Role of Jagged1/STAT3 signalling in platinum‐resistant ovarian cancer

Jagged1, the essential ligand of the Notch signalling pathway, is highly expressed in metastatic prostate cancer, and its high expression in breast cancer is linked to poor survival rates. However, the mechanism of Jagged1′s involvement in platinum‐resistant ovarian cancer has not been thoroughly elucidated to date. The purpose of the present study was to investigate the roles of Jagged1 in the platinum resistance of ovarian cancer and its possible mechanisms. Compared with a platinum responsive group of ovarian epithelial cell carcinomas, we found the positive staining intensity of Notch1, Notch2, Jagged1, STAT3 and Epithelial‐mesenchymal transition (EMT) proteins were lower in a platinum‐resistant group. The DDP‐resistant ovarian cancer cell line (C13K) had a higher IC50 of DDP than its parental cell line (OV2008) (P < 0.05) and acquired an EMT phenotype and invasive characteristics. Inhibiting or knockdown of Jagged1 expression could not only reduce its capacity of migration and invasion but also reverse EMT and down‐regulate the expression of serine 727‐phosphorylated STAT3 (pS727) at the protein level but not total STAT3 or tyrosine 705‐phosphorylated STAT3 (pY705) in C13K cells. Furthermore, it was found that crosstalk between the Jagged1/Notch and JAK/STAT3 signalling pathways were involved in Jagged1‐promoting EMT in C13K cells. Experiments in vivo showed a reduced micrometastatic tumour burden in the lung, liver and spleen of mice implanted with C13K cells with knocked‐down Jagged1 compared with mice implanted with control cells. All of these results demonstrate that Jagged1 can crosstalk with the JAK/STAT3 pathway, and they all cooperate to promote the aberrant occurrence of EMT, further reinforcing the abilities of invasion and migration of platinum‐resistant ovarian cancer in vivo and in vitro.     

Angiotensin II contributes to renal fibrosis independently of Notch pathway activation

Recent studies have described that the Notch signaling pathway is activated in a wide range of renal diseases. Angiotensin II (AngII) plays a key role in the progression of kidney diseases. AngII contributes to renal fibrosis by upregulation of profibrotic factors, induction of epithelial mesenchymal transition and accumulation of extracellular matrix proteins. In cultured human tubular epithelial cells the Notch activation by transforming growth factor-β1 (TGF-β1) has been involved in epithelial mesenchymal transition. AngII mimics many profibrotic actions of TGF-β1. For these reasons, our aim was to investigate whether AngII could regulate the Notch/Jagged system in the kidney, and its potential role in AngII-induced responses. In cultured human tubular epithelial cells, TGF-β1, but not AngII, increased the Notch pathway-related gene expression, Jagged-1 synthesis, and caused nuclear translocation of the activated Notch. In podocytes and renal fibroblasts, AngII did not modulate the Notch pathway. In tubular epithelial cells, pharmacological Notch inhibition did not modify AngII-induced changes in epithelial mesenchymal markers, profibrotic factors and extracellular matrix proteins. Systemic infusion of AngII into rats for 2 weeks caused tubulointerstitial fibrosis, but did not upregulate renal expression of activated Notch-1 or Jagged-1, as observed in spontaneously hypertensive rats. Moreover, the Notch/Jagged system was not modulated by AngII type I receptor blockade in the model of unilateral ureteral obstruction in mice. These data clearly indicate that AngII does not regulate the Notch/Jagged signaling system in the kidney, in vivo and in vitro. Our findings showing that the Notch pathway is not involved in AngII-induced fibrosis could provide important information to understand the complex role of Notch system in the regulation of renal regeneration vs damage progression.     

