Dual Deletion of Keap1 and Rbpjκ Genes in Liver Leads to Hepatomegaly and Hypercholesterolemia.

The hepatic deletion of Rbpjκ (RbpjF/F::AlbCre) in the mouse leads to exhibition of the Alagille syndrome phenotype during early postnatal liver development with hyperlipidemia and cholestasis due to attenuated disruption of NOTCH signaling. Given the roles of NRF2 signaling in the regulation of lipid metabolism and bile ductal formation, it was anticipated that these symptoms could be alleviated by enhancing NRF2 signaling in the RbpjF/F::AlbCre mouse by hepatic deletion of Keap1 in compound Keap1F/F::RbpjF/F::AlbCre mice. Unexpectedly, these mice developed higher hepatic and plasma cholesterol levels with more severe cholestatic liver damage during the pre-weaning period than in the RbpjF/F::AlbCre mice. In addition, hypercholesterolemia and hepatic damage were sustained throughout the growth period unlike in the RbpjF/F::AlbCre mouse. These enhanced abnormalities in lipid metabolism appear to be due to NRF2-dependent changes in gene expression related to cholesterol synthetic and subsequent bile acid production pathways. Notably, the hepatic expression of Cyp1A7 and Abcb11 genes involved in bile acid homeostasis was significantly reduced in Keap1F/F::RbpjF/F::AlbCre compared to RbpjF/F::AlbCre mice. The accumulation of liver cholesterol and the weakened capacity for bile excretion during the 3 pre-weaning weeks in the Keap1F/F::RbpjF/F::AlbCre mice may aggravate hepatocellular damage level caused by both excessive cholesterol and residual bile acid toxicity in hepatocytes. These results indicate that a tuned balance of NOTCH and NRF2 signaling is of biological importance for early liver development after birth.

NOTCH2 sensitizes the chondrocyte to the inflammatory response of tumor necrosis factor α.

Notch regulates the immune and inflammatory response and has been associated with the pathogenesis of osteoarthritis in humans and preclinical models of the disease. Notch2tm1.1Ecan mice harbor a NOTCH2 gain-of-function and are sensitized to osteoarthritis, but the mechanisms have not been explored. We examined the effects of tumor necrosis factor α (TNFα) in chondrocytes from Notch2tm1.1Ecan mice and found that NOTCH2 enhanced the effect of TNFα on Il6 and Il1b expression. Similar results were obtained in cells from a conditional model of NOTCH2 gain-of-function, Notch22.1Ecan mice, and following the expression of the NOTCH2 intracellular domain in vitro. Recombination signal-binding protein for immunoglobulin Kappa J region partners with the NOTCH2 intracellular domain to activate transcription; in the absence of Notch signaling it inhibits transcription, and Rbpj inactivation in chondrocytes resulted in Il6 induction. Although TNFα induced IL6 to a greater extent in the context of NOTCH2 activation, there was a concomitant inhibition of Notch target genes Hes1, Hey1, Hey2, and Heyl. Electrophoretic mobility shift assay demonstrated displacement of recombination signal-binding protein for immunoglobulin Kappa J region from DNA binding sites by TNFα explaining the increased Il6 expression and the concomitant decrease in Notch target genes. NOTCH2 enhanced the effect of TNFα on NF-κB signaling, and RNA-Seq revealed increased expression of pathways associated with inflammation and the phagosome in NOTCH2 overexpressing cells in the absence and presence of TNFα. Collectively, NOTCH2 has important interactions with TNFα resulting in the enhanced expression of Il6 and inflammatory pathways in chondrocytes.

RBPJ Deficiency Sensitizes Pancreatic Acinar Cells to KRAS-Mediated Pancreatic Intraepithelial Neoplasia Initiation.

