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PubMed2005

Cross talk among Notch3, pre-TCR, and Tal1 in T-cell development and leukemogenesis.

Integrated pathways are believed to determine hematopoietic cell fate and/or neoplastic transformation. Notch signaling has been shown to regulate T-cell differentiation and leukemogenesis. However, specific target genes and molecular partners are not fully elucidated. We show that Notch3 activation sustains aberrant SCL/Tal1 overexpression and phosphorylation in mature thymocytes. Furthermore, we define the role of SCL/Tal1 as a component of an activator complex, including phosphorylated Tal1 and Sp1, that specifically enhances cyclin D1 expression and demonstrate that Tal1/Sp1 specifically co-occupy the D1 promoter in vivo, only in the presence of pre-T-cell receptor (TCR). We therefore conclude not only that cyclin D1 is a target of the Tal1/Sp1 complex, but also that Notch3-dependent activation of pre-TCR/ERK signaling regulates SCL/Tal1 function.

Animals↗

PubMed2003

Delta1-Notch3 interactions bias the functional differentiation of activated CD4+ T cells.

Following activation by antigen, naive CD4+ T helper precursor cells execute distinct genetic programs that result in their differentiation toward the type 1 or type 2 helper T cell (Th1 or Th2) phenotype. Although the differentiation and function of these Th subsets has been well studied, little is known about the contribution to these differentiation events of cell surface receptors other than those for soluble cytokines, such as IL-12 or IL-4. Here, we provide direct evidence that the Delta1 interaction with Notch3 on CD4+ T cells transduces signals, promoting development toward the Th1 phenotype. The positive role of Notch signaling in effector cell differentiation was dose dependent, with high levels of stimulation resulting in reduced T cell activation. Our data revealed a clear contribution of Notch pathways to Th1 versus Th2 fate decisions, while also providing insight into another mechanism for inhibition of CD4+ T cell activation.

Animals↗

PubMed2006

Notch3 signaling initiates choroid plexus tumor formation.

Notch3 has been studied in the context of brain development, but whether it plays a role in the formation of brain tumors is unclear. We demonstrate that the introduction of constitutively active Notch3 into periventricular cells of embryonic day 9.5 mice causes the formation of choroid plexus tumors (CPTs). Tumors arose in the fourth ventricles in 83% of animals and were associated with hydrocephalus. They were microscopically highly similar to choroid plexus papillomas in humans, with an ongoing proliferation rate of 4-6%. Signs of Notch pathway activity were also present in human choroid plexus lesions, and receptor mRNA levels in papillomas were elevated over those in non-neoplastic choroid plexus. Notch2 was overexpressed approximately 500-fold in one case, suggesting that the role of this pathway in CPTs may not be specific to Notch3. Our findings indicate that activated Notch3 can function as an oncogene in the developing brain, and link the Notch pathway to human CPT pathogenesis.

Animals↗

PubMed2002

Combined expression of pTalpha and Notch3 in T cell leukemia identifies the requirement of preTCR for leukemogenesis.

Notch receptors are conserved regulators of cell fate and have been implicated in the regulation of T cell differentiation and lymphomagenesis. However, neither the generality of Notch involvement in leukemia, nor the molecules with which Notch may interact have been clarified. Recently, we showed that transgenic mice expressing the constitutively active intracellular domain of Notch3 in thymocytes and T cells developed early and aggressive T cell neoplasias. Although primarily splenic, the tumors sustained features of immature thymocytes, including expression of pTalpha, a defining component of the pre T cell receptor, known to be a potent signaling complex provoking thymocyte survival, proliferation, and activation. Thus, enforced expression of Notch3, which is ordinarily down-regulated as thymocytes mature, may sustain pre T cell receptor expression, causing dysregulated hyperplasia. This hypothesis has been successfully tested in this article by the observation that deletion of pTalpha in Notch3 transgenic mice abrogates tumor development, indicating a crucial role for pTalpha in T cell leukemogenesis. Parallel observations were made in humans, in that all T cell acute lymphoblastic leukemias examined showed expression of Notch3 and of the Notch target gene HES-1, as well as of pTalpha a and b transcripts, whereas the expression of all these genes was dramatically reduced or absent in remission. Together, these results suggest that the combined expression of Notch3 and pTalpha sustains T cell leukemogenesis and may represent pathognomonic molecular features of human T-ALL.

