Human Jagged1 Research Articles

An Intact Periosteum is Required for Recombinant Human Jagged1 Guided Bone Regeneration in Calvaria Critical-size Defect Healing.

The need to promote calvaria bone healing as a consequence of injury or craniotomy is a major clinical issue. Previous reports tested recombinant human Jagged1 (rhJagged1) treatment for critical-size calvaria defects in the absence of periosteum, and this resulted in significant new bone formation. As the periosteum contributes to healing by serving as a source of progenitor cells, the present study aimed to examine whether significantly more bone is formed when the periosteum is intact for using rhJagged1 to treat critical-size parietal bone defects in mice. Fifteen healthy adult mice, 34 to 65 weeks of age, 26.9 to 48.2 g, were divided into different groups that compared the critical-size defects treated with either phosphate-buffered saline or rhJagged1 protein in either the presence or absence of periosteum. The results indicated that more bone was formed in the presence of periosteum when rhJagged1 is delivered [35% bone volume per tissue volume (BV/TV); P = 0.02] relative to nonperiosteum. Recombinant human Jagged1 protein delivered in the absence of periosteum had the next most new bone formed (25% BV/TV). Defects with phosphate-buffered saline delivered in the absence or presence of periosteum had the least new bone formed (15% and 18% BV/TV, respectively; P = 0.48). The results also show that rhJagged1 does not form ectopic or hypertrophic bone. The usage of rhJagged1 to treat critical-size defects in calvaria is promising clinically, but to maximize clinical efficacy it will require that the periosteum be intact on the noninjured portions of calvaria.

An N-glycan on the C2 domain of JAGGED1 is important for Notch activation.

The canonical members of the Jagged/Serrate and Delta families of transmembrane ligands have an extracellular, amino-terminal C2 domain that binds to phospholipids and is required for optimal activation of the Notch receptor. Somatic mutations that cause amino substitutions in the C2 domain in human JAGGED1 (JAG1) have been identified in tumors. We found in reporter cell assays that mutations affecting an N-glycosylation site reduced the ligand's ability to activate Notch. This N-glycosylation site located in the C2 domain is conserved in the Jagged/Serrate family but is lacking in the Delta family. Site-specific glycan analysis of the JAG1 amino terminus demonstrated that occupancy of this site by either a complex-type or high-mannose N-glycan was required for full Notch activation in reporter cell assays. Similarly to JAG1 variants with defects in Notch binding, N-glycan removal, either by mutagenesis of the glycosylation site or by endoglycosidase treatment, reduced receptor activation. The N-glycan variants also reduced receptor activation in a Notch signaling-dependent vascular smooth muscle cell differentiation assay. Loss of the C2 N-glycan reduced JAG1 binding to liposomes to a similar extent as the loss of the entire C2 domain. Molecular dynamics simulations suggested that the presence of the N-glycan limits the orientation of JAG1 relative to the membrane, thus facilitating Notch binding. These data are consistent with a critical role for the N-glycan in promoting a lipid-binding conformation that is required to orient Jagged at the cell membrane for full Notch activation.

Notch-activated mesenchymal stromal/stem cells enhance the protective effect against acetaminophen-induced acute liver injury by activating AMPK/SIRT1 pathway

BackgroundNotch signaling plays important roles in regulating innate immunity. However, little is known about the role of Notch in mesenchymal stromal/stem cell (MSC)-mediated immunomodulation during liver inflammatory response.MethodsNotch activation in human umbilical cord-derived MSCs was performed by a tissue culture plate coated with Notch ligand, recombinant human Jagged1 (JAG1). Mice were given intravenous injection of Notch-activated MSCs after acetaminophen (APAP)-induced acute liver injury. Liver tissues were collected and analyzed by histology and immunohistochemistry.ResultsMSC administration reduced APAP-induced hepatocellular damage, as manifested by decreased serum ALT levels, intrahepatic macrophage/neutrophil infiltration, hepatocellular apoptosis and proinflammatory mediators. The anti-inflammatory activity and therapeutic effects of MSCs were greatly enhanced by Notch activation via its ligand JAG1. However, Notch2 disruption in MSCs markedly diminished the protective effect of MSCs against APAP-induced acute liver injury, even in the presence of JAG1 pretreatment. Strikingly, Notch-activated MSCs promoted AMP-activated protein kinase (AMPKα) phosphorylation, increased the sirtuins 1 (SIRT1) deacetylase expression, but downregulated spliced X-box-binding protein 1 (XBP1s) expression and consequently reduced NLR family pyrin domain-containing 3 (NLRP3) inflammasome activation. Furthermore, SIRT1 disruption or XBP1s overexpression in macrophages exacerbated APAP-triggered liver inflammation and augmented NLRP3/caspase-1 activity in MSC-administrated mice. Mechanistic studies further demonstrated that JAG1-pretreated MSCs activated Notch2/COX2/PGE2 signaling, which in turn induced macrophage AMPK/SIRT1 activation, leading to XBP1s deacetylation and inhibition of NLRP3 activity.ConclusionsActivation of Notch2 is required for the ability of MSCs to reduce the severity of APAP-induced liver damage in mice. Our findings underscore a novel molecular insights into MSCs-mediated immunomodulation by activating Notch2/COX2/AMPK/SIRT1 pathway and thus provide a new strategy for the treatment of liver inflammatory diseases.

