Notch1 and Notch3 coordinate for pericyte-induced stabilization of vasculature.

The Notch pathway regulates complex patterning events in many species and is critical for the proper formation and function of the vasculature. Despite this importance, how the various components of the Notch pathway work in concert is still not well understood. For example, NOTCH1 stabilizes homotypic endothelial junctions, but the role of NOTCH1 in heterotypic interactions is not entirely clear. NOTCH3, on the other hand, is essential for heterotypic interactions of pericytes with the endothelium, but how NOTCH3 signaling in pericytes impacts the endothelium remains elusive. Here, we use in vitro vascular models to investigate whether pericyte-induced stabilization of the vasculature requires the cooperation of NOTCH1 and NOTCH3. We observe that both pericyte NOTCH3 and endothelial NOTCH1 are required for the stabilization of the endothelium. Loss of either NOTCH3 or NOTCH1 decreases the accumulation of VE-cadherin at endothelial adherens junctions and increases the frequency of wider, more motile junctions. We found that DLL4 was the key ligand for simulating NOTCH1 activation in endothelial cells and observed that DLL4 expression in pericytes is dependent on NOTCH3. Altogether, these data suggest that an interplay between pericyte NOTCH3 and endothelial NOTCH1 is critical for pericyte-induced vascular stabilization.

Blockade of Dopamine D2 Receptors as a Novel Approach to Stimulation of Notch1+ Endothelial Progenitor Cells and Angiogenesis in C57BL/6 Mice with Pulmonary Emphysema Induced by Proteases and Deficiency of α1-Antitrypsin.

We studied the effects of spiperone, a selective blocker of dopamine D2 receptors, on the model of pulmonary emphysema provoked by administration of elastase and D-galactosamine hydrochloride to female C57BL/6 mice and characterized by activation of proteases in the lungs and systemic deficiency of its inhibitor α1-antitrypsin. In this model, spiperone prevented the development of inflammatory reaction and reduced the area of emphysematous expanded alveolar tissue. The expression of angiogenic marker CD31 in the lungs increased under these conditions. Regeneration of the damaged microvascular bed under the action of spiperone resulted from recruiting of Notch1+ endothelial progenitor cells (CD45-CD31+CD34+) into the lungs and blockade of the inhibitory effect of dopamine on phosphorylation of VEGF-2 receptors in endothelial cells of different maturity. In addition, spiperone produced a protective effect on hepatocytes and restored the production and secretion of α1-antitrypsin by these cells.

CBX4 exhibits oncogenic activities in breast cancer via Notch1 signaling.

Polycomb chromobox (CBX) proteins are involved in gene silencing to function as oncogenes or tumor suppressors through the polycomb repressive complex (PRC1). CBX4 has been implicated in the progression of human cancers, but its role and clinical significance in breast cancer remain unclear. Here, we show that CBX4 is up-regulated in breast cancer and exerts oncogenic activities via miR-137-mediated activation of Notch1 signaling pathway. CBX4 expression was increased in breast cancer, compared with the nontumorous tissues. High CBX4 expression was closely correlated with tumor metastasis, advanced clinical stage and poor overall survival in a cohort of 179 patients with breast cancer. In vitro studies demonstrated that CBX4 overexpression enhanced, whereas CBX4 knockdown inhibited cell growth and migration. Mechanistically, in a PRC1-dependent manner, CBX4 inhibited the promoter activity of miR-137 and suppressed its expression. miR-137 decreased the expression of Notch1, Jag1 and Hey2 via targeting their 3'-UTRs. The suppression of Notch1 by siRNA or overexpression of miR-137 markedly attenuated CBX4-promoted phenotypes. Collectively, these findings indicate that CBX4 promotes breast cancer via miR-137-mediated Notch1 signaling. Our data, therefore, suggest that CBX4 serve as a prognostic biomarker and that targeting CBX4/miR-137 axis may provide therapeutic potent in the treatment of breast cancer.

Mammalian Numb protein antagonizes Notch by controlling postendocytic trafficking of the Notch ligand Delta-like 4.

