Akt3 activation by R-Ras in an endothelial cell enforces quiescence and barrier stability of neighboring endothelial cells via Jagged1.

Communication between adjacent endothelial cells is important for the homeostasis of blood vessels. We show that quiescent endothelial cells use Jagged1 to instruct neighboring endothelial cells to assume a quiescent phenotype and secure the endothelial barrier. This phenotype enforcement by neighboring cells is operated by R-Ras through activation of Akt3, which results in upregulation of a Notch ligand Jagged1 and consequential upregulation of Notch target genes, such as UNC5B, and VE-cadherin accumulation in the neighboring cells. These signaling events lead to the stable interaction between neighboring endothelial cells to continue to fortify juxtacrine signaling via Jagged1-Notch. This mode of intercellular signaling provides a positive feedback regulation of endothelial cell-cell interactions and cellular quiescence required for the stabilization of the endothelium.

Identifications of three novel alleles of Serrate in Drosophila.

The Notch signaling pathway, an evolutionarily highly conserved pathway, participates in various essential physiological processes in organisms. Activation of Notch signaling in the canonical manner requires the combination of ligand and receptor. There are two ligands of Notch in Drosophila: Delta (Dl) and Serrate (Ser). A mutation mf157 is identified for causing nicks of fly wings in genetic analysis from a mutant library (unpublished) that was established previously. Immunofluorescent staining illustrates that mf157 represses the expression of Cut and Wingless (Wg), the targets of Notch signaling. MARCM cloning analysis reveals that mf157 functions at the same level or the upstream of ligands of Notch in signaling sending cells. Sequencing demonstrates that mf157 is a novel allele of the Ser gene. Subsequently, mf553 and mf167 are also identified as new alleles of Ser from our library. Furthermore, the complementary assays and the examination of transcripts confirm the sequencing results. Besides, the repressed phenotypes of Notch signaling were reverted by transposon excision experiments of mf157. In conclusion, we identify three fresh alleles of Ser. Our works supply additional genetic resources for further study of functions of Ser and Notch signaling regulation.

Sacubitril/valsartan inhibits the proliferation of vascular smooth muscle cells through notch signaling and ERK1/2 pathway.

AIMS: To explore the role and mechanism of Notch signaling and ERK1/2 pathway in the inhibitory effect of sacubitril/valsartan on the proliferation of vascular smooth muscle cells (VSMCs). MAIN METHODS: Human aortic vascular smooth muscle cells (HA-VSMCs) were cultured in vitro. The proliferating VSMCs were divided into three groups as control group, Ang II group and Ang II + sacubitril/valsartan group. Cell proliferation and migration were detected by CCK8 and scratch test respectively. The mRNA and protein expression of PCNA, MMP-9, Notch1 and Jagged-1 were detected by qRT-PCR and Western blot respectively. The p-ERK1/2 expression was detected by Western blot. KEY FINDINGS: Compared with the control group, proliferation and migration of VSMCs and the expression of PCNA, MMP-9, Notch1, Jagged-1 and p-ERK1/2 was increased in Ang II group. Sacubitril/valsartan significantly reduced the proliferation and migration. Additionally, pretreatment with sacubitril/valsartan reduced the PCNA, MMP-9, Notch1, Jagged-1 and p-ERK1/2 expression.

Jagged-mediated development and disease: Mechanistic insights and therapeutic implications for Alagille syndrome.

Notch signaling controls multiple aspects of embryonic development and adult homeostasis. Alagille syndrome is usually caused by a single mutation in the jagged canonical Notch ligand 1 (JAG1), and manifests with liver disease and cardiovascular symptoms that are a direct consequence of JAG1 haploinsufficiency. Recent insights into Jag1/Notch-controlled developmental and homeostatic processes explain how pathology develops in the hepatic and cardiovascular systems and, together with recent elucidation of mechanisms modulating liver regeneration, provide a basis for therapeutic efforts. Importantly, disease presentation can be regulated by genetic modifiers, that may also be therapeutically leverageable. Here, we summarize recent insights into how Jag1 controls processes of relevance to Alagille syndrome, focused on Jag1/Notch functions in hepatic and cardiovascular development and homeostasis.