Notch signaling promotes serrated neoplasia pathway in colorectal cancer through epigenetic modification of EPHB2 and EPHB4.

BackgroundDysregulation of erythropoietin-producing hepatoma (Eph) proteins in human cancers is extensively documented but not clear in colorectal cancer (CRC). In this study, we aimed to investigate the role of Notch signaling pathway and epigenetic modification of EPHB2 and EPHB4 expression in serrated neoplasia development.MethodsThe expression of EPHB2 and EPHB4 in CRC clinical specimens and cell lines were determined by immunohistochemistry, Western blot, and real-time PCR. Cell proliferation and invasion were evaluated by MTT and chamber kits, luciferase assay and co-immunoprecipitation were used to detect the transcriptional regulation and protein–protein interactions, respectively. The immunofluorescence assay was employed to confirm the subcellular location of Notch intracellular domain (NICD), and chromatin immunoprecipitation assay was implied to detect the modification types of H3K4me3 and H3K27me3. Mice xenograft model was used to detect the in vivo effects of EPHB2 and EPHB4 genes on cell growth.ResultsIn CRC clinical specimens and cell lines, we found that EPHB2 was significantly decreased, while EPHB4 was elevated in the CRC tissues, and these aberrant expression manners correlated with worse overall survival rates in the clinic. When the EPHB2 and EPHB4 expressions were manipulated by overexpression or knockdown in the SW620 cells, the cell proliferation and invasion were obviously suppressed, whereas EPHB2 knockdown or EPHB4 overexpression showed the opposite phenotypes. We also found that Notch signaling pathway was abnormally activated and treatment of Notch signaling ligand human Jagged1 peptide downregulated EPHB2 and upregulated EPHB4 in the SW620 cells, as well as promoted the chromatin modification protein Jumonji domain-containing protein-3 (JMJD3) cytonuclear trans-localization with the NICD, which indicated that NICD brought JMJD3 to the EPHB4 enhancer region to decrease the H3K27me3 level.ConclusionTaken together, we provide a new mechanistic option in understanding the role of Notch signaling and the roles of EPHB2 and EPHB4 in CRC.

Jagged-1 Signaling in the Bone Marrow Microenvironment Promotes Endothelial Progenitor Cell Expansion and Commitment of CD133+ Human Cord Blood Cells for Postnatal Vasculogenesis.

Notch signaling is involved in cell fate decisions during murine vascular development and hematopoiesis in the microenvironment of bone marrow. To investigate the close relationship between hematopoietic stem cells and human endothelial progenitor cells (EPCs) in the bone marrow niche, we examined the effects of Notch signals [Jagged-1 and Delta-like ligand (Dll)-1] on the proliferation and differentiation of human CD133+ cell-derived EPCs. We established stromal systems using HESS-5 murine bone marrow cells transfected with human Jagged-1 (hJagged-1) or human Dll-1 (hDll-1). CD133+ cord blood cells were co-cultured with the stromal cells for 7 days, and then their proliferation, differentiation, and EPC colony formation was evaluated. We found that hJagged-1 induced the proliferation and differentiation of CD133+ cord blood EPCs. In contrast, hDll-1 had little effect. CD133+ cells stimulated by hJagged-1 differentiated into CD31+/KDR+ cells, expressed vascular endothelial growth factor-A, and showed enhanced EPC colony formation compared with CD133+ cells stimulated by hDll-1. To evaluate the angiogenic properties of hJagged-1- and hDll-1-stimulated EPCs in vivo, we transplanted these cells into the ischemic hindlimbs of nude mice. Transplantation of EPCs stimulated by hJagged-1, but not hDll-1, increased regional blood flow and capillary density in ischemic hindlimb muscles. This is the first study to show that human Notch signaling influences EPC proliferation and differentiation in the bone marrow microenvironment. Human Jagged-1 induced the proliferation and differentiation of CD133+ cord blood progenitors compared with hDll-1. Thus, hJagged-1 signaling in the bone marrow niche may be used to expand EPCs for therapeutic angiogenesis.