The biological antagonism between the signaling proteins Numb and Notch has been implicated in the regulation of many developmental processes, especially in asymmetric cell division. Mechanistic studies show that Numb inactivates Notch via endocytosis and proteasomal degradation that directly reduce Notch protein levels at the cell surface. However, some aspects of how Numb antagonizes Notch remain unclear. Here, we report a novel mechanism in which Numb acts as a Notch antagonist by controlling the intracellular destination and stability of the Notch ligand Delta-like 4 (Dll4) through a postendocytic-sorting process. We observed that Numb/Numblike knockdown increases the stability and cell-surface accumulation of Dll4. Further study indicated that Numb acts as a sorting switch to control the postendocytic trafficking of Dll4. Of note, the Numb/Numblike knockdown decreased Dll4 delivery to the lysosome, while increasing the recycling of Dll4 to the plasma membrane. Moreover, we demonstrate that this enrichment of Dll4 at the cell surface within Numb/Numblike knockdown cells could activate Notch signaling in neighboring cells. We also provide evidence that Numb negatively controls the Dll4 plasma membrane recycling through a well-documented recycling regulator protein AP1. In conclusion, our study has uncovered a molecular mechanism whereby Numb regulates the endocytic trafficking of the Notch ligand Dll4. Our findings provide a new perspective on how Numb counteracts Notch signaling and sheds additional critical insights into the antagonistic relationship between Numb and Notch signaling.

Estrogen receptor ß-dependent Notch1 activation protects vascular endothelium against tumor necrosis factor α (TNFα)-induced apoptosis.

Unlike age-matched men, premenopausal women benefit from cardiovascular protection. Estrogens protect against apoptosis of endothelial cells (ECs), one of the hallmarks of endothelial dysfunction leading to cardiovascular disorders, but the underlying molecular mechanisms remain poorly understood. The inflammatory cytokine TNFα causes EC apoptosis while dysregulating the Notch pathway, a major contributor to EC survival. We have previously reported that 17ß-estradiol (E2) treatment activates Notch signaling in ECs. Here, we sought to assess whether in TNFα-induced inflammation Notch is involved in E2-mediated protection of the endothelium. We treated human umbilical vein endothelial cells (HUVECs) with E2, TNFα, or both and found that E2 counteracts TNFα-induced apoptosis. When Notch1 was inhibited, this E2-mediated protection was not observed, whereas ectopic overexpression of Notch1 diminished TNFα-induced apoptosis. Moreover, TNFα reduced the levels of active Notch1 protein, which were partially restored by E2 treatment. Moreover, siRNA-mediated knockdown of estrogen receptor ß (ERß), but not ERα, abolished the effect of E2 on apoptosis. Additionally, the E2-mediated regulation of the levels of active Notch1 was abrogated after silencing ERß. In summary, our results indicate that E2 requires active Notch1 through a mechanism involving ERß to protect the endothelium in TNFα-induced inflammation. These findings could be relevant for assessing the efficacy and applicability of menopausal hormone treatment, because they may indicate that in women with impaired Notch signaling, hormone therapy might not effectively protect the endothelium.

Mediator 1 contributes to enamel mineralization as a coactivator for Notch1 signaling and stimulates transcription of the alkaline phosphatase gene.

Tooth enamel is mineralized through the differentiation of multiple dental epithelia including ameloblasts and the stratum intermedium (SI), and this differentiation is controlled by several signaling pathways. Previously, we demonstrated that the transcriptional coactivator Mediator 1 (MED1) plays a critical role in enamel formation. For instance, conditional ablation of Med1 in dental epithelia causes functional changes in incisor-specific dental epithelial stem cells, resulting in mineralization defects in the adult incisors. However, the molecular mechanism by which Med1 deficiency causes these abnormalities is not clear. Here, we demonstrated that Med1 ablation causes early SI differentiation defects resulting in enamel hypoplasia of the Med1-deficient molars. Med1 deletion prevented Notch1-mediated differentiation of the SI cells resulting in decreased alkaline phosphatase (ALPL), which is essential for mineralization. However, it does not affect the ability of ameloblasts to produce enamel matrix proteins. Using the dental epithelial SF2 cell line, we demonstrated that MED1 directly activates transcription of the Alpl gene through the stimulation of Notch1 signaling by forming a complex with cleaved Notch1-RBP-Jk on the Alpl promoter. These results suggest that MED1 may be essential for enamel matrix mineralization by serving as a coactivator for Notch1 signaling regulating transcription of the Alpl gene.

Oral feeding with polyunsaturated fatty acids fosters hematopoiesis and thrombopoiesis in healthy and bone marrow-transplanted mice.

