Physical interaction of Delta1, Jagged1, and Jagged2 with Notch1 and Notch3 receptors

The Delta/Serrate/LAG-2 (DSL) domain-containing proteins, Delta1, Jagged1, and Jagged2, are considered to be ligands for Notch receptors. However, the physical interaction between the three DSL proteins and respective Notch receptors remained largely unknown. In this study, we investigated this issue through the targeting of Notch1 and Notch3 in two experimental systems using fusion proteins comprising their extracellular portions. Cell-binding assays showed that soluble forms of Notch1 and Notch3 proteins physically bound to the three DSL proteins on the cell surface. In solid-phase binding assays using immobilized soluble Notch1 and Notch3 proteins, it was revealed that each DSL protein directly bound to the soluble Notch proteins with different affinities. All interactions between the DSL proteins and soluble Notch proteins were dependent on Ca(2+). Taken together, these results suggest that Delta1, Jagged1, and Jagged2 are ligands for Notch1 and Notch3 receptors.     

Fringe differentially modulates Jagged1 and Delta1 signalling through Notch1 and Notch2

Proteins encoded by the fringe family of genes are required to modulate Notch signalling in a wide range of developmental contexts. Using a cell co-culture assay, we find that mammalian Lunatic fringe (Lfng) inhibits Jagged1-mediated signalling and potentiates Delta1-mediated signalling through Notch1. Lfng localizes to the Golgi, and Lfng-dependent modulation of Notch signalling requires both expression of Lfng in the Notch-responsive cell and the Notch extracellular domain. Lfng does not prevent binding of soluble Jagged1 or Delta1 to Notch1-expressing cells. Lfng potentiates both Jagged1- and Delta1-mediated signalling via Notch2, in contrast to its actions with Notch1. Our data suggest that Fringe-dependent differential modulation of the interaction of Delta/Serrate/Lag2 (DSL) ligands with their Notch receptors is likely to have a significant role in the combinatorial repertoire of Notch signalling in mammals.     

Fringe glycosyltransferases differentially modulate Notch1 proteolysis induced by Delta1 and Jagged1

Fringe O-fucose-beta1,3-N-acetylglucosaminyltransferases modulate Notch signaling by potentiating signaling induced by Delta-like ligands, while inhibiting signaling induced by Serrate/Jagged1 ligands. Based on binding studies, the differential effects of Drosophila fringe (DFng) on Notch signaling are thought to result from alterations in Notch glycosylation that enhance binding of Delta to Notch but reduce Serrate binding. Here, we report that expression of mammalian fringe proteins (Lunatic [LFng], Manic [MFng], or Radical [RFng] Fringe) increased Delta1 binding and activation of Notch1 signaling in 293T and NIH 3T3 cells. Although Jagged1-induced signaling was suppressed by LFng and MFng, RFng enhanced signaling induced by either Delta1 or Jagged1, underscoring the diversity of mammalian fringe glycosyltransferases in regulating signaling downstream of different ligand-receptor combinations. Interestingly, suppression of Jagged1-induced Notch1 signaling did not correlate with changes in Jagged1 binding as found for Delta1. Our data support the idea that fringe glycosylation increases Delta1 binding to potentiate signaling, but we propose that although fringe glycosylation does not reduce Jagged1 binding to Notch1, the resultant ligand-receptor interactions do not effectively promote Notch1 proteolysis required for activation of downstream signaling events.     

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.     