Notch1 and 2 cooperate in limb ectoderm to receive an early Jagged2 signal regulating interdigital apoptosis

Spontaneous and engineered mutations in the Notch ligand Jagged2 produced the Syndactylism phenotype (Jiang, R.L., Lan, Y., Chapman, H.D., Shawber, C., Norton, C.R., Serreze, D.V., Weinmaster, G., Gridley, T., 1998. Defects in limb, craniofacial, and thymic Development in Jagged2 mutant mice. Genes Dev. 12, 1046-1057; Sidow, A., Bulotsky, M.S., Kerrebrock, A.W., Bronson, R.T., Daly, M.J., Reeve, M.P., Hawkins, T.L., Birren, B.W., Jaenisch, R., Lander, E.S., 1997. Serrate2 is disrupted in the mouse limb-development mutant syndactylism. Nature 389, 722-725). Given that additional ligands may be expressed in the developing limb bud, it was possible that loss of Jagged2 disabled only part of Notch function in the limb. In addition, it is not clear from the expression pattern of Jagged2 in the apical ectodermal ridge (AER) whether the ectodermal or mesenchymal compartment of the limb bud receives the Jagged2 signal. To elucidate the requirement for the Notch pathway in limb development, we have analyzed single and compound Notch receptor mutants as well as gamma-secretase-deficient limbs. Floxed alleles were removed either from the developing limb bud ectoderm (using Msx2-Cre) or from the mesenchyme (using Prx1-Cre). Our results confirm that Jagged2 loss describes the contribution of the entire Notch pathway to the mouse limb development and revealed that both Notch1 and 2 are required in the ectoderm to receive the Jagged2 signal. Interestingly, our allelic series allowed us to determine that Notch receives this signal at an early stage in the developmental process and that memory of this event is retained by the mesenchyme, where Notch signaling appears to be dispensable. Thus, Notch signaling plays a non-autonomous role in digit septation.     

Evidence for the notch signaling pathway on the role of estrogen in angiogenesis

We have investigated the molecular mechanisms involved in 17 beta-estradiol-induced angiogenic pathway. We show here that 17 beta-estradiol promoted a 6-fold increase in Jagged1 expression and an 8-fold increase in Notch1 expression by cDNA arrays in breast cancer MCF7 cells. Interestingly, Jagged1 was abrogated by incubation with the estrogen antagonist, ICI182,780. A similar up-regulation of both Notch1 receptor and Jagged1 ligand was found in endothelial cells. Additionally, imperfect estrogen-responsive elements were found in the 5' untranslated region of Notch1 and Jagged1 genes. Treatment with 17 beta-estradiol also led to an activation of Notch signaling in MCF7 cells expressing Notch1 reporter gene or by promoting Jagged1-induced Notch signaling in coculture assays. Inoculation of MCF7 cells in 17 beta-estradiol-treated nude mice resulted in up-regulation of Notch1 expression as well as increased number of tumor microvessels in comparison to placebo-treated mice. Notch1-expressing endothelial cell cultures formed cord-like structures on Matrigel in contrast to cells expressing a dominant-negative form of Notch1, emphasizing the relevance of Notch1 pathway in vessel assembly. Finally, Notch1-expressing MCF7 cells up-regulated hypoxia-inducible factor 1 alpha gene, a well-known angiogenic factor that clustered with Notch1 gene. This study implicates Notch signaling in the cross talk between 17 beta-estradiol and angiogenesis.     

Expression of Notch1 and Jagged2 in the enteric nervous system.

The Notch signaling pathway is a vitally important pathway in regulating brain development. To explore the involvement of the Notch pathway in neuronal cells of adult rat gut, we investigated the expression of Notch1 and Jagged2 by in situ hybridization (ISH) and immunohistochemistry (IHC). In the enteric nervous system, Notch1 and Jagged2 were expressed in ganglia of the submucosal and myenteric plexus. Notch1 was preferentially expressed in cholinergic neurons lacking calretinin or nitric oxide synthase (NOS), whereas Jagged2 was present in most neuron subtypes. We propose that Notch1 and Jagged2 have a continuing role in the maintenance and function of neuronal cells in the adult enteric nervous system.     