Soluble and multivalent Jag1 DNA origami nanopatterns activate Notch without pulling force.

The Notch signaling pathway has fundamental roles in embryonic development and in the nervous system. The current model of receptor activation involves initiation via a force-induced conformational change. Here, we define conditions that reveal pulling force-independent Notch activation using soluble multivalent constructs. We treat neuroepithelial stem-like cells with molecularly precise ligand nanopatterns displayed from solution using DNA origami. Notch signaling follows with clusters of Jag1, and with chimeric structures where most Jag1 proteins are replaced by other binders not targeting Notch. Our data rule out several confounding factors and suggest a model where Jag1 activates Notch upon prolonged binding without appearing to need a pulling force. These findings reveal a distinct mode of activation of Notch and lay the foundation for the development of soluble agonists.

Novel JAG1 variants leading to Alagille syndrome in two Chinese cases.

Alagille Syndrome (ALGS) is a complex genetic disorder characterized by cholestasis, congenital cardiac anomalies, and butterfly vertebrae. The variable phenotypic expression of ALGS can lead to challenges in accurately diagnosing affected infants, potentially resulting in misdiagnoses or underdiagnoses. This study highlights novel JAG1 gene mutations in two cases of ALGS. The first case with a novel p.Pro325Leufs*87 variant was diagnosed at 2 months of age and exhibited a favorable prognosis and an unexpected manifestation of congenital hypothyroidism. Before the age of 2, the second patient was incorrectly diagnosed with liver structural abnormalities, necessitating extensive treatment. In addition, he exhibited delays in language acquisition that may have been a result of SNAP25 haploinsufficiency. The identification of ALGS remains challenging, highlighting the importance of early detection and genetic testing for effective patient management. The variant p.Pro325Leufs*87 is distinct from reported variants linked to congenital hypothyroidism in ALGS patients, thereby further confirming the clinical and genetic complexity of ALGS. This emphasizes the critical need for individualized and innovative approaches to diagnosis and medical interventions, uniquely intended to address the complexity of this syndrome.

Endothelial Jagged1 levels and distribution are post-transcriptionally controlled by ZFP36 decay proteins.

Vascular morphogenesis requires a delicate gradient of Notch signaling controlled, in part, by the distribution of ligands (Dll4 and Jagged1). How Jagged1 (JAG1) expression is compartmentalized in the vascular plexus remains unclear. Here, we show that Jag1 mRNA is a direct target of zinc-finger protein 36 (ZFP36), an RNA-binding protein involved in mRNA decay that we find robustly induced by vascular endothelial growth factor (VEGF). Endothelial cells lacking ZFP36 display high levels of JAG1 and increase angiogenic sprouting in vitro. Furthermore, mice lacking Zfp36 in endothelial cells display mispatterned and increased levels of JAG1 in the developing retinal vascular plexus. Abnormal levels of JAG1 at the sprouting front alters NOTCH1 signaling, increasing the number of tip cells, a phenotype that is rescued by imposing haploinsufficiency of Jag1. Our findings reveal an important feedforward loop whereby VEGF stimulates ZFP36, consequently suppressing Jag1 to enable adequate levels of Notch signaling during sprouting angiogenesis.

Jagged1 contained in MSC-derived small extracellular vesicles promotes squamous differentiation of cervical cancer by activating NOTCH pathway.

PURPOSE: Cervical cancer is the fourth most common cancer in women and poses a major threat to women's health, urgently requiring new treatment methods. METHODS: This study first successfully extracted and identified small extracellular vesicles secreted by human umbilical cord-derived mesenchymal stem cells. We studied the effects of MSC-sEV on the squamous differentiation levels of cervical cancer CaSki cells in vitro, and explored the effects of MSC-sEV on the NOTCH pathway, the growth, proliferation, migration abilities and squamous differentiation levels of cervical cancer cells. The roles of MSC-sEV were also verified in human keratinocyte HaCaT cells. RESULTS: The results showed that Jagged1 protein on MSC-sEV can bind to NOTCH1 on cervical cancer cells, activate NOTCH signaling, and promote squamous differentiation levels in CaSki cells, thus inhibiting the growth, proliferation and migration abilities of CaSki cells. MSC-sEV can also activate the NOTCH pathway in HaCaT cells, but promote the viability of HaCaT cells. CONCLUSION: MSC-sEV can activate the NOTCH pathway to promote squamous differentiation of CaSki cells and inhibit the growth proliferation and migration abilities of CaSki cells which may be a new mechanism for cervical cancer treatment.