Animals↗

PubMed1999

CADASIL: hereditary disease of arteries causing brain infarcts and dementia.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) begins with migraine with aura in approximately one-third of the patients. More severe symptoms of recurrent strokes usually appear at 30-50 years of age. However, well before the first stroke, CADASIL may be diagnosed on the basis of characteristic hyperintensities in T2-weighted magnetic resonance images. Multiple lacunar infarcts located mainly in the basal ganglia and frontal white matter lead to a cognitive decline and finally to dementia. These infarcts result from a thickening and fibrosis of the walls of the small and medium-sized penetrating arteries with consequent obliteration and/or thrombosis. Although the symptoms are almost exclusively neurological, the arteriopathy is generalized. Thus, basophilic, periodic acid-Schiff-positive and, in electron microscopy, osmiophilic material accumulates between degenerating smooth muscle cells. This occurs even in dermal arteries, which renders skin a useful target for diagnostic biopsy. Presently, no specific therapy is available. CADASIL is caused by missense point mutations in the Notch3 gene, which encodes a transmembrane receptor protein. Each gene defect leads to either a gain or loss of a cysteine residue in the extracellular, N-terminal domain of the molecule, which most probably results in conformational alteration. The function of Notch3 in adults and the pathogenesis of CADASIL are still unknown.

Cerebral Arterial Diseases↗

PubMed2004

The role of Notch receptor expression in bile duct development and disease.

Mutations in the Jagged1 gene, a ligand for the Notch signalling pathway, have been implicated in the pathogenesis of Alagille syndrome (AGS), resulting in bile duct paucity. Recently, a mouse model for AGS suggested that abnormalities of the Notch2 receptor, as well as of Jagged1, may be present. Expression patterns of Notch receptors have not been described in the developing human liver or in paediatric liver. The expression of Notch receptors and ligands was examined in fetal, paediatric normal, and diseased human liver by RT-PCR and immunohistochemistry. RT-PCR showed Notch1-4 mRNA expression to be present. In fetal liver, Notch3 protein was expressed on mesenchymal cells, closely adjacent to ductal plate cells that expressed Jagged1. In paediatric normal liver, Notch1 and Notch2 were present on mature bile duct cells. Notch expression was altered in disease, with distinct differences in AGS from extrahepatic biliary atresia (EHBA) and alpha1-anti-trypsin deficiency (alpha1AT). In AGS, where extensive ductular reaction was present, Jagged1 was expressed on ductular reactive cells (DRCs), along with marked Notch2 and Notch3 staining. Where there was ductular paucity, Notch2 and Notch3 were not expressed on remaining biliary epithelial cells. In EHBA and alpha1AT, Notch receptor expression was not seen on DRCs. Instead, Notch2 and Notch3 were expressed by stromal cells. In all diseases, Notch3 was expressed on neovessels in portal tracts and cirrhotic fibrous septa. In conclusion, Notch3 is expressed in close proximity to Jagged1 at the time of ductal plate formation, suggesting that Notch3 is important for bile duct development. The expression of both Notch2 and Notch3 in AGS on DRCs confirms that these receptors may be important in the pathogenesis of this disease. Further studies are required to investigate the presence of Notch2 and Notch3 at other periods in liver development and to clarify the role of Notch signalling in paediatric cholestases.

Adolescent↗

PubMed2005

Autosomal-dominant familial hematuria with retinal arteriolar tortuosity and contractures: a novel syndrome.

