Notch-related Gene Expression Research Articles

The mechanism of intestinal stem cells differentiation after ischemia-reperfusion injury in a rat model.

Notch and Wnt/β-catenin signaling are responsible for regulation of intestinal stem cells (ISCs) proliferation and differentiation. The purpose of the study was to evaluate Wnt/β-catenin and Notch signaling roles in regulation of ISC differentiation following ischemia-reperfusion (IR) injury in a rat. Rats were assigned into two groups: Sham rats underwent laparotomy without vascular intervention and IR rats underwent occlusion of SMA and portal vein for 20min followed by 48h of reperfusion. Wnt/β-catenin and Notch-related gene expression were determined using Real-Time PCR. Enterocyte proliferation, differentiation and Wnt-related proteins were determined by immunohistochemistry. IR rats demonstrated a significant decrease in β-catenin gene expression, a decrease in cyclin D1 and β-catenin positive cells in jejunum and ileum compared to Sham rats. IR rats demonstrated a significant increase in Notch-related gene expression in jejunum and ileum compared to Sham rats. The number of secretory cells was higher mainly in the jejunum and number of absorptive cells was significantly lower in jejunum and lower in ileum in IR rats compared to Sham rats. Intestinal stem-cell differentiation is toward secretory cells 48h after IR injury; however, Wnt/β-catenin pathway inhibition and Notch-related gene expression stimulation suggest crosstalk between pathways.

A comprehensive analysis of notch signaling genes in breast cancer: Expression pattern and prognostic significance

The most recurrent type of cancer among women is breast cancer which is an intricate disease with high intertumoral and intratumoral heterogeneity. Such variability is a key factor in the failure of current treatments and the emergence of resistance. It is crucial to develop novel therapeutic options to enhance the prognosis for breast cancer patients due to the limitations of current therapy and the unavoidable formation of acquired drug resistance (chemo and endocrine) as well as radio resistance. Poor clinical results in the treatment of breast cancer, that is resistance are associated with deregulated Notch signalling within the breast tumor and its tumor microenvironment (TME). In this research, a bioinformatics approach was used to check the expression pattern, the role, as well as the prognostic and diagnostic significance of the deregulated Notch-related genes in BC patients. The various bioinformatic tools include; UCSC XENA, GEPIA 2, UALCAN, bc Genexminer, KM Plotter, ENRICHR, STRING and Cytoscape. The study demonstrates that highly dysregulated genes (NOTCH4, CCND1, JAG1, DLL1, MAML2, and EGFR) can be used as biomarkers to identify breast cancer patients with poor prognosis and as potential targets for therapeutic intervention. The study found that 6 genes—NOTCH4, CCND1, JAG1, DLL1, MAML2, and EGFR—out of 22 tested genes showed a significant log2 fold change. Our study revealed that Luminal Breast Cancer patients display a high expression of the CCND1 gene in comparison to its expression in normal. The results of our study also depicted that the patients with elevated levels of NOTCH-related gene expression displayed better relapse-free survival with p < 0.05. Moreover, we analysed the deregulated notch genes that play an important role in various cellular and molecular processes. The study shows that these highly deregulated screened genes could be utilized as the Biomarkers that help to reveal poor prognosis and could act as targets for treating BC. However, the identification of these dysregulated genes involved in notch signallibng through insilico approach is not sufficient.

A novel stem cell type at the basal side of the subventricular zone maintains adult neurogenesis.

According to the current consensus, murine neural stem cells (NSCs) apically contacting the lateral ventricle generate differentiated progenitors by rare asymmetric divisions or by relocating to the basal side of the ventricular–subventricular zone (V‐SVZ). Both processes will ultimately lead to the generation of adult‐born olfactory bulb (OB) interneurons. In contrast to this view, we here find that adult‐born OB interneurons largely derive from an additional NSC‐type resident in the basal V‐SVZ. Despite being both capable of self‐renewal and long‐term quiescence, apical and basal NSCs differ in Nestin expression, primary cilia extension and frequency of cell division. The expression of Notch‐related genes also differs between the two NSC groups, and Notch activation is greatest in apical NSCs. Apical downregulation of Notch‐effector Hes1 decreases Notch activation while increasing proliferation across the niche and neurogenesis from apical NSCs. Underscoring their different roles in neurogenesis, lactation‐dependent increase in neurogenesis is paralleled by extra activation of basal but not apical NSCs. Thus, basal NSCs support OB neurogenesis, whereas apical NSCs impart Notch‐mediated lateral inhibition across the V‐SVZ.

