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.

Comprehensive genomic features indicative for Notch responsiveness.

Transcription factor RBPJ is the central component in Notch signal transduction and directly forms a coactivator complex together with the Notch intracellular domain (NICD). While RBPJ protein levels remain constant in most tissues, dynamic expression of Notch target genes varies depending on the given cell-type and the Notch activity state. To elucidate dynamic RBPJ binding genome-wide, we investigated RBPJ occupancy by ChIP-Seq. Surprisingly, only a small set of the total RBPJ sites show a dynamic binding behavior in response to Notch signaling. Compared to static RBPJ sites, dynamic sites differ in regard to their chromatin state, binding strength and enhancer positioning. Dynamic RBPJ sites are predominantly located distal to transcriptional start sites (TSSs), while most static sites are found in promoter-proximal regions. Importantly, gene responsiveness is preferentially associated with dynamic RBPJ binding sites and this static and dynamic binding behavior is repeatedly observed across different cell types and species. Based on the above findings we used a machine-learning algorithm to predict Notch responsiveness with high confidence in different cellular contexts. Our results strongly support the notion that the combination of binding strength and enhancer positioning are indicative of Notch responsiveness.

Hypoxic environment promotes angiogenesis and bone bridge formation by activating Notch/RBPJ signaling pathway in HUVECs.

After epiphyseal fracture, the epiphyseal plate is prone to ischemia and hypoxia, leading to the formation of bone bridge and deformity. However, the exact mechanism controlling the bone bridge formation remains unclear. Notch/RBPJ signaling axis has been indicated to regulate angiogenesis and osteogenic differentiation. Our study aims to investigate the mechanism of bone bridge formation after epiphyseal plate injury, and to provide a theoretical basis for new therapeutic approaches to prevent the bone bridge formation. The expression of DLL4 and RBPJ was significantly up-regulated in HUVECs after ischemia and hypoxia treatment. Notch/RBPJ pathway positively regulated the osteogenic differentiation of BMSCs. HUVECs can induce osteogenic differentiation of BMSCs under ischemia and hypoxia. Notch/RBPJ pathway is involved in the regulation of the trans-epiphyseal bridge formation. Notch/RBPJ in HUVECs is associated with osteogenic differentiation of BMSCs and may participate in the regulation of the bone bridge formation across the epiphyseal plate.

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.

Rbpj Deficiency Disrupts Vascular Remodeling via Abnormal Apelin and Cdc42 (Cell Division Cycle 42) Activity in Brain Arteriovenous Malformation.

BACKGROUND: Brain arteriovenous malformations (bAVM) are characterized by enlarged blood vessels, which direct blood through arteriovenous shunts, bypassing the artery-capillary-vein network and disrupting blood flow. Clinically, bAVM treatments are invasive and not routinely applicable. There is critical need to understand mechanisms of bAVM pathologies and develop pharmacological therapies. METHODS: We used an in vivo mouse model of Rbpj-mediated bAVM, which develops pathologies in the early postnatal period and an siRNA in vitro system to knockdown RBPJ in human brain microvascular endothelial cells (ECs). To understand molecular events regulated by endothelial Rbpj, we conducted RNA-Seq and chromatin immunoprecipitation-Seq analyses from isolated brain ECs. RESULTS: Rbpj-deficient (mutant) brain ECs acquired abnormally rounded shape (with no change to cell area), altered basement membrane dynamics, and increased endothelial cell density along arteriovenous shunts, compared to controls, suggesting impaired remodeling of neonatal brain vasculature. Consistent with impaired endothelial cell dynamics, we found increased Cdc42 (cell division cycle 42) activity in isolated mutant ECs, suggesting that Rbpj regulates small GTPase (guanosine triphosphate hydrolase)-mediated cellular functions in brain ECs. siRNA-treated, RBPJ-deficient human brain ECs displayed increased Cdc42 activity, disrupted cell polarity and focal adhesion properties, and impaired migration in vitro. RNA-Seq analysis from isolated brain ECs identified differentially expressed genes in mutants, including Apelin, which encodes a ligand for G protein-coupled receptor signaling known to influence small GTPase activity. Chromatin immunoprecipitation-Seq analysis revealed chromatin loci occupied by Rbpj in brain ECs that corresponded to G-protein and Apelin signaling molecules. In vivo administration of a competitive peptide antagonist against the Apelin receptor (Aplnr/Apj) attenuated Cdc42 activity and restored endothelial cell morphology and arteriovenous connection diameter in Rbpj-mutant brain vessels. CONCLUSIONS: Our data suggest that endothelial Rbpj promotes rearrangement of brain ECs during cerebrovascular remodeling, through Apelin/Apj-mediated small GTPase activity, and prevents bAVM. By inhibiting Apelin/Apj signaling in vivo, we demonstrated pharmacological prevention of Rbpj-mediated bAVM.