BACKGROUND: Autosomal-dominant forms of hematuria have been mostly related to mutations in the COL4A3/COL4A4 genes. Patients with thin basement membrane (BM) disease do not have extrarenal manifestations, while those with Alport syndrome often present with hearing loss, anterior lenticonus, and dot-and-fleck retinopathy. METHODS: We performed a phenotypic study and a candidate gene approach in a four-generation family presenting with autosomal-dominant hematuria associated with extrarenal manifestations. Renal biopsy was analyzed for determination of BM thickness and expression of chains of type IV collagen. Linkage to 18 candidate genes/loci was investigated using polymorphic microsatellite markers. RESULTS: In all affected patients, hematuria without proteinuria was associated with muscular contractures and retinal arterial tortuosities responsible for retinal hemorrhages. Cardiac arrhythmia, Raynaud phenomena, and brain MRI abnormalities were also observed. Despite the presence of red cells in tubule sections, no glomerular abnormalities were found by electron microscopy. Expression of type IV collagen chains and glomerular BM thickness was normal. We searched for a molecular defect affecting either BM or angiogenesis. Linkage analyses of genes encoding BM components (COL4A3/COL4A4, COL6A1, COL6A2, COL6A3, FBLN1), and angiogenic factors or their receptors (VHL, ANPT1, ANPT2, TIE, TEK, NOTCH2, NOTCH3, NOTCH4, DLL4, JAG1, JAG2) and of the facio-sapulo-humeral dystrophy and 3q21 loci failed to show segregation of the disease with those gene loci. CONCLUSION: We have identified a new inherited hematuria syndrome associated with retinal vessel tortuosities and contractures. We recommend performing a fundus examination in patients with familial hematuria and episodes of visual impairment, as well as a urinary analysis in patients with retinal arterial tortuosity or congenital muscular contractures.

Collagen Type IV↗

PubMed2000

[CADASIL: genetics and physiopathology].

CADASIL, an autosomal dominant adult onset arteriopathy causing stroke and dementia in humans, is underlaid by a non atherosclerotic non amyloid angiopathy involving mainly the media of small cerebral arteries; it is characterized by major lesions of vascular smooth muscle cells. Using a positional cloning approach, we mapped CADASIL locus on chromosome 19 and identified the mutated gene as being Notch3. This gene, previously unknown in humans, encodes for a large transmembrane receptor belonging to the Notch/lin12 gene family which are known to be involved in cell fate specification during development. Genetic analysis of more than 120 CADASIL unrelated families allowed us to show that these mutations are highly stereotyped and affect only the extra cellular domain of the protein. On the basis of these data, a molecular diagnostic test has been set up and is now widely required by clinicians involved in the diagnosis of vascular leukoencephalopathies. Using this test, we recently showed that CADASIL can also occur in patients who do not have any affected relative due to the existence of notch3 de novo mutations. As a first step to investigate the molecular and cellular mechanisms leading from Notch3 mutations to CADASIL phenotype, we analyzed by in-situ hybridization and immunohistochemistry the pattern of expression of this gene. Notch3 expression is highly restricted to the vascular smooth muscle cell in normal human adults. In CADASIL tissues there is a dramatic accumulation of the extracellular domain of the protein which suggests that one of the main mechanisms of CADASIL involves anomalies in the proteolytical cleavage and clearance of this protein. These data provide important clues to the mechanisms of this condition and current work should lead in the next future to a complete understanding of CADASIL and set up the basis of a rational therapeutical approach of this condition.

Dementia, Multi-Infarct↗

PubMed2002

Coordinate Notch3-hairy-related transcription factor pathway regulation in response to arterial injury. Mediator role of platelet-derived growth factor and ERK.

The Notch family of receptors and downstream effectors plays a critical role in cell fate determination during vascular ontogeny. Moreover, the human cerebral autosomal dominant artriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) syndrome of premature stroke and dementia is a heritable arteriopathy with alterations in vascular smooth muscle cells (VSMCs) resulting from mutations within Notch3. However, the expression and regulation of the Notch and hairy-related transcription factor (HRT) pathway in adult VSMCs in vitro and in vivo remain poorly characterized. The present study documents that the well-described modulation of VSMC fate in response to vascular injury and growth factor activation involves a coordinate regulation of the Notch and HRT pathways. Furthermore, platelet-derived growth factor promotes a similar coordinate down-regulation of the Notch receptors and HRT genes in cultured VSMCs via an ERK-dependent signaling pathway. Moreover, we established that HRT1 and HRT2 are direct downstream target genes of Notch3 signaling in VSMCs and determined that the activity of the nuclear protein RBP-Jk is essential for their regulation. These findings provide initial insight into the context- and cell type-dependent coordinate regulation of Notch3 and downstream HRT transcriptional pathway effector genes in VSMCs in vitro and in vivo that may have important implications for understanding the role of Notch signaling in human health and vascular disease.