Effect of NTN and Lmx1α on the Notch Signaling Pathway during the Differentiation of Human Bone Marrow Mesenchymal Stem Cells into Dopaminergic Neuron-Like Cells.

Human bone marrow mesenchymal stem cells (h-BMSCs) have the potential to differentiate into dopaminergic neuron-like cells to treat Parkinson's disease. The Notch signaling pathway has been implicated in the regulation of cell fate decisions such as differentiation of BMSCs. This study investigated changes in the expression of Notch-related genes in the differentiation of BMSCs in vitro into dopaminergic (DA) neuron-like cells. BMSCs transfected with empty lentiviral vectors served as the control group and those transfected with NTN and Lmx1α recombinant lentiviral vectors served as the experimental group. After induction and culture of NTN and Lmx1α-transfected h-BMSCs for 21 days, the cells exhibited features of dopaminergic neuron-like cells, which were observed by transmission and scanning electron microscopy and verified by immunofluorescence of tyrosine hydroxylase (TH) and dopamine transporter (DAT). These induced cells could secrete dopamine and had basic action potentials. Expression of the neural stem cell (NSC) markers, including octamer-binding protein (Oct4), paired box gene 6 (Pax6), and sex determining region Y-box 1 (SOX1), increased on day 14 of induction and decreased on day 21 of induction during differentiation. The human Notch signaling pathway PCR array showed a differential expression of Notch-related genes during the differentiation of h-BMSCs into DA neuron-like cells in vitro relative to that in the control group. In conclusion, h-BMSCs overexpressing NTN and Lmx1α can successfully be induced to differentiate into dopaminergic neuron-like cells with a neuronal phenotype exhibiting fundamental biological functions in vitro, and NTN and Lmx1α may affect the expression of Notch-related genes during differentiation.

Differential expression of Notch related genes in dental pulp stem cells and stem cells isolated from apical papilla

Although dental pulp and apical papilla are originated from neural crest cells, these tissues exhibit distinct characteristics. Notch signaling is one of the known signaling pathways regulating stemness and behaviors of stem cells. The aim of this study was to examine Notch signaling related gene expression profile comparing between coronal pulp tissues and apical pulp complex. Results demonstrated that coronal pulp tissue had higher expression levels of various genes in Notch pathway. However, NOTCH2, MAML2, DTX4, and NEDD4 mRNA levels were significantly lower in coronal pulp tissue than those of apical pulp complex. Furthermore, dental pulp stem cells (DPSCs) and stem cells isolated from apical papilla (SCAPs) were isolated and characterized. These two cell types exhibited similar mesenchymal stem cell surface markers. DPSCs expressed higher mRNA levels of NOTCH3, NOTCH4, DLL1, and HES1. In addition, SCAPs demonstrated higher colony formation and cell proliferation than DPSCs. In summary, cells and tissues from dental pulp and apical papilla exhibited the distinct gene expression profile of Notch related genes. This could be of one the signaling participated in control of DPSCs and SCAPs cells behaviors.

Effects of bisphenol A and its alternative bisphenol F on Notch signaling and intestinal development: A novel signaling by which bisphenols disrupt vertebrate development