Targeted inhibition of RBPJ transcription complex alleviates the exhaustion of CD8+ T cells in hepatocellular carcinoma.

Impaired function of CD8+ T cells in hepatocellular carcinoma (HCC) is an important reason for acquired resistance. Compared with single-target inhibitors, small-molecule compounds that could both inhibit tumor cells and alleviate T cell exhaustion are more promising to reduce resistance. In this study, we screened immunosuppressive targets in HCC by combining cancer-immunity cycle score with weighted gene co-expression network and system analysis. Through in vitro and in vivo validation experiments, we found that one of the screened molecules, recombination signal binding protein for immunoglobulin kappa J region (RBPJ), was negatively correlated with CD8+ T cell mediated killing function. More importantly, its transcription complex inhibitor RIN1 not only inhibited the malignant biological behaviors of HCC cells by inhibiting mTOR pathway, but also reduced the expression of PD-L1 and L-kynurenine synthesis in HCC cells, thus alleviating T cell exhaustion. Meanwhile, the combination of RIN1 and anti-PD-1/PD-L1 antibodies could further activate CD8+ T cells. In short, RBPJ is an important factor regulating the function of T cells. Target inhibition of RBPJ transcription complex by small molecule compound may be a new strategy for immunotherapy of HCC.

Endothelial Rbpj Is Required for Cerebellar Morphogenesis and Motor Control in the Early Postnatal Mouse Brain.

Intercellular influences are necessary for coordinated development and function of vascular and neural components in the brain. In the early postnatal period after birth, the mammalian cerebellum undergoes extensive morphogenesis - developing its characteristic lobules, organizing its diverse cell types into defined cellular layers, and establishing neural circuits that support cerebellar function, such as coordinated movement. In parallel, the cerebellar vasculature undergoes extensive postnatal growth and maturation, keeping pace with the expanding neural compartment. Endothelial deletion of Rbpj leads to neurovascular abnormalities in mice, including arteriovenous (AV) shunts that supplant capillaries and instead direct high-pressure/high-flow arterial blood directly to veins. Gross and histopathological cerebellar abnormalities, associated with these Rbpj-mediated brain AV malformations (AVMs), led to our hypothesis that early postnatal morphogenesis and lamination of cerebellum was perturbed in mice harboring endothelial Rbpj deficiency from birth. Here, we show that endothelial Rbpj-mutant mice developed enlarged vascular malformations on the cerebellar surface, by 2-week post-Rbpj deletion. In addition, outgrowth of cerebellar lobules was impaired through decreased cell proliferation, but not increased apoptosis, in the external granule layer. Molecular layer thickness was reduced, and the Purkinje layer was affected, by decreased Purkinje cell number, primary dendrite length, and dendritic arbor density. Endothelial deletion of Rbpj also led to impaired motor behaviors, consistent with abnormal cerebellar morphogenesis and lamination. Thus, our data suggest that Rbpj is required, in early postnatal vascular endothelium, to ensure proper cerebellar outgrowth, morphogenesis, and function in mice.

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.

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.

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.

Exosomal hsa_circ_0004658 derived from RBPJ overexpressed-macrophages inhibits hepatocellular carcinoma progression via miR-499b-5p/JAM3.