Hematopoietic stem cells play the vital role of maintaining appropriate levels of cells in blood. Therefore, regulation of their fate is essential for their effective therapeutic use. Here we report the role of polyunsaturated fatty acids (PUFAs) in regulating hematopoiesis which has not been explored well so far. Mice were fed daily for 10 days with n-6/n-3 PUFAs, viz. linoleic acid (LA), arachidonic acid (AA), alpha-linolenic acid and docosahexanoic acid (DHA) in four separate test groups with phosphate-buffered saline fed mice as control set. The bone marrow cells of PUFA-fed mice showed a significantly higher hematopoiesis as assessed using side population, Lin-Sca-1+ckit+, colony-forming unit (CFU), long-term culture, CFU-spleen assay and engraftment potential as compared to the control set. Thrombopoiesis was also stimulated in PUFA-fed mice. A combination of DHA and AA was found to be more effective than when either was fed individually. Higher incorporation of PUFAs as well as products of their metabolism was observed in the bone marrow cells of PUFA-fed mice. A stimulation of the Wnt, CXCR4 and Notch1 pathways was observed in PUFA-fed mice. The clinical relevance of this study was evident when bone marrow-transplanted recipient mice, which were fed with PUFAs, showed higher engraftment of donor cells, suggesting that the bone marrow microenvironment may also be stimulated by feeding with PUFAs. These data indicate that oral administration of PUFAs in mice stimulates hematopoiesis and thrombopoiesis and could serve as a valuable supplemental therapy in situations of hematopoietic failure.

Notch1 signalling inhibits apoptosis of human dental follicle stem cells via both the cytoplasmic mitochondrial pathway and nuclear transcription regulation.

Dental follicle stem cells (DFSCs) have been considered as promising candidate cells for periodontal tissue regeneration. Understanding the signalling pathways underlying the apoptosis of DFSCs will facilitate its biomedical application. Here we showed that Notch1 signalling could inhibit DFSCs apoptosis because the constitutive overexpression of the intracellular domain of Notch1 (ICN1) promoted proliferation and suppressed apoptosis by inhibiting cytoplasmic mitochondrial membrane depolarization, cytochrome c release and activation of caspase-9 and caspase-3. The survival-promoting effect of Notch1 was also accomplished by up-regulation of the anti-apoptotic proteins Bcl-2 and Mcl-1, down-regulation of the pro-apoptotic proteins Bax and Bad, and blockade of Bax multimerization. Moreover, p-Akt (S473) was significantly increased after ectopic Notch 1 activation. The expression of p53 was also inhibited in Notch1-overexpressing DFSCs, while the ectopic expression of p53 promoted apoptosis even when Notch1 was overexpressed. Meanwhile, all of the opposite phenomena were observed in Notch1 shRNA-silenced DFSCs. Our data strongly suggested that Notch1 signalling inhibited the apoptosis of DFSCs via the cytoplasmic mitochondrial pathway and ICN-Akt signalling pathway, together with nuclear gene expression regulation. These findings would provide molecular cues for the further medical application of DFSCs.

Inflammation increases NOTCH1 activity via MMP9 and is counteracted by Eicosapentaenoic Acid-free fatty acid in colon cancer cells.

Aberrant NOTCH1 signalling is critically involved in multiple models of colorectal cancer (CRC) and a prominent role of NOTCH1 activity during inflammation has emerged. Epithelial to Mesenchymal Transition (EMT), a crucial event promoting malignant transformation, is regulated by inflammation and Metalloproteinase-9 (MMP9) plays an important role in this process. Eicosapentaenoic Acid (EPA), an omega-3 polyunsaturated fatty acid, was shown to prevent colonic tumors in different settings. We recently found that an extra-pure formulation of EPA as Free Fatty Acid (EPA-FFA) protects from colon cancer development in a mouse model of Colitis-Associated Cancer (CAC) through modulation of NOTCH1 signalling. In this study, we exposed colon cancer cells to an inflammatory stimulus represented by a cytokine-enriched Conditioned Medium (CM), obtained from THP1-differentiated macrophages. We found, for the first time, that CM strongly up-regulated NOTCH1 signalling and EMT markers, leading to increased invasiveness. Importantly, NOTCH1 signalling was dependent on MMP9 activity, upon CM exposure. We show that a non-cytotoxic pre-treatment with EPA-FFA antagonizes the effect of inflammation on NOTCH1 signalling, with reduction of MMP9 activity and invasiveness. In conclusion, our data suggest that, in CRC cells, inflammation induces NOTCH1 activity through MMP9 up-regulation and that this mechanism can be counteracted by EPA-FFA.