We previously found bisphenol A (BPA) alternative, bisphenol F (BPF) upregulated Notch-related gene expression in intestines of the African clawed frog Xenopus laevis, suggesting an agonistic action on Notch signaling, a crucial signaling in multiple biological processes during development. Here, we aimed to confirm the actions of BPA and BPF on Notch signaling and to reveal their effects on intestinal development. Using X. laevis, an excellent model for developmental biology, we found that 10–1000 nM BPA and BPF significantly elevated Notch-related gene expression in a concentration-dependent manner. Subsequently, exceptional cell proliferation as well as intestinal histological changes were observed in treated intestines. Importantly, Notch inhibitor markedly suppressed the effects of BPA and BPF described above. Furthermore, we employed rat intestinal epithelium cells (IEC-6), an ideal in vitro model of intestinal epithelial cell differentiation, to confirm the effects of bisphenols. As expected, BPA and BPF upregulated Notch-related gene expression and induced the translocation of the Notch intracellular domain to the nucleus, followed by exceptional cell proliferation and differentiation, whereas Notch inhibitor antagonized the effects caused by BPA and BPF. All results strongly demonstrate that both BPA and BPF activate Notch signaling and subsequently disrupt intestinal development in vertebrates. Given its fundamental roles in multiple developmental processes, we propose that Notch signaling is an important and general target signaling of bisphenols in many developing tissues of vertebrates including humans.

Inhibition of NOTCH signaling pathway chemosensitizes HCC CD133+ cells to vincristine and 5-fluorouracil through upregulation of BBC3

In hepatocellular carcinoma (HCC), the poor response to the chemotherapeutic agents is partially attributed to the chemoresistance property of cancer stem cells (CSCs). NOTCH signaling pathway plays a crucial role in the chemoresistance through the maintenance of the CSCs. We observed that the NOTCH pathway was activated in HCC CD133+ cells treated with vincristine (VIN)1 and 5-fluorouracil (5-FU)2. Therefore, we examined whether inhibition of the NOTCH can improve sensitization of HCC CD133+ cells to VIN and 5-FU. The Huh7 cell line was pre-incubated γ-secretase DAPT, as a NOTCH inhibitor, and then treated with IC50 dose of VIN or 5-FU. The CD133+ cells were then isolated and analyzed for the cell viability, apoptosis, migration and spheroid formation capacities, and gene and protein expression. It was observed that pre-incubation with DAPT significantly downregulated the expression of NOTCH-related genes and led to a significant reduction in VIN- and 5-FU-CD133+ population. In addition, DAPT pre-incubated VIN- and 5-FU-treated-CD133+ cells formed fewer spheroids in 3D culture and had a lesser migration capacity in 2D culture. Importantly, DAPT enhanced the apoptosis rate of VIN- and 5-FU-treated CD133+ cells for 3- and 2-fold, which was correlated with the enhanced expression of pro-apoptotic BBC3 (BCL-2-binding component 3) and decreased expression of HES1 that was reported to regulate BBC3 negatively. Collectively, it was observed that NOTCH inhibition sensitized the HCC CD133+ cells to VIN and 5-FU through enhancing BBC3-mediated apoptosis. The results highlighted the role of NOTCH/HES1/BBC3 axis in resistance of CD133+ cells to VIN and 5-FU. Understanding the molecular mechanisms underlying chemoresistance in HCC CD133+ cells may help in designing the novel targeted therapies to chemosensitize them.

Low Concentrations of Tetrabromobisphenol A Disrupt Notch Signaling and Intestinal Development in in Vitro and in Vivo Models.

Tetrabromobisphenol A (TBBPA) was recently reported to upregulate Notch target gene expression in embryonic stem cells differentiating to neurons in vitro, implying activation on Notch signaling, a crucial signaling involved in multiple organ development and homeostasis.The present study aimed to determine whether TBBPA at low concentrations can disrupt Notch signaling in the intestine and subsequently its development using in vitro and in vivo models, given TBBPA uptake mainly via the intestine. In rat intestinal epithelium cells (IEC-6), an in vitro model for intestinal development and homeostasis, we found 5-500 nM TBBPA upregulated Notch-related gene expression and stimulated cell proliferation as well as the growth of microvilli in a linear concentration-dependent manner. When Notch inhibitor DAPT had no obvious effects on all end points, DAPT significantly antagonized all changes caused by TBBPA, indicating that TBBPA activated Notch signaling in IEC-6 cells and subsequently stimulated cell proliferation and differentiation. Then we employed Xenopus laevis, an ideal model species for intestinal development with the strong similarities to mammals, to further confirm the action of TBBPA in vivo. Expectedly, we observed the stimulatory effects of TBBPA on Notch signaling and cell proliferation and differentiation in X. laevis intestines, which agrees with the results in vitro. Antagonistic actions of Notch inhibitor DBZ on TBBPA-caused intestinal changes show that TBBPA affected intestinal development via disrupting Notch signaling. Interestingly, TBBPA stimulated cell differentiation into secretory cells, which is generally believed to be regulated by Wnt signaling, suggesting disruption of Wnt signaling besides Notch signaling. All the results for the first time demonstrate that TBBPA at low concentrations, including environmentally relevant concentrations, disrupt Notch signaling and subsequently affect intestinal development by altering cell proliferation and differentiation in vertebrates. Our study highlights the intestine as a new target of TBBPA and broaden our understanding of developmental toxicity of TBBPA.

