Spectrum and frequency of jagged1 (JAG1) mutations in Alagille syndrome patients and their families.

Alagille syndrome (AGS) is a dominantly inherited disorder characterized by liver disease in combination with heart, skeletal, ocular, facial, renal, and pancreatic abnormalities. We have recently demonstrated that Jagged1 (JAG1) is the AGS gene. JAG1 encodes a ligand in the Notch intercellular signaling pathway. AGS is the first developmental disorder to be associated with this pathway and the first human disorder caused by a Notch ligand. We have screened 54 AGS probands and family members to determine the frequency of mutations in JAG1. Three patients (6%) had deletions of the entire gene. Of the remaining 51 patients, 35 (69%) had mutations within JAG1, identified by SSCP analysis. Of the 35 identified intragenic mutations, all were unique, with the exceptions of a 5-bp deletion in exon 16, seen in two unrelated patients, and a C insertion at base 1618 in exon 9, also seen in two unrelated patients. The 35 intragenic mutations included 9 nonsense mutations (26%); 2 missense mutations (6%); 11 small deletions (31%), 8 small insertions (23%), and 1 complex rearrangement (3%), all leading to frameshifts; and 4 splice-site mutations (11%). The mutations are spread across the coding sequence of the gene within the evolutionarily conserved motifs of the JAG1 protein. There is no phenotypic difference between patients with deletions of the entire JAG1 gene and those with intragenic mutations, which suggests that one mechanism involved in AGS is haploinsufficiency. The two missense mutations occur at the same amino acid residue. The mechanism by which these missense mutations lead to the disease is not yet understood; however, they suggest that mechanisms other than haploinsufficiency may result in the AGS phenotype.     

Analysis of JAG1 gene variant in Chinese patients with Alagille syndrome

Alagille syndrome (AGS) is an autosomal dominant disorder characterized by bile duct paucity. It can be caused by variations in the JAG1 gene encoding a protein of Notch ligand and by variations in the NOTCH2 gene encoding a Notch receptor. In this study we identified 15 different JAG1 gene variations in 17 Chinese patients, nine of which were novel alterations including c.766G > T, c.819delC, c.826delT, c.3099_3100delCA, c.1323_1326delCTGG, c.1771_1775delGTGCGinsT, c.1868delG, c. 2791_2792insA and c.866delG. These alterations were located in the extracellular domain of JAG1, in particular in the DSL and EGF-like repeat domain. All the specific variations in five inheritance cases investigated were de novo. Furthermore, no sequence variation of NOTCH2 was detected in JAG1 alteration negative patients.     

Human Jagged 1 mutants cause liver defect in Alagille syndrome by overexpression of hepatocyte growth factor

Alagille syndrome (AGS, MIM 118450) is an autosomal dominant inherited disease. Paucity of interlobular bile ducts is one of the major abnormalities. To explore the molecular mechanism by which mutation in the human Jagged 1 gene (JAG1, MIM 601920) causes liver defects, we investigated the gene regulation of JAG1 to hepatocyte growth factor gene (HGF). By transfecting wild-type and mutant JAG1 into COS-7 cells in vitro, we found that HGF is a target gene of JAG1 downstream. Wild-type JAG1 is inhibitory for HGF expression and mutant JAG1s relieve the inhibition. Several domain disruptions in mutant JAG1 protein reveal a reduced inhibition to HGF expression at different levels. JAG1 mutations actually result in HGF overexpression. Furthermore, JAG1 controls HGF expression by a dosage-dependent regulation and Notch2 signaling seems to mediate JAG1 function. Given that HGF plays a critical role in differentiation of hepatic stem cells, overexpression of HGF acts on off-balanced cell fate determination in AGS patients. Hepatic stem cells may differentiate towards more hepatocytes but less biliary cells, thus causing the paucity of interlobular bile ducts in liver development of AGS. Our novel findings demonstrated that dosage-dependent regulation by mutations of JAG1 is a fundamental mechanism for liver abnormality in AGS.     