Notch1 endocytosis is induced by ligand and is required for signal transduction.

The Notch signalling pathway is widely utilised during embryogenesis in situations where cell-cell interactions are important for cell fate specification and differentiation. DSL ligand endocytosis into the ligand-expressing cell is an important aspect of Notch signalling because it is thought to supply the force needed to separate the Notch heterodimer to initiate signal transduction. A functional role for receptor endocytosis during Notch signal transduction is more controversial. Here we have used live-cell imaging to examine trafficking of the Notch1 receptor in response to ligand binding. Contact with cells expressing ligands induced internalisation and intracellular trafficking of Notch1. Notch1 endocytosis was accompanied by transendocytosis of ligand into the Notch1-expressing signal-receiving cell. Ligand caused Notch1 endocytosis into SARA-positive endosomes in a manner dependent on clathrin and dynamin function. Moreover, inhibition of endocytosis in the receptor-expressing cell impaired ligand-induced Notch1 signalling. Our findings resolve conflicting observations from mammalian and Drosophila studies by demonstrating that ligand-dependent activation of Notch1 signalling requires receptor endocytosis. Endocytosis of Notch1 may provide a force on the ligand:receptor complex that is important for potent signal transduction.

23814, an Inhibitory Antibody of Ligand-Mediated Notch1 Activation, Modulates Angiogenesis and Inhibits Tumor Growth without Gastrointestinal Toxicity.

Dysregulation of Notch signaling has been implicated in the development of many different types of cancer. Notch inhibitors are being tested in the clinic, but in most cases gastrointestinal and other toxicities have limited the dosage and, therefore, the effectiveness of these therapies. Herein, we describe the generation of a monoclonal antibody against the ligand-binding domain of the Notch1 receptor that specifically blocks ligand-induced activation. This antibody, 23814, recognizes both human and murine Notch1 with similar affinity, enabling examination of the effects on both tumor and host tissue in preclinical models. 23814 blocked Notch1 function in vivo, inhibited functional angiogenesis, and inhibited tumor growth without causing gastrointestinal toxicity. The lack of toxicity allowed for combination of 23814 and the VEGFR inhibitor tivozanib, resulting in significant growth inhibition of several VEGFR inhibitor-resistant tumor models. Analysis of the gene expression profiles of an extensive collection of murine breast tumors enabled the successful prediction of which tumors were most likely to respond to the combination of 23814 and tivozanib. Therefore, the use of a specific Notch1 antibody that does not induce significant toxicity may allow combination treatment with angiogenesis inhibitors or other targeted agents to achieve enhanced therapeutic benefit.

Epidermal growth factor-like domain 7 promotes migration and invasion of human trophoblast cells through activation of MAPK, PI3K and NOTCH signaling pathways.

Epidermal growth factor-like domain 7 (Egfl7) is a gene that encodes a partially secreted protein and whose expression is largely restricted to the endothelia. We recently reported that EGFL7 is also expressed by trophoblast cells in mouse and human placentas. Here, we investigated the molecular pathways that are regulated by EGFL7 in trophoblast cells. Stable EGFL7 overexpression in a Jeg3 human choriocarcinoma cell line resulted in significantly increased cell migration and invasiveness, while cell proliferation was unaffected. Analysis of mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways showed that EGFL7 promotes Jeg3 cell motility by activating both pathways. We show that EGFL7 activates the epidermal growth factor receptor (EGFR) in Jeg3 cells, resulting in downstream activation of extracellular regulated kinases (ERKs). In addition, we provide evidence that EGFL7-triggered migration of Jeg3 cells involves activation of NOTCH signaling. EGFL7 and NOTCH1 are co-expressed in Jeg3 cells, and blocking of NOTCH activation abrogates enhanced migration of Jeg3 cells overexpressing EGFL7. We also demonstrate that signaling through EGFR and NOTCH converged to mediate EGFL7 effects. Reduction of endogenous EGFL7 expression in Jeg3 cells significantly decreased cell migration. We further confirmed that EGFL7 stimulates cell migration by using primary human first trimester trophoblast (PTB) cells overexpressing EGFL7. In conclusion, our data suggest that in trophoblast cells, EGFL7 regulates cell migration and invasion by activating multiple signaling pathways. Our results provide a possible explanation for the correlation between reduced expression of EGFL7 and inadequate trophoblast invasion observed in placentopathies.

