms that regulate NOTCH signaling are still obscure. Herein, we show that deletion of endothelial SENP1 (the major protease of the SUMO [post-translational small ubiquitin-like modifier] system) delays retinal vascularization by prolonging NOTCH1 signaling. Importantly, NOTCH1 SUMOylation is required for NOTCH signal activation to fine-tune endothelial growth and angiogenesis, in which SENP1 selectively functions as the key regulator of deSUMOylation. The current results reveal a novel mechanism for the VEGFR-NOTCH balance in angiogenesis via SUMOylation/deSUMOylation of NOTCH. They also indicate an additional mode of interaction between DLL4 (Delta-like 4)-NOTCH signaling and hypoxia-VEGF/VEGFR signaling in coordination of angiogenesis. These findings may be of significance in understanding other NOTCH-regulated biological events and could lead to the development of novel diagnostic and therapeutic strategies for vascular diseases and tumors.