BACKGROUND AND AIMS: Development of pancreatic ductal adenocarcinoma (PDAC) is a multistep process intensively studied; however, precocious diagnosis and effective therapy still remain unsatisfactory. The role for Notch signaling in PDAC has been discussed controversially, as both cancer-promoting and cancer-antagonizing functions have been described. Thus, an improved understanding of the underlying molecular mechanisms is necessary. Here, we focused on RBPJ, the receiving transcription factor in the Notch pathway, examined its expression pattern in PDAC, and characterized its function in mouse models of pancreatic cancer development and in the regeneration process after acute pancreatitis. METHODS: Conditional transgenic mouse models were used for functional analysis of RBPJ in the adult pancreas, initiation of PDAC precursor lesions, and pancreatic regeneration. Pancreata and primary acinar cells were tested for acinar-to-ductal metaplasia together with immunohistology and comprehensive transcriptional profiling by RNA sequencing. RESULTS: We identified reduced RBPJ expression in a subset of human PDAC specimens. Ptf1α-CreERT-driven depletion of RBPJ in transgenic mice revealed that its function is dispensable for the homeostasis and maintenance of adult acinar cells. However, primary RBPJ-deficient acinar cells underwent acinar-to-ductal differentiation in ex vivo. Importantly, oncogenic KRAS expression in the context of RBPJ deficiency facilitated the development of pancreatic intraepithelial neoplasia lesions with massive fibrotic stroma formation. Interestingly, RNA-sequencing data revealed a transcriptional profile associated with the cytokine/chemokine and extracellular matrix changes. In addition, lack of RBPJ delays the course of acute pancreatitis and critically impairs it in the context of KRASG12D expression. CONCLUSIONS: Our findings imply that downregulation of RBPJ in PDAC patients derepresses Notch targets and promotes KRAS-mediated pancreatic acinar cells transformation and desmoplasia development.

Endothelial Rbpj deletion normalizes Notch4-induced brain arteriovenous malformation in mice.

Upregulation of Notch signaling is associated with brain arteriovenous malformation (bAVM), a disease that lacks pharmacological treatments. Tetracycline (tet)-regulatable endothelial expression of constitutively active Notch4 (Notch4*tetEC) from birth induced bAVMs in 100% of mice by P16. To test whether targeting downstream signaling, while sustaining the causal Notch4*tetEC expression, induces AVM normalization, we deleted Rbpj, a mediator of Notch signaling, in endothelium from P16, by combining tet-repressible Notch4*tetEC with tamoxifen-inducible Rbpj deletion. Established pathologies, including AV connection diameter, AV shunting, vessel tortuosity, intracerebral hemorrhage, tissue hypoxia, life expectancy, and arterial marker expression were improved, compared with Notch4*tetEC mice without Rbpj deletion. Similarly, Rbpj deletion from P21 induced advanced bAVM regression. After complete AVM normalization induced by repression of Notch4*tetEC, virtually no bAVM relapsed, despite Notch4*tetEC re-expression in adults. Thus, inhibition of endothelial Rbpj halted Notch4*tetEC bAVM progression, normalized bAVM abnormalities, and restored microcirculation, providing proof of concept for targeting a downstream mediator to treat AVM pathologies despite a sustained causal molecular lesion.

The structure, binding and function of a Notch transcription complex involving RBPJ and the epigenetic reader protein L3MBTL3.

The Notch pathway transmits signals between neighboring cells to elicit downstream transcriptional programs. Notch is a major regulator of cell fate specification, proliferation, and apoptosis, such that aberrant signaling leads to a pleiotropy of human diseases, including developmental disorders and cancers. The pathway signals through the transcription factor CSL (RBPJ in mammals), which forms an activation complex with the intracellular domain of the Notch receptor and the coactivator Mastermind. CSL can also function as a transcriptional repressor by forming complexes with one of several different corepressor proteins, such as FHL1 or SHARP in mammals and Hairless in Drosophila. Recently, we identified L3MBTL3 as a bona fide RBPJ-binding corepressor that recruits the repressive lysine demethylase LSD1/KDM1A to Notch target genes. Here, we define the RBPJ-interacting domain of L3MBTL3 and report the 2.06 Å crystal structure of the RBPJ-L3MBTL3-DNA complex. The structure reveals that L3MBTL3 interacts with RBPJ via an unusual binding motif compared to other RBPJ binding partners, which we comprehensively analyze with a series of structure-based mutants. We also show that these disruptive mutations affect RBPJ and L3MBTL3 function in cells, providing further insights into Notch mediated transcriptional regulation.