Dysregulation of Notch signaling in cardiac mesenchymal cells of patients with tetralogy of Fallot.

Tetralogy of Fallot (TF) is a severe congenital defect of heart development. Fine-tuned sequential activation of Notch signaling genes is responsible for proper heart chamber development. Mutations in Notch genes have been associated with TF. The aim of this study was to analyze the activity of the Notch pathway in cardiac mesenchymal cells derived from ventricular tissue of TF patients. Cardiac mesenchymal cells were isolated from 42 TF patients and from 14 patients with ventricular septal defects (VSDs), used as a comparison group. The Notch pathway was analyzed by estimating the expression of Notch-related genes by qPCR. Differentiation and proliferation capacity of the cells was estimated. The TF-derived cells demonstrated a dysregulated pattern of Notch-related gene expression comparing to VSD-derived cells. Correlation of Notch signaling activation level by HEY1/HES1 expression level with proliferation and cardiogenic-like differentiation of cardiac mesenchymal cells was observed but not with clinical parameters nor with the age of the patients. The data suggest a contribution of dysregulated Notch signaling to the pathogenesis of tetralogy of Fallot and importance of Notch signaling level for the functional state of cardiac mesenchymal cells, which could be critical considering these cells for potential cell therapy approaches.

Abstract 416: Approach of mRNA Delivery by Using Nanoparticles to Recover Endocardial Notch Signaling

Notch signaling plays a significant role in cardiac development and morphogenesis, especially for cardiac chamber development. Failure of mechanotransduction of endocardial Notch signaling in ventricular chamber causes lack of trabeculation. Advent of nanoparticle (NP) technology offers a therapeutic approach of targeted delivery. Although delivering mRNA is an effective method, it is challenging due to stability and cell defense mechanisms. To overcome the limitations and effectively deliver mRNA to endocardium, we used Poly(lactic-co-glycolic acid) (PLGA) synthesized NPs, which have been used in various drug delivery systems due to its biocompatibility, efficient encapsulation of hydrophilic and hydrophobic materials, specific targeting effects, and lower payload concentration. We hypothesized our PLGA NPs are able to deliver NICD mRNA to target endocardium to overexpress Notch signaling in a Notch inhibited zebrafish animal model. Using double emulsion, we synthesized PLGA NPs at 55–65 kDa and performed in vitro test on HUVECs. 69.8 ± 4.3% of Rhodamine B was successfully loaded into the NPs, and after incubation with HUVECs the signal lasted for 28 days after burst release. Hemolysis and whole blood clotting assays proved that NPs did not interfere with the blood clotting process or lyse blood cells. We characterized mRNA incorporated PLGA nanoparticles and incubated with HUVECs after antibody conjugation. To test in vivo study, we injected gata1a morpholino oligomer (MO) to Tg(fli1:gfp) transgenic line to inhibit hematopoiesis, which reduces wall shear stress (WSS) of ventricular chamber leading lack of trabeculae. At 2 days post fertilization (dpf), NICD mRNA incorporated nanoparticles were injected into the circulatory system of gata1a MO injected zebrafish to rescue trabeculation. At 5 dpf, zebrafish hearts were harvested and analyzed via qRT-PCR to compare Notch related gene expression among control, gata1a MO injected, and rescue group. The NICD mRNA injected group successfully rescued the expression level of Notch components despite a lack of WSS and mechanosensitive Notch signaling via gata1a MO injection. This finding will provide an initial therapeutic approach of NICD mRNA for trabecular related congenital heart disease.

