Notch-Jagged signaling complex defined by an interaction mosaic.

The Notch signaling system links cellular fate to that of its neighbors, driving proliferation, apoptosis, and cell differentiation in metazoans, whereas dysfunction leads to debilitating developmental disorders and cancers. Other than a five-by-five domain complex, it is unclear how the 40 extracellular domains of the Notch1 receptor collectively engage the 19 domains of its canonical ligand, Jagged1, to activate Notch1 signaling. Here, using cross-linking mass spectrometry (XL-MS), biophysical, and structural techniques on the full extracellular complex and targeted sites, we identify five distinct regions, two on Notch1 and three on Jagged1, that form an interaction network. The Notch1 membrane-proximal regulatory region individually binds to the established Notch1 epidermal growth factor (EGF) 8-EGF13 and Jagged1 C2-EGF3 activation sites as well as to two additional Jagged1 regions, EGF8-EGF11 and cysteine-rich domain. XL-MS and quantitative interaction experiments show that the three Notch1-binding sites on Jagged1 also engage intramolecularly. These interactions, together with Notch1 and Jagged1 ectodomain dimensions and flexibility, determined by small-angle X-ray scattering, support the formation of nonlinear architectures. Combined, the data suggest that critical Notch1 and Jagged1 regions are not distal but engage directly to control Notch1 signaling, thereby redefining the Notch1-Jagged1 activation mechanism and indicating routes for therapeutic applications.

Jagged1 intracellular domain modulates steroidogenesis in testicular Leydig cells.

Leydig cells represent the steroidogenic lineage of mammalian testis, which produces testosterone. Genetic evidence indicates the requirement of Notch signaling in maintaining a balance between differentiated Leydig cells and their progenitors during fetal development. In primary Leydig cells, Notch1 expression decreases with testicular development, while the expression of its ligand, Jagged1, remains relatively unchanged, suggesting that the roles of Jagged1 extend beyond Notch signaling. In addition, Jagged1 is known to be processed into its intracellular domain, which then translocate to the nucleus. In this study, we investigated the effect of Jagged1 intracellular domain (JICD) on steroidogenesis in Leydig cells. The independent overexpression of JICD in MA-10 Leydig cells was found to inhibit the activity of cAMP-induced Nur77 promoter. In addition, JICD suppressed Nur77 transactivation of the promoter of steroidogenic genes such as P450scc, P450c17, StAR, and 3ß-HSD. Further, adenovirus-mediated overexpression of JICD in primary Leydig cells repressed the expression of steroidogenic genes, consequently lowering testosterone production. These results collectively suggest that steroidogenesis in testicular Leydig cells, which is regulated by LH/cAMP signaling, is fine-tuned by Jagged1 during testis development.

Development of Therapeutic Anti-JAGGED1 Antibodies for Cancer Therapy.

The role of Notch signaling and its ligand JAGGED1 (JAG1) in tumor biology has been firmly established, making them appealing therapeutic targets for cancer treatment. Here, we report the development and characterization of human/rat-specific JAG1-neutralizing mAbs. Epitope mapping identified their binding to the Notch receptor interaction site within the JAG1 Delta/Serrate/Lag2 domain, where E228D substitution prevented effective binding to the murine Jag1 ortholog. These antibodies were able to specifically inhibit JAG1-Notch binding in vitro, downregulate Notch signaling in cancer cells, and block the heterotypic JAG1-mediated Notch signaling between endothelial and vascular smooth muscle cells. Functionally, in vitro treatment impaired three-dimensional growth of breast cancer cell spheroids, in association with a reduction in cancer stem cell number. In vivo testing showed variable effects on human xenograft growth when only tumor-expressed JAG1 was targeted (mouse models) but a more robust effect when stromal-expressed Jag1 was also targeted (rat MDA-MB-231 xenograft model). Importantly, treatment of established triple receptor-negative breast cancer brain metastasis in rats showed a significant reduction in neoplastic growth. MRI imaging demonstrated that this was associated with a substantial improvement in blood-brain barrier function and tumor perfusion. Lastly, JAG1-targeting antibody treatment did not cause any detectable toxicity, further supporting its clinical potential for cancer therapy.

In silico analysis, seasonal variation and gonadotropic regulation of jag1 and its receptor notch1 in testis of spotted snakehead Channa punctatus.