Endothelial RBPJ Is Essential for the Education of Tumor-Associated Macrophages.

Epithelial ovarian cancer (EOC) is one of the most lethal gynecologic cancers worldwide. EOC cells educate tumor-associated macrophages (TAM) through CD44-mediated cholesterol depletion to generate an immunosuppressive tumor microenvironment (TME). In addition, tumor cells frequently activate Notch1 receptors on endothelial cells (EC) to facilitate metastasis. However, further work is required to establish whether the endothelium also influences the education of recruited monocytes. Here, we report that canonical Notch signaling through RBPJ in ECs is an important player in the education of TAMs and EOC progression. Deletion of Rbpj in the endothelium of adult mice reduced infiltration of monocyte-derived macrophages into the TME of EOC and prevented the acquisition of a typical TAM gene signature; this was associated with stronger cytotoxic activity of T cells and decreased tumor burden. Mechanistically, CXCL2 was identified as a novel Notch/RBPJ target gene that regulated the expression of CD44 on monocytes and subsequent cholesterol depletion of TAMs. Bioinformatic analysis of ovarian cancer patient data showed that increased CXCL2 expression is accompanied by higher expression of CD44 and TAM education. Together, these findings indicate that EOC cells induce the tumor endothelium to secrete CXCL2 to establish an immunosuppressive microenvironment. SIGNIFICANCE: Endothelial Notch signaling favors immunosuppression by increasing CXCL2 secretion to stimulate CD44 expression in macrophages, facilitating their education by tumor cells.

Notch-dependent and -independent functions of transcription factor RBPJ.

Signal transduction pathways often involve transcription factors that promote activation of defined target gene sets. The transcription factor RBPJ is the central player in Notch signaling and either forms an activator complex with the Notch intracellular domain (NICD) or a repressor complex with corepressors like KYOT2/FHL1. The balance between these two antagonizing RBPJ-complexes depends on the activation state of the Notch receptor regulated by cell-to-cell interaction, ligand binding and proteolytic cleavage events. Here, we depleted RBPJ in mature T-cells lacking active Notch signaling and performed RNA-Seq, ChIP-Seq and ATAC-seq analyses. RBPJ depletion leads to upregulation of many Notch target genes. Ectopic expression of NICD1 activates several Notch target genes and enhances RBPJ occupancy. Based on gene expression changes and RBPJ occupancy we define four different clusters, either RBPJ- and/or Notch-regulated genes. Importantly, we identify early (Hes1 and Hey1) and late Notch-responsive genes (IL2ra). Similarly, to RBPJ depletion, interfering with transcriptional repression by squelching with cofactor KYOT2/FHL1, leads to upregulation of Notch target genes. Taken together, RBPJ is not only an essential part of the Notch co-activator complex but also functions as a repressor in a Notch-independent manner.

Interaction between BEND5 and RBPJ suppresses breast cancer growth and metastasis via inhibiting Notch signaling.

High frequent metastasis is the major cause of breast cancer (BC) mortality among women. However, the molecular mechanisms underlying BC metastasis remain largely unknown. Here, we identified six hub BC metastasis driver genes (BEND5, HSD11B1, NEDD9, SAA2, SH2D2A and TNFSF4) through bioinformatics analysis, among which BEND5 is the most significant gene. Low BEND5 expression predicted advanced stage and shorter overall survival in BC patients. Functional experiments showed that BEND5 could suppress BC growth and metastasis in vitro and in vivo. Mechanistically, BEND5 inhibits Notch signaling via directly interacting with transcription factor RBPJ/CSL. BEN domain of BEND5 interacts with the N-terminal domain (NTD) domain of RBPJ, thus preventing mastermind like transcriptional coactivator (MAML) from forming a transcription activation complex with RBPJ. Our study provides a novel insight into regulatory mechanisms underlying Notch signaling and suggests that BEND5 may become a promising target for BC therapy.