Abstract B061: Targeting lymphomas with the novel first-in-class pan-NOTCH transcription inhibitor CB-103

Abstract Background: NOTCH pathway is recurrently activated in human cancers, including lymphomas, and overactivation of the pathway has been correlated to a worse prognosis in patients with solid tumors, leukemias, and lymphomas, making it an attractive target for treatment (Rossi et al, JEM 2012; Rossi et al, Blood 2012; Kridel et al, Blood 2012). However, so far, pharmacologic approaches to inhibit its activity are limited to the use of nonspecific gamma secretase inhibitors and blocking antibodies with limited application. None of these therapeutic agents have been approved yet for cancer treatment. CB-103 is a novel first-in-class pan-NOTCH inhibitor that blocks signaling by directly and specifically targeting the NOTCH transcription complex, and it has shown preclinical activity in a variety of solid tumor and leukemia models (Lehal et al, AACR 2017). Here, we present the very first results with CB-103 in a large panel of B and T cell lymphoma cell lines. Methods: 58 human lymphoma cell lines [27 diffuse large B cell lymphomas (DLBCL); 9 mantle cell lymphomas; 6 marginal zone lymphomas; 9 T-cell lymphomas; 7 others], 2 murine and 1 canine, were exposed to increasing doses of CB-103 for 72h. Cell proliferation was measured by using the MTT assay. Transcriptome analysis was performed using the Illumina HumanHT 12 Expression BeadChips and with the HTG EdgeSeq Oncology Biomarker Panel, a targeted RNA-Seq investigating over 2500 genes, followed by a limma moderate t-test, and GSEA. Results: CB-103 presented a median IC50 above 20 μM across the whole panel of 61 lymphoma cell lines (range from 400 nM to &amp;gt; 20 μM), without significant differences among lymphoma subtypes. Importantly, 13/58 cell lines had an IC50 &amp;lt;10 μM and were defined as sensitive. These cell lines were derived from ABC DLBCL (3/7, 43%), GCB DLBCL (5/20, 25%), MCL (3/9, 33%), marginal zone lymphoma (1/6, 17%), and canine DLBCL (1/1, 100%). The sensitive cell lines, when compared with those presenting IC50 &amp;gt;10 μM, presented a gene expression signature significantly enriched with genes involved in the epithelial-mesenchymal transition (FDR 0.0014), a Notch-related process. Indeed, the transcripts higher in the sensitive cell lines included, among others, DLL1 (P 0.010; Log Fold Change 2.39), BAMBI (P 0.046; Log Fold Change 0.92), MCM7 (P 0.015; Log Fold Change 1), and ULK1 (P 0.045; Log Fold Change 2.39), all connected to NOTCH pathway. Finally, 10 of the CB-103 sensitive human cell lines were also exposed to another NOTCH inhibitor, the gamma secretase inhibitor LY-3039187, which resulted active only in one MCL cell line, while it had IC50 &amp;gt;20 μM in the other cell lines tested. Conclusions: CB-103 presented in vitro antilymphoma activity in a restricted number of B cell lymphoma cell lines, which were characterized by a NOTCH-related gene expression signature. Further studies with the compound are ongoing and a phase I is planned to start in Q3 2017 (EUDRACT Number 2017-001491-35). Citation Format: Filippo Spriano, Chiara Tarantelli, Alberto Arribas, Eugenio Gaudio, Luciano Cascione, Luca Aresu, Emanuele Zucca, Davide Rossi, Anastasios Stathis, Maximilien Murone, Dirk Weber, Rajwinder Lehal, Freddy Radtke, Francesco Bertoni. Targeting lymphomas with the novel first-in-class pan-NOTCH transcription inhibitor CB-103 [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B061.