Significance of NotchScore and JAG1 in predicting prognosis and immune response of low-grade glioma.

Introduction: Low-grade glioma (LGG) is a prevalent malignant tumor in the intracranial region. Despite the advancements in treatment methods for this malignancy over the past decade, significant challenges still persist in the form of drug resistance and tumor recurrence. The Notch signaling pathway plays essential roles in many physiological processes as well as in cancer development. However, the significance of the pathway and family genes in LGG are poorly understood. Methods: We conducted gene expression profiling analysis using the TCGA dataset to investigate the gene set associated with the Notch signaling pathway. we have proposed a metric called "NotchScore" that quantifies the strength of the Notch signaling pathway and enables us to assess its significance in predicting prognosis and immune response in LGG. We downregulated JAG1 in low-grade gliomas to assess its influence on the proliferation and migration of these tumors. Ultimately, we determined the impact of the transcription factor VDR on the transcription of PDL1 through chip-seq data analysis. Results: Our findings indicate that tumors with a higher NotchScore, exhibit poorer prognosis, potentially due to their ability to evade the anti-tumor effects of immune cells by expressing immune checkpoints. Among the genes involved in the Notch signaling pathway, JAG1 has emerged as the most representative in terms of capturing the characteristics of both NotchScore and Notch pathways. The experimental results demonstrate that silencing JAG1 yielded a significant decrease in tumor cell proliferation in LGG cell lines. Our study revealed mechanisms by which tumors evade the immune system through the modulation of PDL1 transcription levels via the PI3K-Akt signaling pathway. Additionally, JAG1 potentially influences PDL1 in LGG by regulating the PI3K-Akt signaling pathway and the expression of the transcription factor VDR. Discussion: These findings contribute to our understanding of immune evasion by tumors in LGG. The insights gained from this research may have implications for the development of therapeutic interventions for LGG.

The Potential of JAG Ligands as Therapeutic Targets and Predictive Biomarkers in Multiple Myeloma.

The NOTCH ligands JAG1 and JAG2 have been correlated in vitro with multiple myeloma (MM) cell proliferation, drug resistance, self-renewal and a pathological crosstalk with the tumor microenvironment resulting in angiogenesis and osteoclastogenesis. These findings suggest that a therapeutic approach targeting JAG ligands might be helpful for the care of MM patients and lead us to explore the role of JAG1 and JAG2 in a MM in vivo model and primary patient samples. JAG1 and JAG2 protein expression represents a common feature in MM cell lines; therefore, we assessed their function through JAG1/2 conditional silencing in a MM xenograft model. We observed that JAG1 and JAG2 showed potential as therapeutic targets in MM, as their silencing resulted in a reduction in the tumor burden. Moreover, JAG1 and JAG2 protein expression in MM patients was positively correlated with the presence of MM cells in patients' bone marrow biopsies. Finally, taking advantage of the Multiple Myeloma Research Foundation (MMRF) CoMMpass global dataset, we showed that JAG2 gene expression level was a predictive biomarker associated with patients' overall survival and progression-free survival, independently from other main molecular or clinical features. Overall, these results strengthened the rationale for the development of a JAG1/2-tailored approach and the use of JAG2 as a predictive biomarker in MM.

Generation of an Alagille syndrome (ALGS) patient-derived induced pluripotent stem cell line (TRNDi032-A) carrying a heterozygous mutation (p.Cys682Leufs*7) in the JAG1 gene.

Alagille syndrome (ALGS) is an autosomal dominant, multisystemic disorder due to haploinsufficiency in either the JAG1 gene (ALGS type 1) or the NOTCH2 gene (ALGS type 2). The disease has been difficult to diagnose and treat due to its muti-system clinical presentation, variable expressivity, and prenatal onset for some of the features. The generation of this iPSC line (TRNDi032-A) carrying a heterozygous mutation, p.Cys682Leufs*7 (c.2044dup), in the JAG1 gene provides a means of studying the disease and developing novel therapeutics towards patient treatment.