The present study in seasonally breeding spotted snakehead Channa punctatus, for the first time in nonmammalian vertebrates, demonstrated correlation between reproductive phase-dependent testicular expression of ligand Jag1/receptor Notch1 and spermatogenic events. Testicular transcriptome sequencing data from our earlier study in C. punctatus was used in the present study to select the best transcript for jag1 (cpjag1) and notch1 (cpnotch1). The transcripts cpjag1 and cpnotch1 encoded full-length putative proteins of 1215 (cpJag1) and 2475 (cpNotch1) amino acids, respectively. A marked homology in the extracellular domains of Jag1 and Notch1 was observed following their alignment with respective proteins from different vertebrates, suggesting conservation in ligand-receptor interaction in C. punctatus. Both cpJag1 and cpNotch1 showed phylogenetic closeness with their teleostean counterparts, especially with that of Perciformes. Temporal expression of cpjag1 and cpnotch1 in testis depending on reproductive phases showed an appreciably high expression during spermatogenically inactive resting and postspawning phases when seminiferous lobules consisted of spermatogonial stem cells and undifferentiated spermatogonia. Their expression sharply declined during spermatogenically active preparatory and spawning phases. It appears that involvement of cpjag1/cpnotch1 is restricted to inactive phases when spermatogonial stem cells renew themselves and replenish undifferentiated spermatogonia. This assumption is ascertained by an experimental study in which high level of testicular cpjag1/cpnotch1 expression in control fish of resting phase markedly decreased after administration of human chorionic gonadotropin that is known to induce proliferation and differentiation of spermatogonia and spawning of spermatozoa.

Characterization of a bioactive Jagged1-coated polycaprolactone-based membrane for guided tissue regeneration.

OBJECTIVE: The aim of the present study was to develop a Jagged1-coated polycaprolactone (PCL) membrane and to evaluate the response of human periodontal ligament cells (hPDL) on this membrane in vitro. METHODS: Membranes were prepared from PCL and PCL-incorporated hydroxyapatite (PCL/HA). The membranes' surface roughness, surface wettability, and mechanical properties were examined. An indirect affinity immobilization technique was used to coat the membranes with Jagged1. Membrane cytotoxicity was evaluated using LIVE/DEAD and MTT assays. The morphology of the cells on the membranes was observed using scanning electron microscopy. hPDL alkaline phosphatase (ALP) enzymatic activity and mineral deposition were examined using an ALP assay and Alizarin Red S staining, respectively. Notch target gene mRNA expression was determined using real-time polymerase chain reaction. RESULTS: The PCL/HA membranes exhibited a significantly reduced surface contact angle, decreased maximum tensile strain, and ultimate tensile stress. However, the surface roughness parameters were significantly increased. The PCL and PCL/HA membranes were not cytotoxic to hPDL in vitro. hPDLs attached and spread on both membrane types. Further, indirect affinity immobilized Jagged1 on the membranes upregulated hPDL Notch target gene expression. After culturing in osteogenic medium, Jagged1-immobilized PCL/HA membranes significantly enhanced hPDL ALP enzymatic activity. CONCLUSION: Indirect immobilized Jagged1 PCL/HA membranes could be further developed as an alternative guided tissue regeneration membrane to promote osteogenic differentiation in periodontal defects.

Notch-Jagged complex structure implicates a catch bond in tuning ligand sensitivity.

Notch receptor activation initiates cell fate decisions and is distinctive in its reliance on mechanical force and protein glycosylation. The 2.5-angstrom-resolution crystal structure of the extracellular interacting region of Notch1 complexed with an engineered, high-affinity variant of Jagged1 (Jag1) reveals a binding interface that extends ~120 angstroms along five consecutive domains of each protein. O-Linked fucose modifications on Notch1 epidermal growth factor-like (EGF) domains 8 and 12 engage the EGF3 and C2 domains of Jag1, respectively, and different Notch1 domains are favored in binding to Jag1 than those that bind to the Delta-like 4 ligand. Jag1 undergoes conformational changes upon Notch binding, exhibiting catch bond behavior that prolongs interactions in the range of forces required for Notch activation. This mechanism enables cellular forces to regulate binding, discriminate among Notch ligands, and potentiate Notch signaling.

Identification of Domains for Efficient Notch Signaling Activity in Immobilized Notch Ligand Proteins.

Notch is a critical signaling pathway that controls cell fate and tissue homeostasis, but the functional characterization of Notch ligand domains that activate Notch receptors remains incomplete. Here, we established a method for immobilizing Notch ligand proteins onto beads to measure time-dependent Notch activity after the addition of Notch ligand-coated beads. A comparison between activities by the Notch ligand found on the cell surface to that of the ligand immobilized on beads showed that immobilized Notch ligand protein produces comparable signal activity during the first 10 h. Follow-up truncation studies showed that the N-terminal epidermal growth factor (EGF) repeat three region of delta like canonical Notch ligand 4 (DLL4) or jagged 1 (JAG1) is the minimum region for activating Notch signaling, and the DLL4 EGF repeat three domain may have a role in activation through a mechanism other than by increasing binding affinity. In addition, we found that reconstruction of the DLL4 delta and OSM-11 (DOS) motif (N257P) resulted in an increase in both binding affinity and signaling activity, which suggests that the role of the DOS motif is conserved among Notch ligands. Furthermore, active DLL4 protein on beads promoted T cell differentiation or inhibited B cell differentiation in vitro, whereas JAG1 proteins on beads did not have any effect. Taken together, our findings provide unambiguous evidence for the role of different Notch ligands and their domains in Notch signal activation, and may be potential tools for controlling Notch signaling activation. J. Cell. Biochem. 118: 785-796, 2017. © 2016 Wiley Periodicals, Inc.