Different Notch signaling in cells from calcified bicuspid and tricuspid aortic valves

AimsCalcific aortic valve disease is the most common heart valve disease in the Western world. Bicuspid and tricuspid aortic valve calcifications are traditionally considered together although the dynamics of the disease progression is different between the two groups of patients. Notch signaling is critical for bicuspid valve development and NOTCH1 mutations are associated with bicuspid valve and calcification. We hypothesized that Notch-dependent mechanisms of valve mineralization might be different in the two groups. Methods and resultsWe used aortic valve interstitial cells and valve endothelial cells from patients with calcific aortic stenosis with bicuspid or tricuspid aortic valve. Expression of Notch-related genes in valve interstitial cells by qPCR was different between bicuspid and tricuspid groups. Discriminant analysis of gene expression pattern in the interstitial cells revealed that the cells from calcified bicuspid valves formed a separate group from calcified tricuspid and control cells. Interstitial cells from bicuspid calcified valves demonstrated significantly higher sensitivity to stimuli at early stages of induced proosteogenic differentiation and were significantly more sensitive to the activation of proosteogenic OPN, ALP and POSTIN expression by Notch activation. Notch-activated endothelial-to-mesenchymal transition and the corresponding expression of HEY1 and SLUG were also more prominent in bicuspid valve derived endothelial cells compared to the cells from calcified tricuspid and healthy valves. ConclusionEarly signaling events including Notch-dependent mechanisms that are responsible for the initiation of aortic valve calcification are different between the patients with bicuspid and tricuspid aortic valves.

Activation and dynamic expression of Notch signalling in dental pulp cells after injury invitro and invivo.

To investigate the expression pattern of Notch signalling in odontoblast-like cells stimulated by lipopolysaccharide (LPS) invitro, and in injured rat dental pulp invivo. Mouse odontoblast-like cells (MDPC-23) were exposed to LPS. Expression of Notch-related genes was detected by real-time PCR. A rat pulpitis model was established by mechanical injury and LPS plus mechanical injury was followed by the analysis of expression of Notch2 by immunohistochemical staining. One-way analysis of variance (anova) was performed to examine the effect of differing concentrations of LPS on cell proliferation, and least significant difference test was used for paired comparisons. For independent sample, t-test was performed to compare the expression of Notch signalling genes between LPS group and control group invitro. The invitro study revealed the proliferation of MDPC-23 cells on exposure to 10ngmL-1 to 1μgmL-1 LPS. Expression of Notch1 and Notch2 was significantly higher in the LPS group than that in the control group on day 1 and day 3 (P˂0.05). The levels of both Delta1 and Jagged1 were higher in the study group than in the control group on day 3 (P=0.019 and P=0.034) and day 5 (P˂0.001 and P=0.046), respectively. In addition, Hes1 levels were significantly higher in the study group than in the control group on day 5 (P=0.005). The invivo study demonstrated positive staining for Notch2, both in the mechanical injury (MI) group and in the LPS plus mechanical injury (LMI) group from day 3 to day 7, which showed very weak or absent staining on day 14, thereby demonstrating the dynamic nature of the change. Both invitro and invivo activation and dynamic expression of Notch signalling in dental pulp cells after injury were found. Notch signalling activation by LPS stimulation or mechanical injury showed a similar pattern invivo.

Determination of Relative Notch1 and γ-Secretase-Related Gene Expression in Puromycin-Treated Microdissected Rat Kidneys

Notch signaling pathways are involved in the regulation of cell differentiation and are highly conserved across species. Notch ligand binding leads to gamma-secretase-mediated proteolytic cleavage of the Notch receptor releasing the Notch intracellular domain, resulting in its subsequent translocation into the nucleus and gene expression regulation. To investigate the level of expression of Notch signaling pathway components in microanatomic regions following renal injury, kidneys from untreated, vehicle control, and puromycin aminonucleoside (PA, 150 mg/kg)-treated rats were evaluated. Frozen tissue sections from rats were microdissected using laser capture microdissection (LCM) to obtain glomeruli, cortical (proximal) tubules, and collecting ducts, and relative gene expression levels of Presenilin1, Notch1 and Hes1 were determined. In untreated rats, the Notch1 expression in glomeruli was higher than in the proximal tubules and similar to that in collecting ducts, whereas Presenilin1 and Hes1 expressions were highest in the collecting ducts, followed by cortical tubules and glomeruli. Following PA-induced renal injury, Hes1 gene expression increased significantly in the glomeruli and tubules compared to the collecting ducts where no injury was observed microscopically. Although these data present some evidence of change in Notch signaling related to injury, the expression of Presenilin1, Notch1, and Hes1 in the microanatomic regions of the kidney following PA treatment were not significantly different when compared to controls. These results demonstrate that there are differences in Notch-related gene expression in the different microanatomic regions of the kidneys in rats and suggest a minimal role for Notch in renal injury induced by PA. In addition, this work shows that LCM coupled with the RT-PCR can be used to determine the relative differences in target gene expression within regions of a complex organ.