Annexin A2 Stabilizes Oncogenic JAG1 Intracellular Domain by Inhibiting Proteasomal Degradation in Glioblastoma Cells.

Glioblastoma (GBM) is the most lethal brain cancer, causing inevitable deaths of patients owing to frequent relapses of cancer stem cells (CSCs). The significance of the NOTCH signaling pathway in CSCs has been well recognized; however, there is no NOTCH-selective treatment applicable to patients with GBM. We recently reported that Jagged1 (JAG1), a NOTCH ligand, drives a NOTCH receptor-independent signaling pathway via JAG1 intracellular domain (JICD1) as a crucial signal that renders CSC properties. Therefore, mechanisms regulating the JICD1 signaling pathway should be elucidated to further develop a selective therapeutic regimen. Here, we identified annexin A2 (ANXA2) as an essential modulator to stabilize intrinsically disordered JICD1. The binding of ANXA2 to JICD1 prevents the proteasomal degradation of JICD1 by heat shock protein-70/90 and carboxy-terminus of Hsc70 interacting protein E3 ligase. Furthermore, JICD1-driven propagation and tumor aggressiveness were inhibited by ANXA2 knockdown. Taken together, our findings show that ANXA2 maintains the function of the NOTCH receptor-independent JICD1 signaling pathway by stabilizing JICD1, and the targeted suppression of JICD1-driven CSC properties can be achieved by blocking its interaction with ANXA2.

Characterization of an induced pluripotent stem cell line NCHi011-A from a 23-year-old female with Alagille Syndrome harboring a heterozygous JAG1 pathogenic variant.

Alagille syndrome (ALGS) is a multisystem disease with high variability in clinical features. ALGS is predominantly caused by pathogenic variants in the Notch ligand JAG1. An iPSC line, NCHi011-A, was generated from a ALGS patient with complex cardiac phenotypes consisting of pulmonic valve and branch pulmonary artery stenosis. NCHi011-A is heterozygous for a single base duplication causing a frameshift in the JAG1 gene. This iPSC line demonstrates normal cellular morphology, expression of pluripotency markers, trilineage differentiation potential, and identity to the source patient. NCHi011-A provides a resource for modeling ALGS and investigating the role of Notch signaling in the disease.

Generation of iPSC line NCHi012-A from a patient with Alagille syndrome and heterozygous pathogenic variant in the JAG1 gene.

Alagille syndrome (ALGS) is an autosomal dominant disease affecting the liver, heart and other organs with high variability. About 95% of ALGS cases are associated with pathogenic variants in JAG1, encoding the Jagged1 ligand that binds to Notch receptors. The iPSC line NCHi012-A was derived from an ALGS patient with cholestatic liver disease and mild pulmonary stenosis, who is heterozygous for a 2 bp deletion in the JAG1 coding sequence. We report here an initial characterization of NCHi012-A to evaluate its morphology, pluripotency, differentiation potential, genotype, karyotype and identity to the source patient.

Clinical Characterization of Alagille Syndrome in Patients with Cholestatic Liver Disease.

Alagille syndrome (ALGS) is a multisystem condition characterized by cholestasis and bile duct paucity on liver biopsy and variable involvement of the heart, skeleton, eyes, kidneys, and face and caused by pathogenic variants in the JAG1 or NOTCH2 gene. The variable expressivity of the clinical phenotype and the lack of genotype-phenotype correlations lead to significant diagnostic difficulties. Here we present an analysis of 18 patients with cholestasis who were diagnosed with ALGS. We used an NGS panel targeting coding exons of 52 genes, including the JAG1 and NOTCH2 genes. Sanger sequencing was used to verify the mutation in the affected individuals and family members. The specific facial phenotype was seen in 16/18 (88.9%). Heart defects were seen in 8/18 (44.4%) patients (pulmonary stenosis in 7/8). Butterfly vertebrae were seen in 5/14 (35.7%) patients. Renal involvement was detected in 2/18 (11.1%) cases-one patient had renal cysts, and one had obstructive hydronephrosis. An ophthalmology examination was performed on 12 children, and only one had posterior embryotoxon (8.3%). A percutaneous liver biopsy was performed in nine cases. Bile duct paucity was detected in six/nine cases (66.7%). Two patients required liver transplantation because of cirrhosis. We identified nine novel variants in the JAG1 gene-eight frameshift variants (c.1619_1622dupGCTA (p.Tyr541X), c.1160delG (p.Gly387fs), c.964dupT (p.C322fs), c.120delG (p.L40fs), c.1984dupG (p.Ala662Glyfs), c.3168_3169delAG (p.R1056Sfs*51), c.2688delG (p.896CysfsTer49), c.164dupG (p.Cys55fs)) and one missense variant, c.2806T > G (p.Cys936Gly). None of the patients presented with NOTCH2 variants. In accordance with the classical criteria, only six patients could meet the diagnostic criteria in our cohort without genetic analysis. Genetic testing is important in the diagnosis of ALGS and can help differentiate it from other types of cholestasis.