Animals↗

PubMed2001

Expression patterns of Notch1, Notch2, and Notch3 suggest multiple functional roles for the Notch-DSL signaling system during brain development.

The Notch-DSL signaling system consists of multiple receptors and ligands, and plays many roles in development. The function of Notch receptors and ligands in mammalian brain, however, is poorly understood. In the current study, we examined the expression patterns for three receptors of this system, Notch1, 2, and 3, in late embryonic and postnatal rat brain by in situ hybridization. The three receptors have overlapping but different patterns of expression. Messenger RNA for all three proteins is found in postnatal central nervous system (CNS) germinal zones and, in early postnatal life, within numerous cells throughout the CNS. Within zones of cellular proliferation of the postnatal brain, Notch1 mRNA is found in both the subventricular and the ventricular germinal zones, whereas Notch2 and Notch3 mRNAs are more highly localized to the ventricular zones. Both Notch1 and Notch3 mRNAs are expressed along the inner aspect of the dentate gyrus, a site of adult neurogenesis. Notch2 mRNA is expressed in the external granule cell layer of the developing cerebellum. In several brain areas, Notch1 and Notch2 mRNAs are relatively concentrated in white matter, whereas Notch3 mRNA is not. Neurosphere cultures (which contain CNS stem cells), purified astrocyte cultures, and striatal neuron-enriched cultures express Notch1 mRNA. However, in these latter cultures, Notch1 mRNA is produced by nestin-containing cells, rather than by postmitotic neurons. Taken together, these results support multiple roles for Notch1, 2, and 3 receptor activation during CNS development, particularly during gliogenesis.

Animals↗

PubMed2004

Differential expression of notch signaling-related transcripts accompanies pro-thymocyte proliferation and phenotype transition induced by epidermal growth factor plus insulin in fetal thymus organ cultures.

Thymus regression upon stressing stimuli, such as infectious diseases, is followed by organ reconstitution, paralleling its development in ontogeny. A narrow window of thymus development was here studied, encompassing the pro-T lymphoid precursor expansion during specification stages, by the use of epidermal growth factor plus insulin (INS) in murine fetal thymus organ cultures. Aiming to disclose signaling pathways related to these stages, cultured thymus lobes had their RNA extracted, for the search of transcripts differentially expressed using RNAse protection assays and reverse transcriptase-polymerase chain reactions. We found no difference that could explain INS-driven thymocyte growth, in the pattern of transcripts for death/proliferation mediators, or for a series of growth factor receptors and transcriptional regulators known as essential for thymus development. Thymocyte suspensions from cultured lobes, stained for phenotype analysis by fluorescence activated cell sorting, showed a decreased staining for Notch1 protein at cell surfaces upon INS addition. We analyzed the expression of Notch-related elements, and observed the recruitment of a specific set of transcripts simultaneous and compatible with INS-driven thymocyte growth, namely, transcripts for Notch3, for its ligand Jagged2, and for Deltex1, a mediator of a poorly characterized alternative pathway downstream of the Notch receptor.

Animals↗

PubMed2003

Long-lasting induction of Notch2 in the hippocampus of kainate-treated adult mice.

Notch proteins are involved in cell fate specification during development in tissues including brain. Little is known about their function in adulthood. Recently, Notch receptors have been hypothesized to play a role in neurodegeneration and in particular in Alzheimer's disease (Notch1) and CADASIL (Notch3). Here we show that another family member (Notch2) is constitutively expressed in adult mouse hippocampus in DG and not in CA1 and CA3 neurons. Treatment with kainic acid resulted in marked Notch2 induction in pyramidal neurons of CA1 and in a subpopulation of CA3 neurons surviving the lesion and protein expression was still detectable 6 weeks after drug treatment. These results suggest Notch2 involvement in the response of postmitotic neurons to excitotoxic stimuli.

Animals↗

PubMed2006

Gene expression signatures differentiate ovarian/peritoneal serous carcinoma from diffuse malignant peritoneal mesothelioma.

