Characterization of Notch3-deficient mice: normal embryonic development and absence of genetic interactions with a Notch1 mutation

The Notch signaling pathway is an evolutionarily conserved signaling mechanism and mutations in its components disrupt cell fate specification and embryonic development in many organisms. To analyze the in vivo role of the Notch3 gene in mice, we created a deletion allele by gene targeting. Embryos homozygous for this mutation developed normally and homozygous mutant adults were viable and fertile. We also examined whether we could detect genetic interactions during early embryogenesis between the Notch3 mutation and a targeted mutation of the Notch1 gene. Double homozygous mutant embryos exhibited defects normally observed in Notch1-deficient embryos, but we detected no obvious synergistic effects in the double mutants. These data demonstrate that the Notch3 gene is not essential for embryonic development or fertility in mice, and does not have a redundant function with the Notch1 gene during early embryogenesis.     

Notch signaling is required for lateral induction of Jagged1 during FGF-induced lens fiber differentiation

Previous studies of the developing lens have shown that Notch signaling regulates differentiation of lens fiber cells by maintaining a proliferating precursor pool in the anterior epithelium. However, whether Notch signaling is further required after the onset of fiber cell differentiation is not clear. This work investigates the role of Notch2 and Jagged1 (Jag1) in secondary fiber cell differentiation using rat lens epithelial explants undergoing FGF-2 dependent differentiation in vitro. FGF induced Jag1 expression and Notch2 signaling (as judged by the appearance of activated Notch2 Intracellular Domain (N2ICD)) within 12-24 h. These changes were correlated with induction of the Notch effector, Hes5, upregulation of N-cadherin (N-cad), and downregulation of E-cadherin (E-cad), a cadherin switch characteristic of fiber cell differentiation. Induction of Jag1 was efficiently blocked by U0126, a specific inhibitor of MAPK/ERK signaling, indicating a requirement for signaling through this pathway downstream of the FGF receptor. Other growth factors that activate MAPK/ERK signaling (EGF, PDGF, IGF) did not induce Jag1. Inhibition of Notch signaling using gamma secretase inhibitors DAPT and L-685,458 or anti-Jag1 antibody markedly decreased FGF-dependent expression of Jag1 demonstrating Notch-dependent lateral induction. In addition, inhibition of Notch signaling reduced expression of N-cad, and the cyclin dependent kinase inhibitor, p57Kip2, indicating a direct role for Notch signaling in secondary fiber cell differentiation. These results demonstrate that Notch-mediated lateral induction of Jag1 is an essential component of FGF-dependent lens fiber cell differentiation.     

尿源干细胞对神经源性膀胱大鼠膀胱功能及Notch1、Jagged1蛋白表达的影响

目的:研究尿源干细胞对神经源性膀胱大鼠膀胱功能及Notch1、Jagged1蛋白表达的影响。方法:纳入60只健康雌性清洁SD大鼠作为实验对象,将其按照随机抽签法分为正常对照组、研究组以及损伤组,每组各20只。其中研究组和损伤组大鼠均建立神经源性膀胱模型,研究组在造模成功后予以尿源性干细胞尾静脉注射。28 d后,比较三组大鼠膀胱功能相关指标水平,膀胱湿质量和膀胱湿质量/体质量,Notch1、Jagged1蛋白表达水平。结果:损伤组收缩时间、排尿量、膀胱峰压均低于正常对照组,而研究组收缩时间、排尿量、膀胱峰压均高于损伤组(均P0.05);损伤组膀胱基压高于正常对照组,而研究组膀胱基压低于损伤组(均P0.05)。损伤组膀胱湿质量和膀胱湿质量/体质量均高于正常对照组,而研究组膀胱湿质量和膀胱湿质量/体质量低于损伤组(均P0.05)。损伤组Notch1、Jagged1蛋白表达水平均高于正常对照组,而研究组Notch1、Jagged1蛋白表达水平低于损伤组(均P0.05)。结论:尿源干细胞的应用可显著改善神经源性膀胱大鼠膀胱功能,同时可下调Notch1、Jagged1蛋白表达水平。     

Expression of Jagged1 and its association with hepatitis B virus X protein in hepatocellular carcinoma

Jagged1 is one of the ligands of Notch signaling pathway, which controls cellular proliferation and differentiation, and also plays important roles in various malignant tumors. However, the expression of Jagged1 in hepatocellular carcinoma (HCC) has not been elucidated, nor whether it is associated with hepatitis B virus X protein (HBx). In this study, we found that Jagged1 was highly expressed in 79.2% (42/53) of HCC tissues compared with adjacent nontumor liver (P <0.05), and its expression was found to be closely related with HBx (rs=0.522, P <0.001) in HCC tissues. Our in vitro study also showed that alteration of HBx expression in HCC cell lines led to a consistent change of Jagged1. Moreover, Jagged1 was found to co-localize and directly interact with HBx in HCC tissues and HBx expressed HCC cell lines. Our results reveal that Jagged1, which is regulated by HBx, may contribute to the development of HCC.     