[MiR-203a-5p Inhibits Multiple Myeloma Cell Proliferation and Cell Cycle Progression via Targeting JAG1].

OBJECTIVE: To investigate the biological function of miR-203a-5p and the underlying mechanism in multiple myeloma (MM). METHODS: Three miRNA expression profiles (GSE16558, GSE24371 and GSE17498) were downloaded from the GEO database. The three miRNA expression profiles contained 131 MM samples and 17 normal plasmacyte samples. The robust rank aggregation (RRA) method was used to identify the differentially expressed miRNAs between MM and normal plasmacytes. In order to carry out cytological experiments, MM cell line with stable over-expression of miR-203a-5p was constructed with lentivirus. Expression levels of miR-203a-5p in MM cells were quantified by qRT-PCR. The effects of miR-203a-5p on MM cells were investigated using assays of cell viability and cell cycle. Cell proliferation was measured using the Cell Counting kit (CCK)8 assay. The percentage of cells in each cell cycle was measured with a FACSCalibur system. Xenograft tumor models were established to evaluate the role of miR-203a-5p in tumorigenesis in vivo . To elucidate the underlying molecular mechanisms of miR-203a-5p in mediating cell proliferation inhibition and cell cycle arrest in MM, we used TargetScan and miRanda to predict the candidate targets of miR-203a-5p. The potential target of miR-203a-5p in MM cells was explored using the luciferase reporter assay, qRT-PCR, and Western blot. RESULTS: An integrated analysis of three MM miRNA expression datasets showed that the levels of miR-203a-5p in MM were notably downregulated compared with those in normal plasmacytes. Accordingly, the relative expression levels of miR-203a-5p were decreased in MM cell lines. In addition, overexpression of miR-203a-5p inhibited the proliferation and cell cycle progression of RPMI8226 and U266 cells. In vivo experiments demonstrated that upregulation of miR-203a-5p expression could significantly inhibit the tumorigenesis of subcutaneous myeloma xenografts in nude mice. Mechanistic investigation led to the identification of Jagged 1 (JAG1) as a novel and direct downstream target of miR-203a-5p. Interestingly, the reintroduction of JAG1 abrogated miR-203a-5p-induced MM cell growth inhibition and cell cycle arrest. CONCLUSION: Our data demonstrate that miR-203a-5p inhibits cell proliferation and cell cycle progression in MM cells by targeting JAG1, supporting the utility of miR-203a-5p as a novel and potential therapeutic agent for miRNA-based MM therapy.

Generation of JAG1 gene c.1615C > T heterozygous mutation human embryonic stem cell line (SDQLCHe001-A) using cytosine base editor.

Pathogenic variants in Jagged-1 (JAG1), which encodes the ligand of the Notch receptor, had been demonstrated to cause Alagille syndrome. However, there is no evidence to support any genotype-phenotype correlations. Here, we generated a gene-edited human embryonic stem cell (hESC) line (H9) carrying the c.1615C > T mutation in JAG1 that was identified in a patient with Alagille syndrome (ALGS). This modified cell line was accomplished by using cytosine base editor (CBE), and may serve as a valuable model for JAG1 mutaion related disease, and facilitate to gain more insight into the biological function of JAG1.

ASO silencing of a glycosyltransferase, Poglut1 , improves the liver phenotypes in mouse models of Alagille syndrome.