Notch effector recombination signal binding protein for immunoglobulin kappa J signaling is required for the initiation of endometrial stromal cell decidualization†.

The Notch signaling pathway is required for reproductive success. This pathway activates its transcriptional effector, recombination signal binding protein for immunoglobulin kappa J (Rbpj), to induce transcription of its target genes. This signaling pathway is required for successful decidualization, implantation, and uterine repair following parturition. To identify the compartmental specific roles of the Notch signaling pathway in the establishment of pregnancy, we generated epithelial and decidual stromal cell specific knockouts of Rbpj utilizing lactoferrin iCre and Prl8A2 iCre, respectively. Both conditional knockout mouse models were fertile. The Rbpj epithelial knockout mice displayed 27% resorption sites at E15.5, but this did not significantly impact the number of live born pups compared with controls. In addition, the Rbpj epithelial knockout mice displayed increased estrogen signaling in their stromal compartment. Given that both mouse models exhibited fertility comparable to control animals, the epithelial and stromal specific nature of the iCre recombinases utilized, and previously published Rbpj total uterine knockout mouse models, we conclude that Notch effector Rbpj signaling is required at the initiation of pregnancy to support decidualization in stromal cells, but that Rbpj is not required in the epithelial compartment nor is it required for post-implantation pregnancy success.

RBP-J deficiency promoted the proliferation and differentiation of CD133-positive cells in both in vitro and in vivo studies.

Although Notch signalling pathway could control the proliferation and differentiation of neural stem cells (NSCs), it is largely unknown about the effect of Notch signalling pathway on the neurogenesis of CD133-positive cells. By using the primary cultured ependymal cells and the transgenic mouse, we found that CD133 immunoreactivity was exclusively localized in the ependymal layer of ventricles; moreover, most CD133-positive cells were co-labelled with Nestin. In addition, recombination signal binding protein J (RBP-J), a key nuclear effector of Notch signalling pathway, was highly active in CD133-positive cells. CD133-positive cells can differentiate into the immature and mature neurons; in particular, the number of CD133-positive cells differentiating into the immature and mature neurons was significantly increased following the deficiency or interference of RBP-J in vivo or in vitro. By using real-time qPCR and Western blot, we found that RBP-J and Hes1 were downregulated, whereas Notch1 was upregulated in the expression levels of mRNAs and proteins following the deficiency or interference of RBP-J. These results demonstrated RBP-J deficiency promoted the proliferation and differentiation of CD133-positive cells. Therefore, we speculated that RBP-J could maintain CD133-positive cells in the characteristics of NSCs possibly by regulating Notch1/RBP-J/Hes1 pathway. It will provide a novel molecular insight into the function of RBP-J as well as facilitate a future investigation of CD133-positive cells with respect to their potential application in neurodegenerative disorder.

Analyzing the Interaction of RBPJ with Mitotic Chromatin and Its Impact on Transcription Reactivation upon Mitotic Exit.

The sequence-specific transcription factor RBPJ, also known as CSL (CBF1, Su(H), Lag1), is an evolutionarily conserved protein that mediates Notch signaling to guide cell fates. When cells enter mitosis, DNA is condensed and most transcription factors dissociate from chromatin; however, a few, select transcription factors, termed bookmarking factors, remain associated. These mitotic chromatin-bound factors are believed to play important roles in maintaining cell fates through cell division. RBPJ is one such factor that remains mitotic chromatin associated and therefore could function as a bookmarking factor. Here, we describe how to obtain highly purified mitotic cells from the mouse embryonal carcinoma cell line F9, perform chromatin immunoprecipitation with mitotic cells, and measure the first run of RNA synthesis upon mitotic exit. These methods serve as basis to understand the roles of mitotic bookmarking by RBPJ in propagating Notch signals through cell division.

RITA1 drives the growth of bladder cancer cells by recruiting TRIM25 to facilitate the proteasomal degradation of RBPJ.