Effect of active Notch signaling system on the early repair of rat sciatic nerve injury.

It is all known that dedifferentiated Schwann cells (SCs) play an important role in neural regeneration, and Notch signaling has complex and extensive regulatory functions in dedifferentiated SCs. So studies have focused on how to improve peripheral nerve repair by regulating proliferation and dedifferentiation in SCs with Notch signaling meloculars.We have found SCs can be activated when adding Recombinant rat jagged1/FC chimera (an activator of the Notch signaling system) in vivo. Compared with that of the control groups, at 4 weeks post-surgery nerve regeneration and functional rehabilitation in the Recombinant rat jagged1/FC chimera group were advanced significantly, and the expression of neurotrophic factors in the regenerated nerves was elevated largely. These results indicated that SCs activated by Notch signaling could promote nerve repair effectively in the early regenerative stage, suggesting the possible clinical application for the treatment of peripheral nerve defects.

Notch1 is an important mediator for enhancing of B-cell activation and antibody secretion by Notch ligand.

The roles of Notch1 and Notch2 in T-cell function have been well studied, but the functional roles of Notch in B cells have not been extensively investigated, except for Notch2 involvement in peripheral marginal zone B-cell differentiation. This study examined the roles of Notch1 in murine primary B cells. During B-cell activation by B-cell receptor ligation, Notch1 was up-regulated while Notch2 was not. In addition, Notch1 up-regulation itself did not contribute to the further activation of B cells, but the Notch ligand was important for Notch1-mediated further B-cell activation. Moreover, Notch1 deficiency significantly decreased B-cell activation and antibody secretion under the presence of Notch ligand. These data suggest that Notch1 is an important mediator for enhancing B-cell activation and antibody secretion by Notch ligand.

Notch1 activation is a poor prognostic factor in patients with gastric cancer.

BACKGROUND: Aberrant Notch1 activation has been studied in many malignant tumours, but its role in gastric cancer remains unknown. This study is aimed to investigate the prognostic significance of Notch1 activation in patients with gastric cancer. METHODS: In this study, we prospectively enrolled two independent sets of patients with gastric cancer from China and defined the activation of Notch1 by immunohistochemical staining of its active form, intracellular domain of Notch1 (ICN1). The prognostic value of Notch1 activation and clinical outcomes in gastric cancer were evaluated. RESULTS: Expression of ICN1 is elevated in gastric cancer tissues and is predominately localised in the cell cytoplasm and/or membrane. High ICN1 expression positively correlates with tumour invasion depth (P=0.032), lymph node metastasis (P<0.001), advanced TNM stage (P=0.003) and reduced overall survival (P=0.0004) in the training set. Multivariate Cox regression analysis identified ICN1 as an independent prognostic factor (P=0.031), which could be incorporated into the TNM system to generate a better predictive model for patient outcomes. The c-index was 0.7375 when assessed with the TNM stage and improved to 0.8037 when ICN1 expression was added in the training set. These results were validated in the validation set. CONCLUSIONS: Notch1 activation was correlated with gastric cancer progression and defined as a novel independent prognostic factor. Combining ICN1 expression with TNM stage may provide a better predictive model for outcomes of gastric cancer patients.

The pathological significance of Notch1 in oral squamous cell carcinoma.

Notch signaling has been reported to be involved in several types of malignant tumors; however, the role and activation mechanism of Notch signaling in oral squamous cell carcinoma (OSCC) remains poorly characterized. The purpose of this study was to elucidate the pathological significance of Notch signaling and its activation mechanism in the development and progression of OSCC. In this study, we showed that the expression of Notch1 and intracellular Notch domain (NICD) are upregulated in OSCCs. In addition, Notch1 and NICD were found to be characteristically localized at the invasive tumor front. TNF-α, a major inflammatory cytokine, significantly activated Notch signaling in vitro. In a clinicopathological analysis, Notch1 expression correlated with both the T-stage and the clinical stage. Furthermore, loss of Notch1 expression correlated with the inhibition of cell proliferation and TNF-α-dependent invasiveness in an OSCC cell line. In addition, γ-secretase inhibitor (GSI) prevented cell proliferation and TNF-α-dependent invasion of OSCC cells in vitro. These results indicate that altered expression of Notch1 is associated with increased cancer progression and that Notch1 regulates the steps involved in cell metastasis in OSCC. Moreover, inactivating Notch signaling with GSI could therefore be a useful approach for treating patients with OSCC.