PURPOSE: Ovarian/primary peritoneal serous carcinoma (OC/PPC) and diffuse peritoneal malignant mesothelioma (DMPM) are highly aggressive tumors that are closely related morphologically and histogenetically. It remains unclear whether both tumors are molecularly distinct neoplasms. The current study compared global gene expression patterns in OC/PPC and DMPM. EXPERIMENTAL DESIGN: Ten OC/PPC and five DMPM effusions were analyzed for gene expression profiles using the Affymetrix U133 Plus 2 arrays and the dCHIP analysis program. Differentially expressed candidate genes were validated using quantitative real-time PCR and immunohistochemistry. RESULTS: Unsupervised hierarchical clustering using all 54,675 genes in the array classified the samples into two groups: DMPM specimens versus OC/PPC specimens. A total of 189 genes that were differentially expressed in these two groups were selected based on statistical significance. Genes overexpressed in DMPM (n = 68) included calretinin, vitronectin, claudin 15, alpha4 laminin, hyaluronan synthase 1, cadherin 11, RAB7, v-maf, and the epidermal growth factor-containing fibulin-like extracellular matrix protein 1. Genes overexpressed in OC/PPC (n = 121) included insulin-like growth factor II (IGF-II); IGF-II binding protein 3; cyclin E1; folate receptors 1 and 3; RAB25; MUC4; endothelin-1; CD24; kallikreins 6, 7, and 8; claudins 3, 4, and 6; Notch3; and MMP-7. Quantitative real-time PCR validated the differential expression of 13 genes, and immunohistochemistry confirmed the differences for four gene products. CONCLUSIONS: Expression profiling separates OC/PPC from DMPM and identifies a number of genes that are differentially expressed in these tumors. The molecular signatures unique to OC/PPC and DMPM should provide a molecular basis to study both tumors and new potential markers for facilitating their differential diagnosis.

Carcinoma↗

PubMed2004

Constitutively active Notch4 promotes early human hematopoietic progenitor cell maintenance while inhibiting differentiation and causes lymphoid abnormalities in vivo.

Notch transmembrane receptors are known to play a critical role in cell-fate decisions, with Notch1 shown to enhance self-renewal of hematopoietic stem cells and cause T-cell leukemia. Four Notch receptors exist, and the extent of redundancy and overlap in their function is unknown. Notch4 is structurally distinct from Notch1 through Notch3 and has not been extensively studied in hematopoiesis. By polymerase chain reaction (PCR) we find Notch4 transcript expression in human marrow cells and in both CD34(+) and CD34(-) populations. When constitutively active Notch1 or Notch4 was overexpressed in normal human marrow or cord cells, we found reduced colony-forming and short-term proliferative ability while the primitive progenitor content of myeloid long-term cultures was significantly increased. Notch4-intracellular domain (Notch4-IC)-transduced cord cells transplanted into beta(2)-microglobulin(-/-) nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice resulted in significantly higher levels of engraftment of both green fluorescent protein-positive (GFP(+)) and GFP(-) populations as compared with controls. GFP(+) cells in bone marrow and spleen of animals that had received transplants gave rise to an immature CD4(+)CD8(+) T-cell population, whereas B-cell development was blocked. These results indicate that activation of Notch4 results in enhanced stem cell activity, reduced differentiation, and altered lymphoid development, suggesting it may influence both stem cells and the fate of the common lymphoid progenitor.

Animals↗

PubMed1998

Delta-notch signaling in odontogenesis: correlation with cytodifferentiation and evidence for feedback regulation.

Recent data suggest that dental cells utilize the evolutonarily conserved Notch-mediated intercellular signaling pathway to regulate their fates. Here we report on the expression and regulation of Delta1, a transmembrane ligand of the Notch receptors, during mouse odontogenesis. Delta1 is weakly expressed in dental epithelium during tooth initiation and morphogenesis, but during cytodifferentiation, expression is upregulated in the epithelium-derived ameloblasts and the mesenchyme-derived odontoblasts. The expression pattern of Delta1 in ameloblasts and odontoblasts is complementary to Notch1, Notch2, and Notch3 expression in adjacent epithelial and mesenchymal cells. Notch1 and Notch2 are upregulated in explants of dental mesenchyme adjacent to implanted cells expressing Delta1, suggesting that feedback regulation by Delta-Notch signaling ensures the spatial segregation of Notch receptors and ligands. TGFbeta1 and BMPs induce Delta1 expression in dental mesenchyme explants at the stage at which Delta1 is upregulated in vivo, but not at earlier stages. In contrast to the Notch family receptors and their ligand Jagged1, expression of Delta1 in the tooth germ is not affected by epithelial-mesenchymal interactions, showing that the Notch receptors and their two ligands Jagged1 and Delta1 are subject to different regulations.