Bladder cancer (BC) is one of the most prevalent malignancies worldwide, but it lacks effective targeted therapy due to its elusive molecular mechanism. Therefore, it is important to further investigate the molecular mechanisms that mediate BC progression. By performing a tumor tissue-based gene microarray and shRNA library screening, we found that recombination signal binding protein for immunoglobulin kappa J region (RBPJ) interacting and tubulin associated 1 (RITA1) is crucial for the growth of BC cells. Moreover, RITA1 is aberrantly highly expressed in BC tissues and is also correlated with poor prognosis in patients with BC. Mechanistically, we determined that RITA1 recruits tripartite motif containing 25 (TRIM25) to ubiquitinate RBPJ to accelerate its degradation via proteasome, which leads to the transcriptional inhibition of Notch1 downstream targets. Our results suggest that aberrant high expression of RITA1 drives the growth of BC cells via the RITA1/TRIM25/RBPJ axis and RITA1 may serve as a promising therapeutic target for BC.

Notch signaling is a novel regulator of visceral smooth muscle cell differentiation in the murine ureter.

The contractile phenotype of smooth muscle cells (SMCs) is transcriptionally controlled by a complex of the DNA-binding protein SRF and the transcriptional co-activator MYOCD. The pathways that activate expression of Myocd and of SMC structural genes in mesenchymal progenitors are diverse, reflecting different intrinsic and extrinsic signaling inputs. Taking the ureter as a model, we analyzed whether Notch signaling, a pathway previously implicated in vascular SMC development, also affects visceral SMC differentiation. We show that mice with a conditional deletion of the unique Notch mediator RBPJ in the undifferentiated ureteric mesenchyme exhibit altered ureter peristalsis with a delayed onset, and decreased contraction frequency and intensity at fetal stages. They also develop hydroureter 2 weeks after birth. Notch signaling is required for precise temporal activation of Myocd expression and, independently, for expression of a group of late SMC structural genes. Based on additional expression analyses, we suggest that a mesenchymal JAG1-NOTCH2/NOTCH3 module regulates visceral SMC differentiation in the ureter in a biphasic and bimodal manner, and that its molecular function differs from that in the vascular system.

Noncanonical Notch signals have opposing roles during cardiac development.

The Notch pathway is an ancient intercellular signaling system with crucial roles in numerous cell-fate decision processes across species. While the canonical pathway is activated by ligand-induced cleavage and nuclear localization of membrane-bound Notch, Notch can also exert its activity in a ligand/transcription-independent fashion, which is conserved in Drosophila, Xenopus, and mammals. However, the noncanonical role remains poorly understood in in vivo processes. Here we show that increased levels of the Notch intracellular domain (NICD) in the early mesoderm inhibit heart development, potentially through impaired induction of the second heart field (SHF), independently of the transcriptional effector RBP-J. Similarly, inhibiting Notch cleavage, shown to increase noncanonical Notch activity, suppressed SHF induction in embryonic stem cell (ESC)-derived mesodermal cells. In contrast, NICD overexpression in late cardiac progenitor cells lacking RBP-J resulted in an increase in heart size. Our study suggests that noncanonical Notch signaling has stage-specific roles during cardiac development.

NACK and INTEGRATOR act coordinately to activate Notch-mediated transcription in tumorigenesis.