Non-degradative ubiquitination of the Notch1 receptor by the E3 ligase MDM2 activates the Notch signalling pathway.

The Notch receptor is necessary for modulating cell fate decisions throughout development, and aberrant activation of Notch signalling has been associated with many diseases, including tumorigenesis. The E3 ligase MDM2 (murine double minute 2) plays a role in regulating the Notch signalling pathway through its interaction with NUMB. In the present study we report that MDM2 can also exert its oncogenic effects on the Notch signalling pathway by directly interacting with the Notch 1 receptor through dual-site binding. This involves both the N-terminal and acidic domains of MDM2 and the RAM [RBP-Jκ (recombination signal-binding protein 1 for Jκ)-associated molecule] and ANK (ankyrin) domains of Notch 1. Although the interaction between Notch1 and MDM2 results in ubiquitination of Notch1, this does not result in degradation of Notch1, but instead leads to activation of the intracellular domain of Notch1. Furthermore, MDM2 can synergize with Notch1 to inhibit apoptosis and promote proliferation. This highlights yet another target for MDM2-mediated ubiquitination that results in activation of the protein rather than degradation and makes MDM2 an attractive target for drug discovery for both the p53 and Notch signalling pathways.

Hesperetin, a potential therapy for carcinoid cancer.

BACKGROUND: The investigators' laboratory has demonstrated that the Notch1 signaling pathway acts as a tumor suppressor in carcinoid tumors. The aim of this study was to examine hesperetin, a flavonoid, as a potential Notch1 activator and carcinoid tumor suppressor. METHODS: A high-throughput drug screen revealed hesperetin as a Notch1 activator. Human gastrointestinal carcinoid (BON) cell growth after hesperetin treatment was assessed with a 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide assay. Western blots were used to measure neuroendocrine tumor markers, human achaete-scute complex-like 1, and chromogranin A. Notch1 expression was measured using western blot analysis and real-time polymerase chain reaction. RESULTS: Hesperetin induced cell death in a dose-dependent manner and reduced achaete-scute complex-like 1 and chromogranin A expression, with a concomitant rise in Notch1 levels. It also induced Notch1 messenger ribonucleic acid, indicating regulation at the transcriptional level. CONCLUSION: Hesperetin induces Notch1 expression in carcinoid cells, subsequently suppressing tumor cell proliferation and bioactive hormone production. This provides evidence for further study into hesperetin as a potential treatment for carcinoid cancer.

Notch signaling promotes airway mucous metaplasia and inhibits alveolar development.

The airways are conduits that transport atmospheric oxygen to the distal alveolus. Normally, airway mucous cells are rare. However, diseases of the airway are often characterized by mucous metaplasia, in which there are dramatic increases in mucous cell numbers. As the Notch pathway is known to regulate cell fate in many contexts, we misexpressed the active intracellular domain of the mouse Notch1 receptor in lung epithelium. Notch misexpression resulted in an increase in mucous cells and a decrease in ciliated cells in the airway. Similarly, mouse embryonic tracheal explants and adult human airway epithelium treated with Notch agonists displayed increased mucous cell numbers and decreased ciliated cell numbers. Notch antagonists had the opposite effect. Notably, Notch antagonists blocked IL13-induced mucous metaplasia. IL13 has a well-established role as an inflammatory mediator of mucous metaplasia and functions through Stat6-mediated gene transcription. We found that Notch ligands, however, are able to cause mucous metaplasia in Stat6-null cultured trachea, thus identifying a novel pathway that stimulates mucous metaplasia. Notch signaling may therefore play an important role in airway disease and, by extension, Notch antagonists may have therapeutic value. Conversely, in the distal lung, Notch misexpression prevented the differentiation of alveolar cell types. Instead, the distal lung formed cysts composed of cells that were devoid of alveolar markers but that expressed some, but not all, markers of proximal airway epithelium. Occasional distal cystic cells appeared to differentiate into normal proximal airway cells, suggesting that ectopic Notch signaling arrests the normal differentiation of distal lung progenitors before they initiate an alveolar program.