Conditional JAG1 mutation shows the developing heart is more sensitive than developing liver to JAG1 dosage

Mutations of Jagged 1 (JAG1), a ligand in the Notch signaling pathway, cause Alagille syndrome (AGS). AGS is an autosomal dominant, multisystem disorder with variable expressivity, characterized by bile duct paucity and resultant liver disease in combination with cardiac, ocular, skeletal, and facial findings. JAG1 mutations in AGS include gene deletions and protein truncating, splicing, and missense mutations, suggesting that haploinsufficiency is the mechanism of disease causation. With limited exceptions, there is no genotype-phenotype correlation. We have studied a JAG1 missense mutation (JAG1-G274D) that was previously identified in 13 individuals from an extended family with cardiac defects of the type seen in patients with AGS (e.g., peripheral pulmonic stenosis and tetralogy of Fallot) in the absence of liver dysfunction. Our data indicate that this mutation is "leaky." Two populations of proteins are produced from this allele. One population is abnormally glycosylated and is retained intracellularly rather than being transported to the cell surface. A second population is normally glycosylated and is transported to the cell surface, where it is able to signal to the Notch receptor. The JAG1-G274D protein is temperature sensitive, with more abnormally glycosylated (and nonfunctional) molecules produced at higher temperatures. Carriers of this mutation therefore have >50% but <100% of the normal concentration of JAG1 molecules on the cell surface. The cardiac-specific phenotype associated with this mutation suggests that the developing heart is more sensitive than the developing liver to decreased dosage of JAG1.     

Spectrum of <Emphasis Type="Italic">JAG1</Emphasis> gene mutations in Polish patients with Alagille syndrome

Alagille syndrome (ALGS) is an autosomal dominant disorder characterized by developmental abnormalities in several organs including the liver, heart, eyes, vertebrae, kidneys, and face. The majority (90-94 %) of ALGS cases are caused by mutations in the JAG1 (JAGGED1) gene, and in a small percent of patients (～1 %) mutations in the NOTCH2 gene have been described. Both genes are involved in the Notch signaling pathway. To date, over 440 different JAG1 gene mutations and ten NOTCH2 mutations have been identified in ALGS patients. The present study was conducted on a group of 35 Polish ALGS patients and revealed JAG1 gene mutations in 26 of them. Twenty-three different mutations were detected including 13 novel point mutations and six large deletions affecting the JAG1 gene. Review of all mutations identified to date in individuals from Poland allowed us to propose an effective diagnostic strategy based on the mutations identified in the reported patients of Polish descent. However, the distribution of mutations seen in this cohort was not substantively different than the mutation distribution in other reported populations.     

Notch signaling in human development and disease

Mutations in Notch signaling pathway members cause developmental phenotypes that affect the liver, skeleton, heart, eye, face, kidney, and vasculature. Notch associated disorders include the autosomal dominant, multi-system, Alagille syndrome caused by mutations in both a ligand (Jagged1 (JAG1)) and receptor (NOTCH2) and autosomal recessive spondylocostal dysostosis, caused by mutations in a ligand (Delta-like-3 (DLL3)), as well as several other members of the Notch signaling pathway. Mutations in NOTCH2 have also recently been connected to Hajdu-Cheney syndrome, a dominant disorder causing focal bone destruction, osteoporosis, craniofacial morphology and renal cysts. Mutations in the NOTCH1 receptor are associated with several types of cardiac disease and mutations in NOTCH3 cause the dominant adult onset disorder CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy), a vascular disorder with onset in the 4th or 5th decades. Studies of these human disorders and their inheritance patterns and types of mutations reveal insights into the mechanisms of Notch signaling.     