Expression of Notch signalling-related genes in normal and differentiating rat dental pulp cells

Notch signalling is of fundamental importance to various processes during embryonic development and in adults. The possible role of Hey1, an important Notch signalling component, in odontoblast differentiation was evaluated in this study. Primary cultured dental pulp cells, derived from upper incisors of 5-week-old Wistar rats, were placed in alpha-modification of Eagle's minimal essential medium supplemented with 10% Fetal Bovine Serum (FBS), and ascorbic acid (AA) and beta-glycerophosphate (beta-GP), with or without dexamethasone, and cultured on dishes coated with collagen type IA for 7 days. Conventional and real-time Polymerase Chain Reaction (PCR) was performed to determine the expression of Notch-related genes and dentin sialophosphoprotein as a marker of odontoblast differentiation. Dentin sialophosphoprotein and Hey1 expression was significantly increased and decreased in the presence of AA + beta-GP compared with controls, respectively. These findings suggest that Hey1 may be a negative regulator in odontoblast differentiation.

Notch Signaling Influences Neuroprotective and Proliferative Properties of Mature Müller Glia

Notch signaling is known to play important roles during retinal development. Recently, Notch signaling has been shown to be active in proliferating Müller glia in acutely damaged chick retina (Hayes et al., 2007). However, the roles of Notch in mature, undamaged retina remain unknown. Thus, the purpose of this study was to determine the role of the Notch-signaling pathway in the postnatal retina. Here we show that components of the Notch-signaling pathway are expressed in most Müller glia at low levels in undamaged retina. The expression of Notch-related genes varies during early postnatal development and across regions, with higher expression in peripheral versus central retina. Blockade of Notch activity with a small molecule inhibitor before damage was protective to retinal interneurons (amacrine and bipolar cells) and projection neurons (ganglion cells). In the absence of damage, Notch is upregulated in retinas treated with insulin and FGF2; the combination of these factors is known to stimulate the proliferation and dedifferentiation of Müller glia (Fischer et al., 2002b). Inhibition of Notch signaling during FGF2 treatment reduces levels of the downstream effectors of the MAPK-signaling pathway-p38 MAPK and pCREB in Müller glia. Further, inhibition of Notch activity potently inhibits FGF2-induced proliferation of Müller glia. Together, our data indicate that Notch signaling is downstream of, and is required for, FGF2/MAPK signaling to drive the proliferation of Müller glia. In addition, our data suggest that low levels of Notch signaling in Müller glia diminish the neuroprotective activities of these glial cells.

Mesenchymal stem cells regulate the proliferation and differentiation of neural stem cells through Notch signaling

The effects of mesenchymal stem cells (MSCs) on proliferation and cell fate determination of neural stem cells (NSCs) have been investigated. NSCs were co-cultured with MSCs or NIH3T3 cells using an in vitro transwell system. After 4 days, immunofluorescence staining showed that the number of cells positive for the cell proliferation antigen, ki-67, in neurospheres in MSCs was greater than in NIH3T3 cells. In some experiments, the top-layers of MSCs and NIH3T3 cells were removed to induce NSCs differentiation. Seven days after initiating differentiation, the levels of the neuronal marker, NSE, were higher in NSCs in MSCs co-culture group, and those of glial fibrillary acidic protein (GFAP) were lower, compared with NIH3T3 cells co-culture group. These were confirmed by immunofluorescence. The role of the Notch signaling pathway analyzed with the specific inhibitor, DAPT, and by examining the expression of Notch-related genes using RT-PCR showed that after co-culturing with MSCs for 24h, NSCs expressed much higher levels of ki-67, Notch1, and Hes1 than did NSCs co-cultured with NIH3T3 cells. Treatment with DAPT decreased ki-67, Notch1 and Hes1 expression in NCSs, and increased Mash1 expression. The data indicate that the interactions between MSCs and NSCs promote NSCs proliferation and are involved in specifying neuronal fate, mediated in part by Notch signaling.