γ‐secretase inhibitor DAPT mitigates cisplatin‐induced acute kidney injury by suppressing Notch1 signaling

Organ toxicity, including kidney injury, limits the use of cisplatin for the treatment of multiple human cancers. Hence, interventions to alleviate cisplatin‐induced nephropathy are of benefit to cancer patients. Recent studies have demonstrated that pharmacological inhibition of the Notch signaling pathway enhances cisplatin efficacy against several cancer cells. However, whether augmentation of the anti‐cancer effect of cisplatin by Notch inhibition comes at the cost of increased kidney injury is unclear. We show here that treatment of mice with cisplatin resulted in a significant increase in Notch ligand Delta‐like 1 (Dll1) and Notch1 intracellular domain (N1ICD) protein expression levels in the kidneys. N‐[N‐(3,5‐difluorophenacetyl)‐L‐alanyl]‐S‐phenylglycine t‐butyl ester (DAPT), a γ‐secretase inhibitor reversed cisplatin‐induced increase in renal N1ICD expression and plasma or urinary levels of predictive biomarkers of acute kidney injury (AKI). DAPT also mitigated cisplatin‐induced tubular injury and reduction in glomerular filtration rate. Real‐time multiphoton microscopy revealed marked necrosis and peritubular vascular dysfunction in the kidneys of cisplatin‐treated mice which were abrogated by DAPT. Cisplatin‐induced Dll1/Notch1 signaling was recapitulated in a human proximal tubule epithelial cell line (HK‐2). siRNA‐mediated Dll1 knockdown and DAPT attenuated cisplatin‐induced Notch1 cleavage and cytotoxicity in HK‐2 cells. These data suggest that Dll1‐mediated Notch1 signaling contributes to cisplatin‐induced AKI. Hence, the Notch signaling pathway could be a potential therapeutic target to alleviate renal complications associated with cisplatin chemotherapy.     

Constitutive notch signaling in adult transgenic mice inhibits bFGF‐induced angiogenesis and blocks ovarian follicle development

Notch signaling is important in angiogenesis during embryonic development. However, the embryonic lethal phenotypes of knock‐out and transgenic mice have precluded studies of the role of Notch post‐natally. To develop a mouse model that would bypass the embryonic lethal phenotype and investigate the possible role of Notch signaling in adult vessel growth, we developed transgenic mice with Cre‐conditional expression of the constitutively active intracellular domain of Notch1 (IC‐Notch1). Double transgenic IC‐Notch1/Tie2‐Cre embryos with endothelial specific IC‐Notch1 expression died at embryonic day 9.5. They displayed collapsed and leaky blood vessels and defects in angiogenesis development. A tetracycline‐inducible system was used to express Cre recombinase postnatally in endothelial cells. In adult mice, IC‐Notch1 expression inhibited bFGF‐induced neovascularization and female mice lacked mature ovarian follicles, which may reflect the block in bFGF‐induced angiogenesis required for follicle growth. Our results demonstrate that Notch signaling is important for both embryonic and adult angiogenesis and indicate that the Notch signaling pathway may be a useful target for angiogenic therapies. genesis 52:809–816, 2014. © 2014 Wiley Periodicals, Inc.     

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

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

Critical regulation of bone morphogenetic protein-induced osteoblastic differentiation by Delta1/Jagged1-activated Notch1 signaling

Functional involvement of the Notch pathway in osteoblastic differentiation has been previously investigated using the truncated intracellular domain, which mimics Notch signaling by interacting with the DNA-binding protein CBF-1. However, it is unclear whether Notch ligands Delta1 and Jagged1 also induce an identical cellular response in osteoblastic differentiation. We have shown that both Delta1 and Jagged1 were expressed concomitantly with Notch1 in maturating osteoblastic cells during bone regeneration and that overexpressed and immobilized recombinant Delta1 and Jagged1 alone did not alter the differentiated state of MC3T3-E1 and C2C12 cells. However, they augmented bone morphogenetic protein-2 (BMP2)-induced alkaline phosphatase activity and the expression of several differentiation markers, except for osteocalcin, and ultimately enhanced calcified nodule and in vivo ectopic bone formation of MC3T3-E1. In addition, both ligands transmitted signal through the CBF-1-dependent pathway and stimulated the expression of HES-1, a direct target of Notch pathway. To test the necessity of Notch signaling in BMP2-induced differentiation, Notch signaling was inhibited by the dominant negative extracellular domain of Notch1, specific inhibitor, or small interference RNA. These treatments decreased alkaline phosphatase activity as well as the expression of other differentiation markers and inhibited the promoter activity of Id-1, a target gene of the BMP pathway. These results indicate the functional redundancy between Delta1 and Jagged1 in osteoblastic differentiation whereby Delta1/Jagged1-activated Notch1 enhances BMP2-induced differentiation through the identical signaling pathway. Furthermore, our data also suggest that functional Notch signaling is essential not only for BMP2-induced osteoblast differentiation but also for BMP signaling itself.     