Jagged1蛋白在膝关节骨性关节炎软骨组织中的表达

目的:研究Jagged1蛋白在膝关节骨性关节炎关节软骨中表达的特点,探讨Jagged1蛋白的表达对关节软骨病理改变的影响。方法:选取宁夏医科大学总医院骨科2018年11月1日至2019年10月31日34例因膝关节骨性关节炎行全膝关节置换的患者,取术中去除的股骨髁软骨组织,磨损较重的一侧为负重区(实验组),磨损较轻的一侧为非负重区(对照组)。通过番红、HE染色观察软骨组织形态学变化,通过免疫组织化学检测Jagged1蛋白在不同软骨组织中的表达强度,对比分析Jagged1蛋白表达的差异对关节软骨病变的影响。结果:外观照可见实验组软骨面色泽暗淡,表面粗糙,有大量软骨缺失,对照组软骨面较平整,呈白色,未见软骨明显缺失;HE染色可见实验组软骨面不整,软骨细胞排列紊乱,呈簇状分布,对照组软骨面平整,软骨细胞数量多,胞质丰富;番红染色见实验组软骨组织残留较少,软骨层结构模糊不清,软骨细胞较少,对照组软骨层次结构清晰,软骨细胞及软骨下骨细胞排列整齐;免疫组化可见Jagged1蛋白实验组软骨中有较多阳性表达,在对照组中表达较少,差异有统计学意义(P0.05)。结论:Jagged1蛋白在软骨中的表达强度与关节软骨的磨损程度呈正相关,在膝关节骨性关节炎的病理变化过程中有着重要的作用。     

Silencing of Jagged1 inhibits cell growth and invasion in colorectal cancer

Dysregulated Notch signaling has a critical role in the tumorigenesis. Jagged1, a Notch ligand, is overexpressed in various human cancers. Recent studies revealed the involvement of Jagged1 in colorectal cancer (CRC) development. These basic studies provide a promising potential for inhibition of the Notch pathway for the treatment of CRC. Herein, we aimed to investigate the consequences of targeting Jagged1 using shRNA on CRC both in vitro and in vivo to test their potential to inhibit this key element for CRC treatment. We found that downregulation of Jagged1 with lentiviral Jagged1-shRNA resulted in decreased colon cancer cell viability in vitro, most likely mediated through reduced cell proliferation. Importantly, Jagged1 knockdown induced G₀/G₁ phase cell cycle arrest, with reduced Cyclin D1, Cyclin E and c-Myc expression. Silencing of Jagged1 reduced the migration and invasive capacity of the colon cancer cells in vitro. Furthermore, colon cancer cells with knockdown of Jagged1 had much slower growth rate than control cells in a xenograft mouse model in vivo, with a marked downregulation of cell proliferation markers (PCNA, Ki-67, and c-Myc) and metastasis markers (MMP-2 and MMP-9). These findings rationalize a mechanistic approach to CRC treatment based on Jagged1-targeted therapeutic development.     

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

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

Jagged1在树突状细胞诱导免疫耐受中的作用

Jagged1是Notch信号通路中的一个配体,近来许多研究表明,它能诱导树突状细胞的成熟,并通过促使T淋巴细胞分化成调节性T细胞或II型T辅助细胞,从而诱导免疫耐受。     

The Notch pathway in prostate development and cancer

Abstract The Notch family of transmembrane receptors are important mediators of cell fate determination. Accordingly, Notch signaling is intimately involved in the development of numerous tissues. Recent findings have highlighted a critical role for Notch signaling in normal prostate development. Notch signaling is required for embryonic and postnatal prostatic growth and development, for proper cell lineage specification within the prostate, as well as for adult prostate maintenance and regeneration following castration and hormone replacement. Evidence for Notch as a regulator of prostate cancer development, progression, and metastasis has also emerged. This review summarizes our current understanding of the role of Notch pathway elements, including members of the Jagged, Delta-like, hairy/enhancer-of-split, and hairy/enhancer-of-split related with YRPW motif families, in prostate development and tumorigenesis. Data supporting Notch pathway elements as oncogenes and tumor suppressors in prostate tumors, as well as data implicating Notch receptors and ligands as potential markers of normal prostate stem/progenitor cells and prostate cancer stem/initiating cells, are also presented.     