Down-regulation of the Notch Pathway in Human Airway Epithelium in Association with Smoking and Chronic Obstructive Pulmonary Disease

The airway epithelium of smokers is subject to a variety of mechanisms of injury with consequent modulation of epithelial regeneration and disordered differentiation. Several signaling pathways, including the Notch pathway, control epithelial differentiation in lung morphogenesis, but little is known about the role of these pathways in adults. We tested the hypotheses that Notch-related genes are expressed in the normal nonsmoker small airway epithelium of human adults, and that Notch-related gene expression is down-regulated in healthy smokers and smokers with chronic obstructive pulmonary disease (COPD). We used microarray technology to evaluate the expression of 55 Notch-related genes in the small airway epithelium of nonsmokers. We used TaqMan quantitative polymerase chain reaction (PCR) to confirm the expression of key genes and we used immunohistochemistry to assess the expression of Notch-related proteins in the airway epithelium. Changes in expression of Notch genes in healthy smokers and smokers with COPD compared with nonsmokers were evaluated by PCR. Microarray analysis demonstrated that 45 of 55 Notch-related genes are expressed in the small airway epithelium of adults. TaqMan PCR confirmed the expression of key genes with highest expression of the ligand DLL1, the receptor NOTCH2, and the downstream effector HES1. Immunohistochemistry demonstrated the expression of Jag1, Notch2, Hes1, and Hes5 in airway epithelium. Several Notch ligands, receptors, and downstream effector genes were down-regulated in smokers, with more genes down-regulated in smokers with COPD than in healthy smokers. These observations are consistent with the hypothesis that the Notch pathway likely plays a role in the human adult airway epithelium, with down-regulation of Notch pathway gene expression in association with smoking and COPD.

Localisation of members of the notch system and the differentiation of vibrissa hair follicles: receptors, ligands, and fringe modulators.

Hair vibrissa follicle morphogenesis involves several cell segregation phases, in the dermis as well as in the epidermis. The expression of Notch-related genes, which are well established mediators of multiple cell segregation events in Drosophila development, was studied by in situ hybridisation during embryonic mouse vibrissa follicle morphogenesis and the first adult hair cycle. The results show that two receptors, Notch1 and -2, three ligands, Delta1, Serrate1, and -2, and the three Fringe regulators, Lunatic, Manic, and Radical, are expressed in different locations and morphogenetic stages. First, the appearance of hair vibrissa primordia involves the expression of complementary patterns of Notch2, Delta1, and Lunatic Fringe in the dermis and of Notch1, Serrate2, and Lunatic Fringe in the epidermis. Second, this expression pattern is no longer found after stage 3 in the dermis. Meanwhile, in the epidermis, the expression of Notch1, Serrate2, and Lunatic Fringe before the formation of the placode may be involved in determining two populations of epidermal cells in the developing follicle. Third, complementary expression patterns for Notch1, Manic, and Lunatic Fringe, as well as Serrate1 and -2 as previously shown (Powell et al., 1998), are progressively established from stage 4 of embryonic development both in the outer root sheath and in the hair matrix. These patterns are consistent with the one found in the adult anagen phase. During the hair vibrissa cycle, Notch1 and Manic Fringe display temporal and spatial changes of expression, suggesting that they may intervene as modulators of trichocyte activities.

Ectopic expression of lunatic Fringe leads to downregulation of Serrate-1 in the developing chick neural tube; analysis using in ovo electroporation transfection technique

Lunatic Fringe ( l-Fng) is one of the vertebrate homologues of Drosophila Fringe, which interacts with the Notch signal pathway and regulates activation of the Notch ligands, Delta and Serrate. To elucidate the roles of l-Fng in vertebrate neurogenesis, we transfected chick l-Fng (C- l-Fng) to chick neural tube using the in ovo electroporation technique and examined the subsequent changes in expression of Notch-related genes. We observed downregulation of C- Serrate-1 by ectopic C- l-Fng expression which implied that C- l-Fng acts on the vertebrate Notch pathway to regulate the expression of its ligand.