Notch signaling genes

Notch intercellular signaling is critical for diverse developmental pathways and for homeostasis in various types of stem cells and progenitor cells. Because Notch gene products need to be precisely regulated spatially and temporally, epigenetics is likely to help control expression of Notch signaling genes. Reduced representation bisulfite sequencing (RRBS) indicated significant hypomethylation in myoblasts, myotubes, and skeletal muscle vs. many nonmuscle samples at intragenic or intergenic regions of the following Notch receptor or ligand genes: NOTCH1, NOTCH2, JAG2, and DLL1. An enzymatic assay of sites in or near these genes revealed unusually high enrichment of 5-hydroxymethylcytosine (up to 81%) in skeletal muscle, heart, and cerebellum. Epigenetics studies and gene expression profiles suggest that hypomethylation and/or hydroxymethylation help control expression of these genes in heart, brain, myoblasts, myotubes, and within skeletal muscle myofibers. Such regulation could promote cell renewal, cell maintenance, homeostasis, and a poised state for repair of tissue damage.     

Notch signaling genes: Myogenic DNA hypomethylation and 5-hydroxymethylcytosine

Notch intercellular signaling is critical for diverse developmental pathways and for homeostasis in various types of stem cells and progenitor cells. Because Notch gene products need to be precisely regulated spatially and temporally, epigenetics is likely to help control expression of Notch signaling genes. Reduced representation bisulfite sequencing (RRBS) indicated significant hypomethylation in myoblasts, myotubes, and skeletal muscle vs. many nonmuscle samples at intragenic or intergenic regions of the following Notch receptor or ligand genes: NOTCH1, NOTCH2, JAG2, and DLL1. An enzymatic assay of sites in or near these genes revealed unusually high enrichment of 5-hydroxymethylcytosine (up to 81%) in skeletal muscle, heart, and cerebellum. Epigenetics studies and gene expression profiles suggest that hypomethylation and/or hydroxymethylation help control expression of these genes in heart, brain, myoblasts, myotubes, and within skeletal muscle myofibers. Such regulation could promote cell renewal, cell maintenance, homeostasis, and a poised state for repair of tissue damage.     

A mouse model of Alagille syndrome: Notch2 as a genetic modifier of Jag1 haploinsufficiency

Alagille syndrome is a human autosomal dominant developmental disorder characterized by liver, heart, eye, skeletal, craniofacial and kidney abnormalities. Alagille syndrome is caused by mutations in the Jagged 1 (JAG1) gene, which encodes a ligand for Notch family receptors. The majority of JAG1 mutations seen in Alagille syndrome patients are null alleles, suggesting JAG1 haploinsufficiency as a primary cause of this disorder. Mice homozygous for a Jag1 null mutation die during embryogenesis and Jag1/+ heterozygous mice exhibit eye defects but do not exhibit other phenotypes characteristic of Alagille syndrome patients ( Xue, Y., Gao, X., Lindsell, C. E., Norton, C. R., Chang, B., Hicks, C., Gendron-Maguire, M., Rand, E. B., Weinmaster, G. and Gridley, T. (1999) HUM: Mol. Genet. 8, 723-730). Here we report that mice doubly heterozygous for the Jag1 null allele and a Notch2 hypomorphic allele exhibit developmental abnormalities characteristic of Alagille syndrome. Double heterozygous mice exhibit jaundice, growth retardation, impaired differentiation of intrahepatic bile ducts and defects in heart, eye and kidney development. The defects in bile duct epithelial cell differentiation and morphogenesis in the double heterozygous mice are similar to defects in epithelial morphogenesis of Notch pathway mutants in Drosophila, suggesting that a role for the Notch signaling pathway in regulating epithelial morphogenesis has been conserved between insects and mammals. This work also demonstrates that the Notch2 and Jag1 mutations interact to create a more representative mouse model of Alagille syndrome and provides a possible explanation of the variable phenotypic expression observed in Alagille syndrome patients.     