Notch ligands with contrasting functions: Jagged1 and Delta1 in the mouse inner ear

Each of the sensory patches in the epithelium of the inner ear is a mosaic of hair cells and supporting cells. Notch signalling is thought to govern this pattern of differentiation through lateral inhibition. Recent experiments in the chick suggest, however, that Notch signalling also has a prior function - inductive rather than inhibitory - in defining the prosensory patches from which the differentiated cells arise. Several Notch ligands are expressed in each patch, but their individual roles in relation to the two functions of Notch signalling are unclear. We have used a Cre-LoxP approach to knock out two of these ligands, Delta1 (Dll1) and Jagged1 (Jag1), in the mouse ear. In the absence of Dll1, auditory hair cells develop early and in excess, in agreement with the lateral inhibition hypothesis. In the absence of Jag1, by contrast, the total number of these cells is strongly reduced, with complete loss of cochlear outer hair cells and some groups of vestibular hair cells, indicating that Jag1 is required for the prosensory inductive function of Notch. The number of cochlear inner hair cells, however, is almost doubled. This correlates with loss of expression of the cell cycle inhibitor p27(Kip1) (Cdkn1b), suggesting that signalling by Jag1 is also needed to limit proliferation of prosensory cells, and that there is a core part of this population whose prosensory character is established independently of Jag1-Notch signalling. Our findings confirm that Notch signalling in the ear has distinct prosensory and lateral-inhibitory functions, for which different ligands are primarily responsible.     

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.     

Jagged 1与Delta1对树突状细胞的影响

Jagged 1与Delta1是Notch信号通路中的两个配体,近来研究表明,它们能影响树突状细胞的分化和成熟,并通过树突状细胞等抗原提呈细胞介导T辅助细胞的不同分化,可能为免疫性疾病的临床治疗提供新的药物靶点.     

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.     

FGFR1 cleavage and nuclear translocation regulates breast cancer cell behavior

FGF-10 and its receptors, FGFR1 and FGFR2, have been implicated in breast cancer susceptibility and progression, suggesting that fibroblast growth factor (FGF) signaling may be co-opted by breast cancer cells. We identify a novel pathway downstream of FGFR1 activation, whereby the receptor is cleaved and traffics to the nucleus, where it can regulate specific target genes. We confirm Granzyme B (GrB) as the protease responsible for cleavage and show that blocking GrB activity stopped FGFR1 trafficking to the nucleus and abrogates the promigratory effect of FGF stimulation. We confirm the in vivo relevance of our findings, showing that FGFR1 localized to the nucleus specifically in invading cells in both clinical material and a three-dimensional model of breast cancer. We identify target genes for FGFR1, which exert significant effects on cell migration and may represent an invasive signature. Our experiments identify a novel mechanism by which FGF signaling can regulate cancer cell behavior and provide a novel therapeutic target for treatment of invasive breast cancer.     

MCL1 nuclear translocation induces chemoresistance in colorectal carcinoma

Colorectal cancer (CRC) is one of the most common and deadliest forms of cancer. Myeloid Cell Leukemia 1 (MCL1), a pro-survival member of the Bcl-2 protein family is associated with chemo-resistance in CRC. The ability of MCL1 to inhibit apoptosis by binding to the BH3 domains of pro-apoptotic Bcl-2 family members is a well-studied means by which this protein confers resistance to multiple anti-cancer therapies. We found that specific DNA damaging chemotherapies promote nuclear MCL1 translocation in CRC models. In p53^null CRC, this process is associated with resistance to chemotherapeutic agents, the mechanism of which is distinct from the classical mitochondrial protection. We previously reported that MCL1 has a noncanonical chemoresistance capability, which requires a novel loop domain that is distinct from the BH3-binding domain associated with anti-apoptotic function. Herein we disclose that upon treatment with specific DNA-damaging chemotherapy, this loop domain binds directly to alpha-enolase which in turn binds to calmodulin; we further show these protein−protein interactions are critical in MCL1’s nuclear import and chemoresistance. We additionally observed that in chemotherapy-treated p53^−/− CRC models, MCL1 nuclear translocation confers sensitivity to Bcl-xL inhibitors, which has significant translational relevance given the co-expression of these proteins in CRC patient samples. Together these findings indicate that chemotherapy-induced MCL1 translocation represents a novel resistance mechanism in CRC, while also exposing an inherent and targetable Bcl-xL co-dependency in these cancers. The combination of chemotherapy and Bcl-xL inhibitors may thus represent a rational means of treating p53^−/− CRC via exploitation of this unique MCL1-based chemoresistance mechanism.Subject terms: Targeted therapies, Senescence     

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.     