Spatiotemporal expression of Notch receptors and ligands in developing mouse placenta

Notch signaling is involved in cell lineage specification in many developing organs. In mice there are four known Notch receptor genes (Notch1–4) and five ligands genes (Dll1, 3, 4 and Jagged1 and 2). Notch2 is essential for development of placenta, an organ that mediates feto-maternal nutrient and gas exchange as well as maternal adaptations to pregnancy. However the role of other Notch receptors and ligands in placentation is not known. In order to gain better insight into the role of Notch signaling in mouse placenta we thoroughly analyzed mRNA expression of all Notch receptors and ligands in all trophoblast cell types from the embryonic day (E) 7.5 to E12.5, the period during which all of the substructures of the placenta develop. Here we show that Notch receptors and ligands are specifically and dynamically expressed in multiple cell layers of developing placenta. We found that the Notch2 receptor and Jagged1 and Jagged2 ligand genes are complementarily expressed in trophoblast cells of the chorion and its later derivatives in the labyrinth. Dll4 and Notch2 expression complement each other in the ectoplacental cone, while Dll1 and Notch2 are expressed in an ectoplacental cone derivative, the junctional zone. Moreover Dll4 and Notch2 are expressed at the ectoplacental cone–decidua interface at early stages of placentation. Additionally we show that Notch2 is dynamically expressed in all trophoblast giant cell subtypes, which is consistent with previous reports. Overall these expression pattern results suggest that Notch signaling may play several diverse roles during placenta development.     

Down‐regulation of Notch‐1 and Jagged‐1 inhibits prostate cancer cell growth,migration and invasion,and induces apoptosis via inactivation of Akt,mTOR, and NF‐κB signaling pathways

Notch signaling is involved in a variety of cellular processes, such as cell fate specification, differentiation, proliferation, and survival. Notch‐1 over‐expression has been reported in prostate cancer metastases. Likewise, Notch ligand Jagged‐1 was found to be over‐expressed in metastatic prostate cancer compared to localized prostate cancer or benign prostatic tissues, suggesting the biological significance of Notch signaling in prostate cancer progression. However, the mechanistic role of Notch signaling and the consequence of its down‐regulation in prostate cancer have not been fully elucidated. Using multiple cellular and molecular approaches such as MTT assay, apoptosis assay, gene transfection, real‐time RT‐PCR, Western blotting, migration, invasion assay and ELISA, we found that down‐regulation of Notch‐1 or Jagged‐1 was mechanistically associated with inhibition of cell growth, migration, invasion and induction of apoptosis in prostate cancer cells, which was mediated via inactivation of Akt, mTOR, and NF‐κB signaling. Consistent with these results, we found that the down‐regulation of Notch‐1 or Jagged‐1 led to decreased expression and the activity of NF‐κB downstream genes such as MMP‐9, VEGF, and uPA, contributing to the inhibition of cell migration and invasion. Taken together, we conclude that the down‐regulation of Notch‐1 or Jagged‐1 mediated inhibition of cell growth, migration and invasion, and the induction of apoptosis was in part due to inactivation of Akt, mTOR, and NF‐κB signaling pathways. Our results further suggest that inactivation of Notch signaling pathways by innovative strategies could be a potential targeted approach for the treatment of metastatic prostate cancer. J. Cell. Biochem. 109: 726–736, 2010. © 2010 Wiley‐Liss, Inc.     

Primordial follicle assembly was regulated by notch signaling pathway in the mice

Notch signaling pathway, a highly conserved cell signaling system, exists in most multicellular organisms. The objective of this study was to examine Notch signaling pathway in germ cell cyst breakdown and primordial follicle formation. The receptor and ligand genes of Notch pathway (Notch1, Notch2, Jagged1, Jagged2 and Hes1) were extremely down-regulated after newborn mouse ovaries were cultured then exposed to DAPT or L-685,458 in vitro (P < 0.01). Since DAPT or L-685,548 inhibits Notch signaling pathway, the expression of protein LHX8 and NOBOX was significantly reduced during the formation of the primordial follicles. Down-regulated mRNA expression of specific genes including Lhx8, Figla, Sohlh2 and Nobox, were also observed. The percentages of female germ cells in germ cell cysts and primordial follicles were counted after culture of newborn ovaries for 3 days in vitro. The result showed female germ cells in cysts was remarkably up-regulated while as the oocytes in primordial follicles was significantly down-regulated (P < 0.05). In conclusion, Notch signaling pathway may regulate the formation of primordial follicle in mice.     