β-Site Amyloid Precursor Protein Cleaving Enzyme 1(BACE1) Regulates Notch Signaling by Controlling the Cleavage of Jagged 1 (Jag1) and Jagged 2 (Jag2) Proteins

BACE1 is a type I transmembrane aspartyl protease that cleaves amyloid precursor protein at the β-secretase site to initiate the release of β-amyloid peptide. As a secretase, BACE1 also cleaves additional membrane-bound molecules by exerting various cellular functions. In this study, we showed that BACE1 can effectively shed the membrane-anchored signaling molecule Jagged 1 (Jag1). We also mapped the cleavage sites of Jag1 by ADAM10 and ADAM17. Although Jag1 shares a high degree of homology with Jag2 in the ectodomain region, BACE1 fails to cleave Jag2 effectively, indicating a selective cleavage of Jag1. Abolished cleavage of Jag1 in BACE1-null mice leads to enhanced astrogenesis and, concomitantly, reduced neurogenesis. This characterization provides biochemical evidence that the Jag1-Notch pathway is under the control of BACE1 activity.     

Effects of chlorpyrifos exposure on kidney Notch2-Jagged1 pathway of early prenatal embryo

Aims: To evaluate the effects of this insecticide on the embryonic development of kidney and to assess the important role of Notch2–Jagged1 pathway in this duration. Methods and results: Chlorpyrifos (CPF) 5 mg/kg/d were administrated on gestation 7.5–11.5 day by subcutaneous injection. On gestation 16.5 day, the normal embryo kidney developed through S shape duration to the original kidney, which had the nephrons and could start to secret the urine. But for the CPF‐treated mice, the embryo kidney developed much more slowly, they did not show the S shape and the nephrons. The Notch2–Jagged1 pathway should be expressed stronger in the normal embryo kidney on gestation 16.5 day, but for the CPF‐treated mice we found the obvious weak pathway staining. Conclusions : CPF broke the Notch2–Jagged1 pathway during the embryo kidney development, and the Notch2–Jagged1 pathway plays an important role in the S shape to original kidney formation duration. Birth Defects Res (Part B) 92:97–101, 2011. © 2011 Wiley‐Liss, Inc.     

Both Notch1 and Notch2 contribute to the regulation of melanocyte homeostasis

Notch signaling affects a variety of mammalian stem cells, but there has been limited evidence that a specific Notch molecule regulates adult stem cells. Recently, it was reported that the reduced Notch signaling initiated at the embryonic stage results in a gradual hair graying phenotype after birth. Here we demonstrate that the oral administration of a gamma-secretase inhibitor (GSI) to wild-type adult C57/Bl6 mice led to a gradual increase in gray spots, which remained unchanged for at least 20 weeks after discontinuing the GSI. In GSI-treated mice, there was a severe decrease in unpigmented melanocytes in the bulge/subbulge region where melanocyte stem cells are located. While we confirmed that Notch1+/-Notch2+/- double heterozygous mice with a C57/Bl6 background were born with a normal hair color phenotype and gradually turned gray after the second hair cycle, in the c-kit mutant Wv background, Notch1+/- and Notch2+/- mice had larger white spots on the first appearance of hair than did the Wv/+ mice, which did not change throughout life. Notch1+/-Notch2+/-Wv/+ mice had white hair virtually all over the body at the first appearance of hair and the depigmentation continued to progress thereafter. Using a neural crest organ culture system, GSI blocked the generation of pigmented melanocytes when added to the culture during the period of melanoblast proliferation, but not during the period of differentiation. These observations imply roles of Notch signaling in both development of melanocyte during embryogenesis and maintenance of melanocyte stem cells in adulthood, while the degree of requirement is distinct in these settings: the latter is more sensitive than the former to the reduced Notch signaling. Furthermore, Notch1 and Notch2 cooperates with c-kit signaling during embryogenesis, and they cooperate with each other to regulate melanocyte homeostasis after birth.     