Unique patterns of Notch1, Notch4 and Jagged1 expression in ovarian vessels during folliculogenesis and corpus luteum formation

Notch signaling functions to regulate cell-fate decisions by modulating differentiation, proliferation, and survival of cells. Notch receptors and ligands are expressed in embryonic vasculature and are required for the remodeling of the primary embryonic vasculature of mice. Here, we characterize the expression patterns of Notch1, Notch4, and Jagged1 proteins during the process of folliculogenesis and corpus luteum formation in the mouse ovary, an organ with dynamic physiological angiogenic growth. These Notch proteins and ligand are expressed in a subset of ovarian vessels, including both mature ovarian vasculature as well as angiogenic neovessels. Their expression in the ovary was found in both endothelial and vascular associated mural cells. Our data suggest a complex regulatory role for the Notch signaling pathway during mouse oogenesis and ovarian neovascularization.     

TGF-alpha induces upregulation and nuclear translocation of Hes1 in glioma cell

Both the Notch-signaling pathway and extracellular signal regulated kinase (ERK) cascade are involved in a wide variety of biological processes, such as proliferation, differentiation, survival, and tumorigenesis. Their dysregulation in recent studies have been shown to be associated with glioma formation. Here, we show that transforming growth factor-alpha (TGF-alpha) stimulated glioma cell line U251 growth and can partly compensate for the inhibitory effect of Notch-signaling inhibitor DAPT. The effect of TGF-alpha on ERK1/2 phosphorylation was prompt and transient and could be inhibited by mitogen-activated/extracellular signal-regulated kinase kinase 1/2 (MEK1/2) specific inhibitor PD98059. Moreover, TGF-alpha was capable of up-regulating Hairy-enhancer of split1 (Hes1) expression which was independent of Notch1 activation, and of introducing Hes1 nuclear import in the presence of ERK1/2 activation. Collectively, our data suggest a potential linkage between ERK activation and the Notch-signaling pathway.     

Suppression of Th2 cell development by Notch ligands Delta1 and Delta4

Notch signaling plays important roles in Th cell activation. We show that in response to TLR ligation, dendritic cells up-regulate expression of Notch ligands Delta1 and Delta4 via a MyD88-dependent pathway. Expression of Delta1 or Delta4 by dendritic cells enhanced their ability to activate naive Th cells and promote Th1 cell development, and allowed them to strongly inhibit Th2 cell development. Promotion of Th1 cell development was dependent on IFN-gamma and T-bet expression by responding Th cells. However, the inhibition of Th2 cell development occurred independently of IFN-gamma or T-bet, and resulted from a block in IL-4-initiated commitment to the Th2 lineage. The promotion of Th1 cell development by Delta is not a reflection of the delivery of pro-Th1 instructional signal, but rather it is the result of a block in the downstream effects initiated by IL-4 signaling.     

DNA damage induces nuclear translocation of parkin

Parkinson's disease (PD) is the second most common form of human degenerative disorder. Mutation of parkin is one of the most prevalent causes of autosomal recessive PD. Parkin is an E3 ubiquitin ligase that acts on a variety of substrates, resulting in polyubiquitination and degradation by the proteasome or monoubiquitination and regulation of biological activity. However, the cellular functions of parkin that relate to its pathological involvement in PD are not well understood. Here I show that parkin translocates into nucleus upon DNA damage. Nuclear translocation of parkin appears to be required to promote DNA repair. These findings suggest that DNA damage induces nuclear translocation of parkin leading to the PCNA interaction and possibly other nuclear proteins involved in DNA repair. These results suggest that parkin promotes DNA repair and protects against genotoxicity, and implicate DNA damage as a potential pathogenic mechanism in parkinsonism.