Maternal exposure to multi-wall carbon nanotubes does not induce embryo-fetal developmental toxicity in rats

BACKGROUND: Although the potential risk of carbon nanotubes (CNTs) to humans has recently increased due to expanding production and widespread use, the potential adverse effects of CNTs on embryo–fetal development have not yet been determined. METHODS: This study investigated the potential effects of multi‐wall CNTs (MWCNTs) on pregnant dams and embryo–fetal development in rats. MWCNTs were administered to pregnant rats by gavage at 0, 40, 200, and 1,000 mg/kg/day. All dams were subjected to Cesarean section on day 20 of gestation, and the fetuses were examined for any morphological abnormalities. RESULTS: All animals survived to the end of the study. A decrease in thymus weight was observed in the high dose group in a dose‐dependent manner. However, maternal body weight, food consumption, and oxidant–antioxidant balance in the liver were not affected by treatment with MWCNTs. No treatment‐related differences in gestation index, fetal deaths, fetal and placental weights, or sex ratio were observed between the groups. Morphological examinations of the fetuses demonstrated no significant difference in incidences of abnormalities between the groups. CONCLUSIONS: The results show that repeated oral doses of MWCNTs during pregnancy induces minimal maternal toxicity and no embryo–fetal toxicity at 1,000 mg/kg/day in rats. The no‐observed‐adverse‐effect level of MWCNTs is considered to be 200 mg/kg/day for dams and 1,000 mg/kg/day for embryo–fetal development. In this study, the dosing formulation was not analyzed to determine the degree of reaggregation (or not), nor were blood levels of CNT's measured in the dosed animals to verify or characterize absorption. Birth Defects Res (Part B) 92:69–76, 2011. © 2011 Wiley‐Liss, Inc.     

Differential regulation of osteoclastogenesis by Notch2/Delta-like 1 and Notch1/Jagged1 axes

Introduction

Osteoclastogenesis plays an important role in the bone erosion of rheumatoid arthritis (RA). Recently, Notch receptors have been implicated in the development of osteoclasts. However, the responsible Notch ligands have not been identified yet. This study was undertaken to determine the role of individual Notch receptors and ligands in osteoclastogenesis.

Methods

Mouse bone marrow-derived macrophages or human peripheral blood monocytes were used as osteoclast precursors and cultured with receptor activator of nuclear factor-kappaB ligand (RANKL) and macrophage-colony stimulating factor (M-CSF) to induce osteoclasts. Osteoclasts were detected by tartrate-resistant acid phosphatase (TRAP) staining. K/BxN serum-induced arthritic mice and ovariectomized mice were treated with anti-mouse Delta-like 1 (Dll1) blocking monoclonal antibody (mAb).

Results

Blockade of a Notch ligand Dll1 with mAb inhibited osteoclastogenesis and, conversely, immobilized Dll1-Fc fusion protein enhanced it in both mice and humans. In contrast, blockade of a Notch ligand Jagged1 enhanced osteoclastogenesis and immobilized Jagged1-Fc suppressed it. Enhancement of osteoclastogenesis by agonistic anti-Notch2 mAb suggested that Dll1 promoted osteoclastogenesis via Notch2, while suppression by agonistic anti-Notch1 mAb suggested that Jagged1 suppressed osteoclastogenesis via Notch1. Inhibition of Notch signaling by a gamma-secretase inhibitor suppressed osteoclastogenesis, implying that Notch2/Dll1-mediated enhancement was dominant. Actually, blockade of Dll1 ameliorated arthritis induced by K/BxN serum transfer, reduced the number of osteoclasts in the affected joints and suppressed ovariectomy-induced bone loss.

Conclusions

The differential regulation of osteoclastogenesis by Notch2/Dll1 and Notch1/Jagged1 axes may be a novel target for amelioration of bone erosion in RA patients.     

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.