Animals↗

PubMed2002

Characterization of Notch receptor expression in the developing mammalian heart and liver.

Alagille syndrome (AGS) is an autosomal dominant disorder characterized by bile duct paucity along with cardiovascular, skeletal, and ophthalmologic defects. The identification of JAG1 as the AGS disease gene revealed the crucial role of the Notch signaling pathway in the development of multiple organ systems in humans. Patients with identical mutations in JAG1 demonstrate extreme clinical variability, suggesting that other factors may influence the severity of the developmental defects in this disorder. We have defined the temporal and spatial expression patterns of the Notch receptor genes in the developing mammalian heart and liver in order to identify potential ligand/receptor interactions during embryogenesis. In the developing heart, both Notch1 and Notch2 are expressed in the outflow tracts and the epicardium, in specific cell populations previously shown to express JAG1. These cells are destined to undergo epithelial-to-mesenchymal transformation. In the newborn mouse liver, Notch2 and Notch3 are expressed in opposing cell populations, suggesting they play different roles in cell fate determination during bile duct development. JAG1 is also expressed in cells adjacent to those expressing Notch2, suggesting a possible ligand receptor relationship. The Notch receptors have distinct roles in cell fate determination in different organ systems.

Animals↗

PubMed2005

Relationship between Notch receptors and hyperoxia-induced lung injury in newborn rats.

To investigate role of Notch1 - 3 in hyperoxia-induced lung injury in newborn rat exposed to 85% O2, SD rat litters born on the 22th day were randomly divided into two groups: room air group and hyperoxia group. The animals were sacrificed 1, 4, 7, 10, 14 and 21 days after continued exposure to oxygen (n = 40, oxygen > 0.85) or room air (n = 40). 6 rats each group were used to assess lung histological changes by HE staining and expression of Notch in lungs by immunohistochemistry. Total RNA was extracted by Trizol reagent from frozen lung tissues. Notch mRNA were measured by reverse transcription polymerase chain reaction (RT-PCR). Our results showed that 7, 14 and 21 days after O2 exposure, hyperoxia group showed lung injury characterized by pulmonary edema, hemorrhage and lung development arrest. Positive staining for Notch1, Notch 2 in hyperoxia group was much lower than those in room air group at all time points (P < 0. 01, P < 0.05), but compared with the controls, the hyperoxia group showed higher expression of Notch3 (P > 0.05). Immunostained cells were typically airways epithelia, alveolar epithelial and inflammatory cells, and fibroblasts in hyperoxia group (P < 0.01). Notch mRNA levels showed similar change as protein level (P < 0.01). It is concluded that the prolonged exposure to 85% O2 resulted in abnormal expression of Notch receptors, which might contribute to the pathogenesis of hyperoxia-induced lung injury in newborn rats. The decreased inhibition of Notch1 might be one of the protective reaction and major mechanisms for proliferation/differentiation of type II alveolar epithelial cells. The up-regulation of Notch3 activity might result in the lung development arrest of the newborn rats.

Aerobiosis↗

PubMed2004

Notch expression in developing olfactory neuroepithelium.

Notch genes encode receptors for signaling pathways that regulate neurogenesis in various tissues. To better understand the roles of Notch genes in olfactory neurogenesis, we studied the expression of Notch family in developing mouse olfactory epithelium. In the earliest stage of olfactory development, Notch1 was observed in the mesenchyme lateral to the olfactory placode. During the developmental stage, Notch1 was mainly observed around the basal membrane, while Notch3 was observed in the lower compartment of the olfactory neuroepithelium. Notch2 was not detected during the entire observation period. As the olfactory neuroepithelium grew mature, both Notch1 and Notch3 gradually disappeared. These results suggest distinct roles of Notch1 and Notch3 in the neurogenesis of the peripheral olfactory system.

Animals↗