KSHV Manipulates Notch Signaling by DLL4 and JAG1 to Alter Cell Cycle Genes in Lymphatic Endothelia

Increased expression of Notch signaling pathway components is observed in Kaposi sarcoma (KS) but the mechanism underlying the manipulation of the canonical Notch pathway by the causative agent of KS, Kaposi sarcoma herpesvirus (KSHV), has not been fully elucidated. Here, we describe the mechanism through which KSHV directly modulates the expression of the Notch ligands JAG1 and DLL4 in lymphatic endothelial cells. Expression of KSHV-encoded vFLIP induces JAG1 through an NFκB-dependent mechanism, while vGPCR upregulates DLL4 through a mechanism dependent on ERK. Both vFLIP and vGPCR instigate functional Notch signalling through NOTCH4. Gene expression profiling showed that JAG1- or DLL4-stimulated signaling results in the suppression of genes associated with the cell cycle in adjacent lymphatic endothelial cells, indicating a role for Notch signaling in inducing cellular quiescence in these cells. Upregulation of JAG1 and DLL4 by KSHV could therefore alter the expression of cell cycle components in neighbouring uninfected cells during latent and lytic phases of viral infection, influencing cellular quiescence and plasticity. In addition, differences in signaling potency between these ligands suggest a possible complementary role for JAG1 and DLL4 in the context of KS.     

Inhibition of Notch signaling enhances transdifferentiation of the esophageal squamous epithelium towards a Barrett's-like metaplasia via KLF4

Barrett's esophagus (BE) is defined as an incomplete intestinal metaplasia characterized generally by the presence of columnar and goblet cells in the formerly stratified squamous epithelium of the esophagus. BE is known as a precursor for esophageal adenocarcinoma. Currently, the cell of origin for human BE has yet to be clearly identified. Therefore, we investigated the role of Notch signaling in the initiation of BE metaplasia. Affymetrix gene expression microarray revealed that BE samples express decreased levels of Notch receptors (NOTCH2 and NOTCH3) and one of the the ligands (JAG1). Furthermore, BE tissue microarray showed decreased expression of NOTCH1 and its downstream target HES1. Therefore, Notch signaling was inhibited in human esophageal epithelial cells by expression of dominant-negative-Mastermind-like (dnMAML), in concert with MYC and CDX1 overexpression. Cell transdifferentiation was then assessed by 3D organotypic culture and evaluation of BE-lineage specific gene expression. Notch inhibition promoted transdifferentiation of esophageal epithelial cells toward columnar-like cells as demonstrated by increased expression of columnar keratins (K8, K18, K19, K20) and glandular mucins (MUC2, MUC3B, MUC5B, MUC17) and decreased expression of squamous keratins (K5, K13, K14). In 3D culture, elongated cells were observed in the basal layer of the epithelium with Notch inhibition. Furthermore, we observed increased expression of KLF4, a potential driver of the changes observed by Notch inhibition. Interestingly, knockdown of KLF4 reversed the effects of Notch inhibition on BE-like metaplasia. Overall, Notch signaling inhibition promotes transdifferentiation of esophageal cells toward BE-like metaplasia in part via upregulation of KLF4. These results support a novel mechanism through which esophageal epithelial transdifferentiation promotes the evolution of BE.     

Distinct Biological Roles for the Notch Ligands Jagged-1 and Jagged-2

Notch signaling is activated in a subset of non-small cell lung cancer cells because of overexpression of Notch3, but the role of Notch ligands has not been fully defined. On the basis of gene expression profiling of a panel of non-small cell lung cancer cell lines, we found that the predominant Notch ligands were JAG1, JAG2, DLL1, and DLL3. Given that Notch ligands reportedly have overlapping receptor binding specificities, we postulated that they have redundant biological roles. Arguing against this hypothesis, we found that JAG1 and JAG2 were differentially regulated; JAG1 expression was dependent upon epidermal growth factor receptor (EGFR) activation in HCC827 cells, which require EGFR for survival, whereas JAG2 expression was EGFR-independent in these cells. Furthermore, HCC827 cells underwent apoptosis following depletion of JAG1 but not JAG2, whereas co-culture experiments revealed that depletion of JAG2, but not JAG1, enhanced the ability of HCC827 cells to chemoattract THP-1 human monocytes. JAG2-depleted HCC827 cells expressed high levels of inflammation-related genes, including interleukin 1 (IL1) and a broad range of IL1-regulated cytokines, which was attenuated by inhibition of IL1 receptor (IL1R). Our findings suggest that JAG1 and JAG2 have distinct biological roles including a previously undiscovered role for JAG2 in regulating the expression of cytokines that can promote antitumor immunity.     