BACKGROUND: Notch signaling drives many aspects of neoplastic phenotype. Here, we report that the Integrator complex (INT) is a new component of the Notch transcriptional supercomplex. Together with Notch Activation Complex Kinase (NACK), INT activates Notch1 target genes by driving RNA polymerase II (RNAPII)-dependent transcription, leading to tumorigenesis. METHODS: Size exclusion chromatography and CBF-1/RBPJ/Suppressor of Hairless/Lag-1 (CSL)-DNA affinity fast protein liquid chromatography (FPLC) was used to purify Notch/CSL-dependent complexes for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Chromatin immunoprecipitation (ChIP) and quantitative polymerase chain reaction (qPCR) were performed to investigate transcriptional regulation of Notch target genes. Transfection of Notch Ternary Complex components into HEK293T cells was used as a recapitulation assay to study Notch-mediated transcriptional mechanisms. Gene knockdown was achieved via RNA interference and the effects of protein depletion on esophageal adenocarcinoma (EAC) proliferation were determined via a colony formation assay and murine xenografts. Western blotting was used to examine expression of INT subunits in EAC cells and evaluate apoptotic proteins upon INT subunit 11 knockdown (INTS11 KD). Gene KD effects were further explored via flow cytometry. RESULTS: We identified the INT complex as part of the Notch transcriptional supercomplex. INT, together with NACK, activates Notch-mediated transcription. While NACK is required for the recruitment of RNAPII to a Notch-dependent promoter, the INT complex is essential for RNAPII phosphorylated at serine 5 (RNAPII-S5P), leading to transcriptional activation. Furthermore, INT subunits are overexpressed in EAC cells and INTS11 KD results in G2/M cell cycle arrest, apoptosis, and cell growth arrest in EAC. CONCLUSIONS: This study identifies the INT complex as a novel co-factor in Notch-mediated transcription that together with NACK activates Notch target genes and leads to cancer cell proliferation. Video abstract.

Functional genomics atlas of synovial fibroblasts defining rheumatoid arthritis heritability.

BACKGROUND: Genome-wide association studies have reported more than 100 risk loci for rheumatoid arthritis (RA). These loci are shown to be enriched in immune cell-specific enhancers, but the analysis so far has excluded stromal cells, such as synovial fibroblasts (FLS), despite their crucial involvement in the pathogenesis of RA. Here we integrate DNA architecture, 3D chromatin interactions, DNA accessibility, and gene expression in FLS, B cells, and T cells with genetic fine mapping of RA loci. RESULTS: We identify putative causal variants, enhancers, genes, and cell types for 30-60% of RA loci and demonstrate that FLS account for up to 24% of RA heritability. TNF stimulation of FLS alters the organization of topologically associating domains, chromatin state, and the expression of putative causal genes such as TNFAIP3 and IFNAR1. Several putative causal genes constitute RA-relevant functional networks in FLS with roles in cellular proliferation and activation. Finally, we demonstrate that risk variants can have joint-specific effects on target gene expression in RA FLS, which may contribute to the development of the characteristic pattern of joint involvement in RA. CONCLUSION: Overall, our research provides the first direct evidence for a causal role of FLS in the genetic susceptibility for RA accounting for up to a quarter of RA heritability.

Fibrous Caps in Atherosclerosis Form by Notch-Dependent Mechanisms Common to Arterial Media Development.

Objective: Atheromatous fibrous caps are produced by smooth muscle cells (SMCs) that are recruited to the subendothelial space. We tested whether the recruitment mechanisms are the same as in embryonic artery development, which relies prominently on Notch signaling to form the subendothelial medial SMC layers. Approach and Results: Notch elements were expressed in regions of fibrous cap in human and mouse plaques. To assess the causal role of Notch signaling in cap formation, we studied atherosclerosis in mice where the Notch pathway was inactivated in SMCs by conditional knockout of the essential effector transcription factor RBPJ (recombination signal-binding protein for immunoglobulin kappa J region). The recruitment of cap SMCs was significantly reduced without major effects on plaque size. Lineage tracing revealed the accumulation of SMC-derived plaque cells in the cap region was unaltered but that Notch-defective cells failed to re-acquire the SMC phenotype in the cap. Conversely, to analyze whether the loss of Notch signaling is required for SMC-derived cells to accumulate in atherogenesis, we studied atherosclerosis in mice with constitutive activation of Notch signaling in SMCs achieved by conditional expression of the Notch intracellular domain. Forced Notch signaling inhibited the ability of medial SMCs to contribute to plaque cells, including both cap SMCs and osteochondrogenic cells, and significantly reduced atherosclerosis development. Conclusions: Sequential loss and gain of Notch signaling is needed to build the cap SMC population. The shared mechanisms with embryonic arterial media assembly suggest that the cap forms as a neo-media that restores the connection between endothelium and subendothelial SMCs, transiently disrupted in early atherogenesis.

Identification and evolution of transcription factors RHR gene family (NFAT and RBPJ) involving lamprey (Lethenteron reissneri) innate immunity.