平喘颗粒对慢性哮喘大鼠Notch通路相关因子的影响

摘要 目的：通过观测各组慢性哮喘大鼠Notch信号通路相关因子Notch2、Jagged1、HES-1的变化，以及肺组织气道重塑的改善状况，探讨平喘颗粒在哮喘的治疗方面所发挥的作用机制。方法：按随机法将60只大鼠均分至空白组、模型组、地塞米松组、平喘颗粒组中。除空白组外，剩余的3组大鼠经卵蛋白+氢氧化铝致敏，复制慢性哮喘大鼠模型，空白组用生理盐水代替致敏液。造模前各组予相应的药物灌胃处理。6周后造模完成，处死大鼠并取其肺组织进行HE染色，在光镜下观察病理变化。使用Real-time PCR法检测Notch2、Jagged1 mRNA，Western blot法检测HES-1蛋白的表达程度。结果：与空白组相比，其余各组肺组织均可观察到明显气道重塑改变，Notch2、Jagged1 mRNA及HES-1蛋白表达明显上升（P＜0.05）；与模型组相比，平喘颗粒和地塞米松可缓解气道重塑，各因子表达情况显著下降（P＜0.05），两组间比较则无明显差异（P＞0.05）。结论：Notch2、Jagged1、HES-1表达程度与气道重塑呈正相关，平喘颗粒通过降低Notch2、Jagged1、HES-1表达改善气道重塑，可能是其防治哮喘发生发展的机制。     

Effect of tamoxifen on the Notch signaling pathway in ovarian follicles of mice

ABSTRACT

We investigated the effect of tamoxifen (TAM) treatment on the Notch signaling pathway in mouse ovary. Mice were randomly divided into four groups. Control group A animals were untreated. Control group B animals were treated with the vehicle only. Animals of the 0.5 TAM group received 0.5 mg/day TAM. Animals of the 1.5 TAM group received 1.5 mg/day of TAM. TAM was injected subcutaneously for 5 days. Body weights were measured at the start and end of the experiment. Sections were stained using Crossman’s modified trichrome to examine general ovarian structure. Other sections were immunostained to demonstrate Jagged 1, Ki 67 and Notch 2. The TUNEL method was used to detect apoptosis. No significant differences in body weight or ovarian weight were found among the experimental groups. The number of primordial follicles was greater in the treatment groups than in the control groups, while the number of antral follicles and corpora lutea were reduced in the treatment groups. Cell proliferation rates were decreased by TAM treatment and cystic follicles were formed in the ovarian stroma. Notch 2 expression in the granulosa cells was increased following TAM administration, but no change was found in Jagged 1 expression. TAM administration suppressed follicular development and exhibited a negative effect on ovarian morphology. Our findings suggest that the Notch pathway participates in the action of TAM. We suggest that it may be useful to use Notch pathway regulators to adjust the effects of TAM on the ovary.     

Jagged1 is necessary for postnatal and adult neurogenesis in the dentate gyrus

Understanding the mechanisms that control the maintenance of neural stem cells is crucial for the study of neurogenesis. In the brain, granule cell neurogenesis occurs during development and adulthood, and the generation of new neurons in the adult subgranular zone of the dentate gyrus contributes to learning. Notch signaling plays an important role during postnatal and adult subgranular zone neurogenesis, and it has been suggested as a potential candidate to couple cell proliferation with stem cell maintenance. Here we show that conditional inactivation of Jagged1 affects neural stem cell maintenance and proliferation during postnatal and adult neurogenesis of the subgranular zone. As a result, granule cell production is severely impaired. Our results provide additional support to the proposal that Notch/Jagged1 activity is required for neural stem cell maintenance during granule cell neurogenesis and suggest a link between maintenance and proliferation of these cells during the early stages of neurogenesis.     

MT1-MMP cleaves Dll1 to negatively regulate Notch signalling to maintain normal B-cell development

Notch signalling controls the differentiation of haematopoietic progenitor cells (HPCs). Here, we show that loss of membrane-type 1 matrix metalloproteinase (MT1-MMP, MMP14), a cell surface protease expressed in bone marrow stromal cells (BMSCs), increases Notch signalling in HPCs and specifically impairs B-lymphocyte development. When co-cultured with BMSCs in vitro, HPCs differentiation towards B lymphocytes is significantly compromised on MT1-MMP-deficient BMSCs and this defect could be completely rescued by DAPT, a specific Notch signalling inhibitor. The defective B-lymphocyte development could also be largely rescued by DAPT in vivo. MT1-MMP interacts with Notch ligand Delta-like 1 (Dll1) and promotes its cleavage on cell surface in BMSCs. Ectopic MT1-MMP cleaves Dll1 and results in diminished Notch signalling in co-cultured cells. In addition, recombinant MT1-MMP cleaves a synthetic Dll1 peptide at the same site where MT1-MMP cleaves Dll1 on the cell surface. Our data suggest that MT1-MMP directly cleaves Dll1 on BMSCs to negatively regulate Notch signalling to specifically maintain normal B-cell development in bone marrow.