BACKGROUND AND AIMS: Paucity of intrahepatic bile ducts (BDs) is caused by various etiologies and often leads to cholestatic liver disease. For example, in patients with Alagille syndrome (ALGS), which is a genetic disease primarily caused by mutations in jagged 1 ( JAG1) , BD paucity often results in severe cholestasis and liver damage. However, no mechanism-based therapy exists to restore the biliary system in ALGS or other diseases associated with BD paucity. Based on previous genetic observations, we investigated whether postnatal knockdown of the glycosyltransferase gene protein O -glucosyltransferase 1 ( Poglut1) can improve the ALGS liver phenotypes in several mouse models generated by removing one copy of Jag1 in the germline with or without reducing the gene dosage of sex-determining region Y-box 9 in the liver. APPROACH AND RESULTS: Using an ASO established in this study, we show that reducing Poglut1 levels in postnatal livers of ALGS mouse models with moderate to profound biliary abnormalities can significantly improve BD development and biliary tree formation. Importantly, ASO injections prevent liver damage in these models without adverse effects. Furthermore, ASO-mediated Poglut1 knockdown improves biliary tree formation in a different mouse model with no Jag1 mutations. Cell-based signaling assays indicate that reducing POGLUT1 levels or mutating POGLUT1 modification sites on JAG1 increases JAG1 protein level and JAG1-mediated signaling, suggesting a likely mechanism for the observed in vivo rescue. CONCLUSIONS: Our preclinical studies establish ASO-mediated POGLUT1 knockdown as a potential therapeutic strategy for ALGS liver disease and possibly other diseases associated with BD paucity.

Alagille-like syndrome with surprising karyotype: a case report.

BACKGROUND: Chromosome 5p partial monosomy (5p-syndrome) and chromosome 6p partial trisomy are chromosomal abnormalities that result in a variety of symptoms, but liver dysfunction is not normally one of them. Alagille syndrome (OMIM #118450) is a multisystem disorder that is defined clinically by hepatic bile duct paucity and cholestasis, in association with cardiac, skeletal, and ophthalmologic manifestations, and characteristic facial features. Alagille syndrome is caused by mutations in JAG1 on chromosome 20 or NOTCH2 on chromosome 1. Here, we report a preterm infant with karyotype 46,XX,der(5)t(5,6)(p15.2;p22.3) and hepatic dysfunction, who was diagnosed as having incomplete Alagille syndrome. CASE PRESENTATION: The Japanese infant was diagnosed based on the cardiac abnormalities, ocular abnormalities, characteristic facial features, and liver pathological findings. Analysis of the JAG1 and NOTCH sequences failed to detect any mutations in these genes. CONCLUSIONS: These results suggest that, besides the genes that are known to be responsible for Alagille syndrome, other genetic mutations also may cause Alagille syndrome.

Premature senescence of the liver in Alagille patients.

INTRODUCTION: Alagille syndrome (ALGS) is an autosomal dominant disease characterized by a multisystem involvement including bile duct paucity and cholestasis, caused by JAG1 or NOTCH2 mutations in most of the cases. Jagged1-Notch2 interactions are known to be crucial for intrahepatic biliary tract development, but the Notch signaling pathway is also involved in the juxtacrine transmission of senescence and in the induction and modulation of the senescence-associated secretory phenotype (SASP). AIM: Our aim was to investigate premature senescence and SASP in ALGS livers. METHODS: Liver tissue from ALGS patients was prospectively obtained at the time of liver transplantation (n = 5) and compared to control livers (n = 5). RESULTS: We evidenced advanced premature senescence in the livers of five JAG1 mutated ALGS pediatric patients through increased senescence-associated beta-galactosidase activity (p<0.05), increased p16 and p21 gene expression (p<0.01), and increased p16 and γH2AX protein expression (p<0.01). Senescence was located in hepatocytes of the whole liver parenchyma as well as in remaining bile ducts. The classical SASP markers TGF-ß1, IL-6, and IL-8 were not overexpressed in the livers of our patients. CONCLUSIONS: We demonstrate for the first time that ALGS livers display important premature senescence despite Jagged1 mutation, underlying the complexity of senescence and SASP development pathways.