Nuclear factor of activated T cells (NFAT) and recombination signal binding protein (RBP) belong to the family of Rel homology region (RHR) transcription factors which regulate the expression of genes involved in different aspects of the immune response. To gain insights into the evolution and characterisation of RHR genes in lampreys, a jawless vertebrate, four RHR genes, including nuclear factor of activated T cells (NFAT) and recombination signal binding protein for immunoglobulin kappa J region (RBPJ), have been identified and cloned from the lamprey (Lethenteron reissneri) database. Evolutionary relationships of NFAT and RBPJ genes among different species were determined through molecular phylogenetic analysis. Motif, genetic structure, and tertiary structure analyses showed that NFATs and RBPJ are conserved and contain RHD and IPT domains. Moreover, synteny analysis showed that the neighbourhood genes of Lr-NFATs and Lr-RBPJ have undergone significant changes compared to jawed vertebrates. Real-time quantitative results demonstrated that the RHR gene family plays a significant role in immune defence. This study provides a new understanding of the origin and evolution of the RHR gene family in different species.

Sox9 and Rbpj differentially regulate endothelial to mesenchymal transition and wound scarring in murine endovascular progenitors.

Endothelial to mesenchymal transition (EndMT) is a leading cause of fibrosis and disease, however its mechanism has yet to be elucidated. The endothelium possesses a profound regenerative capacity to adapt and reorganize that is attributed to a population of vessel-resident endovascular progenitors (EVP) governing an endothelial hierarchy. Here, using fate analysis, we show that two transcription factors SOX9 and RBPJ specifically affect the murine EVP numbers and regulate lineage specification. Conditional knock-out of Sox9 from the vasculature (Sox9fl/fl/Cdh5-CreER RosaYFP) depletes EVP while enhancing Rbpj expression and canonical Notch signalling. Additionally, skin wound analysis from Sox9 conditional knock-out mice demonstrates a significant reduction in pathological EndMT resulting in reduced scar area. The converse is observed with Rbpj conditionally knocked-out from the murine vasculature (Rbpjfl/fl/Cdh5-CreER RosaYFP) or inhibition of Notch signaling in human endothelial colony forming cells, resulting in enhanced Sox9 and EndMT related gene (Snail, Slug, Twist1, Twist2, TGF-ß) expression. Similarly, increased endothelial hedgehog signaling (Ptch1fl/fl/Cdh5-CreER RosaYFP), that upregulates the expression of Sox9 in cells undergoing pathological EndMT, also results in excess fibrosis. Endothelial cells transitioning to a mesenchymal fate express increased Sox9, reduced Rbpj and enhanced EndMT. Importantly, using topical administration of siRNA against Sox9 on skin wounds can substantially reduce scar area by blocking pathological EndMT. Overall, here we report distinct fates of EVPs according to the relative expression of Rbpj or Notch signalling and Sox9, highlighting their potential plasticity and opening exciting avenues for more effective therapies in fibrotic diseases.

Inhibition of Wnt signalling by Notch via two distinct mechanisms.

Notch and Wnt are two essential signalling pathways that help to shape animals during development and to sustain adult tissue homeostasis. Although they are often active at the same time within a tissue, they typically have opposing effects on cell fate decisions. In fact, crosstalk between the two pathways is important in generating the great diversity of cell types that we find in metazoans. Several different mechanisms have been proposed that allow Notch to limit Wnt signalling, driving a Notch-ON/Wnt-OFF state. Here we explore these different mechanisms in human cells and demonstrate two distinct mechanisms by which Notch itself, can limit the transcriptional activity of ß-catenin. At the membrane, independently of DSL ligands, Notch1 can antagonise ß-catenin activity through an endocytic mechanism that requires its interaction with Deltex and sequesters ß-catenin into the membrane fraction. Within the nucleus, the intracellular domain of Notch1 can also limit ß-catenin induced transcription through the formation of a complex that requires its interaction with RBPjκ. We believe these mechanisms contribute to the robustness of cell-fate decisions by sharpening the distinction between opposing Notch/Wnt responses.