Jagged1 in the portal vein mesenchyme regulates intrahepatic bile duct development: insights into Alagille syndrome

Mutations in the human Notch ligand jagged 1 (JAG1) result in a multi-system disorder called Alagille syndrome (AGS). AGS is chiefly characterized by a paucity of intrahepatic bile ducts (IHBD), but also includes cardiac, ocular, skeletal, craniofacial and renal defects. The disease penetration and severity of the affected organs can vary significantly and the molecular basis for this broad spectrum of pathology is unclear. Here, we report that Jag1 inactivation in the portal vein mesenchyme (PVM), but not in the endothelium of mice, leads to the hepatic defects associated with AGS. Loss of Jag1 expression in SM22α-positive cells of the PVM leads to defective bile duct development beyond the initial formation of the ductal plate. Cytokeratin 19-positive cells are detected surrounding the portal vein, yet they are unable to form biliary tubes, revealing an instructive role of the vasculature in liver development. These findings uncover the cellular basis for the defining feature of AGS, identify mesenchymal Jag1-dependent and -independent stages of duct development, and provide mechanistic information for the role of Jag1 in IHBD formation.     

The Notch ligands DLL1 and JAG2 act synergistically to regulate hair cell development in the mammalian inner ear

The mammalian auditory sensory epithelium, the organ of Corti, contains sensory hair cells and nonsensory supporting cells arranged in a highly patterned mosaic. Notch-mediated lateral inhibition is the proposed mechanism for creating this sensory mosaic. Previous work has shown that mice lacking the Notch ligand JAG2 differentiate supernumerary hair cells in the cochlea, consistent with the lateral inhibitory model. However, it was not clear why only relatively modest increases in hair cell production were observed in Jag2 mutant mice. Here, we show that another Notch ligand, DLL1, functions synergistically with JAG2 in regulating hair cell differentiation in the cochlea. We also show by conditional inactivation that these ligands probably signal through the NOTCH1 receptor. Supernumerary hair cells in Dll1/Jag2 double mutants arise primarily through a switch in cell fate, rather than through excess proliferation. Although these results demonstrate an important role for Notch-mediated lateral inhibition during cochlear hair cell patterning, we also detected abnormally prolonged cellular proliferation that preferentially affected supporting cells in the organ of Corti. Our results demonstrate that the Notch pathway plays a dual role in regulating cellular differentiation and patterning in the cochlea, acting both through lateral inhibition and the control of cellular proliferation.     

Context-Dependent Sensitivity to Mutations Disrupting the Structural Integrity of Individual EGF Repeats in the Mouse Notch Ligand DLL1

The highly conserved Notch-signaling pathway mediates cell-to-cell communication and is pivotal for multiple developmental processes and tissue homeostasis in adult organisms. Notch receptors and their ligands are transmembrane proteins with multiple epidermal-growth-factor-like (EGF) repeats in their extracellular domains. In vitro the EGF repeats of mammalian ligands that are essential for Notch activation have been defined. However, in vivo the significance of the structural integrity of each EGF repeat in the ligand ectodomain for ligand function is still unclear. Here, we analyzed the mouse Notch ligand DLL1. We expressed DLL1 proteins with mutations disrupting disulfide bridges in each individual EGF repeat from single-copy transgenes in the HPRT locus of embryonic stem cells. In Notch transactivation assays all mutations impinged on DLL1 function and affected both NOTCH1 and NOTCH2 receptors similarly. An allelic series in mice that carried the same point mutations in endogenous Dll1, generated using a mini-gene strategy, showed that early developmental processes depending on DLL1-mediated NOTCH activation were differently sensitive to mutation of individual EGF repeats in DLL1. Notably, some mutations affected only somite patterning and resulted in vertebral column defects resembling spondylocostal dysostosis. In conclusion, the structural integrity of each individual EGF repeat in the extracellular domain of DLL1 is necessary for full DLL1 activity, and certain mutations in Dll1 might contribute to spondylocostal dysostosis in humans.