Macrophage-derived exosomes (Mφ-Exo) have multidimensional involvement in tumor initiation, progression, and metastasis, but their regulation in hepatocellular carcinoma (HCC) is not fully understood. RBPJ has been implicated in macrophage activation and plasticity. In this study we assess the role of exosomes derived from RBPJ-overexpressed macrophages (RBPJ+/+ Mφ-Exo) in HCC. The circular RNA (circRNA) profiles in RBPJ+/+ Mφ-Exo and THP-1-like macrophages (WT Mφ)-Exo was evaluated using circRNA microarray. CCK-8, Transwell, and flow cytometry analyses were used to evaluate the function of Mφ-Exo-circRNA on HCC cells. Luciferase reporter assays, RNA immunoprecipitation, and Pearson's correlation analysis were used to confirm interactions. A nude mouse xenograft model was used to further analyze the functional significance of Mφ-Exo-cirRNA in vivo. Our results shown that hsa_circ_0004658 is upregulated in RBPJ+/+ Mφ-Exo compared to WT Mφ-Exo. RBPJ+/+ Mφ-Exo and hsa_circ_0004658 inhibits proliferation and promotes apoptosis in HCC cells, whereas hsa_circ_0004658 knockdown stimulated cell proliferation and migration but restrained apoptosis in vitro and promotes tumor growth in vivo. The effects of RBPJ+/+ Mφ-Exo on HCC cells can be reversed by the hsa_circ_0004658 knockdown. Mechanistic investigations revealed that hsa_circ_0004658 acts as a ceRNA of miR-499b-5p, resulting in the de-repression of JAM3. These results indicate that exosome circRNAs secreted from RBPJ+/+ Mφ inhibits tumor progression through the hsa_circ_0004658/miR-499b-5p/JAM3 pathway and hsa_circ_0004658 may be a diagnostic biomarker and potential target for HCC therapy.

Colon cancer cells secreted CXCL11 via RBP-Jκ to facilitated tumour-associated macrophage-induced cancer metastasis.

Metastasis is the main cause of colon cancer-related deaths. RBP-Jκ is involved in colon cancer development, but its function in colon cancer metastasis is still unclear. Tumour-associated macrophages are the main cell components in tumour microenvironments. Here, we aimed to determine the function of RBP-Jκ in colon cancer metastasis and its underlying mechanisms for modulating interactions between colon cancer cell and tumour-associated macrophages. Through bioinformation analysis, we found that RBP-Jκ was overexpressed in colon cancer tissues and associated with advanced colon cancer phenotypes, macrophage infiltration and shorter survival overall as confirmed by our patients' data. And our patients' data show that RBP-Jκ expression and tumour-associated macrophages infiltration are associated with colon cancer metastasis and are independent prognostic factors for colon cancer patients. Tumour-associated macrophages induced colon cancer cell migration, invasion and epithelial-mesenchymal transition through secreting TGF-ß1. Colon cancer cells with high RBP-Jκ expression induced the expression of TGF-ß1 in tumour-associated macrophages by secreting CXCL11. Our research revealed that colon cancer cells secreted CXCL11 via overexpression of RBP-Jκ to enhance the expression of TGF-ß1 in tumour-associated macrophages to further promote metastasis of colon cancer cells.

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.

RBP-J promotes cell growth and metastasis through regulating miR-182-5p-mediated Tiam1/Rac1/p38 MAPK axis in colorectal cancer.

BACKGROUND: RBP-J is involved in number of cellular processes. However, the potential mechanisms of RBP-J on colorectal cancer (CRC) development have not been clearly defined. In this study, we aimed to investigate the role and molecular mechanism of RBP-J in CRC. METHODS: The expression levels of RBP-J and Tiam1 in CRC tissues and cells were evaluated by RT-qPCR or western blot. RBP-J was knocked down with sh-RBP-J or overexpressed by pcDNA3.1-RBP-J in CRC cells. Cell proliferation, migration and invasion abilities were analyzed by MTT, wound healing, and transwell assay, respectively. CHIP-qPCR, RIP and dual luciferase reporter assays were performed to confirm the interaction between miR-182-5p and RBP-J or Tiam1. Expression levels of p-p38 MAPK, p38 MAPK, Slug-1, Twist1 and MMP-9 were analyzed by western blot. G-LISA test was used to detect Rac1 activity. RESULTS: Our results showed that the expression of RBP-J and Tiam1 was significantly up-regulated in CRC tissues and cells. RBP-J overexpression promoted proliferation, migration and invasion of CRC cells. Moreover, RBP-J was found to directly target miR-182-5p promoter and positively regulate the Tiam1/Rac1/p38 MAPK signaling pathway in CRC cells. It was also proved that miR-182-5p can bind Tiam1 directly. Furthermore, experiments revealed that RBP-J could promote CRC cell proliferation, migration and invasion via miR-182-5p-mediated Tiam1/Rac1/p38 MAPK axis. In addition, knockdown of RBP-J reduced tumor growth and metastasis in CRC mice. CONCLUSION: RBP-J regulates CRC cell growth and metastasis through miR-182-5p mediated Tiam1/Rac1/p38 MAPK signaling pathway, implying potential novel therapeutic targets for CRC patients.

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.