Jagged1 heterozygosity in mice results in a congenital cholangiopathy which is reversed by concomitant deletion of one copy of Poglut1 (Rumi).

Haploinsufficiency for the Notch ligand JAG1 in humans results in an autosomal-dominant, multisystem disorder known as Alagille syndrome, which is characterized by a congenital cholangiopathy of variable severity. Here, we show that on a C57BL/6 background, jagged1 heterozygous mice (Jag1(+/-) ) exhibit impaired intrahepatic bile duct (IHBD) development, decreased SOX9 expression, and thinning of the periportal vascular smooth muscle cell (VSMC) layer, which are apparent at embryonic day 18 and the first postnatal week. In contrast, mice double heterozygous for Jag1 and the glycosyltransferase, Poglut1 (Rumi), start showing a significant improvement in IHBD development and VSMC differentiation during the first week. At P30, Jag1(+/-) mice show widespread ductular reactions and ductopenia in liver and a mild, but statistically, significant bilirubinemia. In contrast, P30 Jag1/Rumi double-heterozygous mice show well-developed portal triads around most portal veins, with no elevation of serum bilirubin. Conditional deletion of Rumi in VSMCs results in progressive arborization of the IHBD tree, whereas deletion of Rumi in hepatoblasts frequently results in an increase in the number of hepatic arteries without affecting bile duct formation. Nevertheless, removing one copy of Rumi from either VSMCs or hepatoblasts is sufficient to partially suppress the Jag1(+/-) bile duct defects. Finally, all Rumi target sites of the human JAG1 are efficiently glucosylated, and loss of Rumi in VSMCs results in increased levels of full-length JAG1 and a shorter fragment of JAG1 without affecting Jag1 messenger RNA levels. On a C57BL/6 background, Jag1 haploinsufficiency results in bile duct paucity in mice. Removing one copy of Rumi suppresses the Jag1(+/-) bile duct phenotype, indicating that Rumi opposes JAG1 function in the liver.

NOTCH-Mediated Maintenance and Expansion of Human Bone Marrow Stromal/Stem Cells: A Technology Designed for Orthopedic Regenerative Medicine.

Human bone marrow-derived stromal/stem cells (BMSCs) have great therapeutic potential for treating skeletal disease and facilitating skeletal repair, although maintaining their multipotency and expanding these cells ex vivo have proven difficult. Because most stem cell-based applications to skeletal regeneration and repair in the clinic would require large numbers of functional BMSCs, recent research has focused on methods for the appropriate selection, expansion, and maintenance of BMSC populations during long-term culture. We describe here a novel biological method that entails selection of human BMSCs based on NOTCH2 expression and activation of the NOTCH signaling pathway in cultured BMSCs via a tissue culture plate coated with recombinant human JAGGED1 (JAG1) ligand. We demonstrate that transient JAG1-mediated NOTCH signaling promotes human BMSC maintenance and expansion while increasing their skeletogenic differentiation capacity, both ex vivo and in vivo. This study is the first of its kind to describe a NOTCH-mediated methodology for the maintenance and expansion of human BMSCs and will serve as a platform for future clinical or translational studies aimed at skeletal regeneration and repair.

Abstract 2664: An anti-Jagged-1/-2 Probody demonstrates inhibition of Jagged-dependent Notch signaling and is activated in multiple types of tumors

Abstract Probodies represent a unique class of antibody therapeutics that specifically target therapeutic activity to diseased tissues, such as cancer. A Probody is a fully recombinant protein that contains a masking peptide that blocks the antibody's antigen-binding site and is linked to the light chain of the antibody by a protease-cleavable peptide substrate. Probodies are designed to be inactive in circulation and in healthy tissues but activated within the tumor microenvironment. The Probody™ approach is designed to widen the therapeutic index of antibodies to promising therapeutic targets that may be limited by significant toxicity. One such promising set of targets are components of the Notch pathway, which is involved in both oncogenic and healthy tissue signaling. We have developed a fully human IgG1 monoclonal antibody that binds and inhibits the activity of both human and mouse Jagged-1 and Jagged-2, two key ligands in the Notch signaling pathway. A Probody produced from this antibody induced dose-dependent inhibition of Notch signaling, resulting in anti-tumor activity in in vivo tumor models without significant toxicity. To further explore the clinical relevance of the anti-Jagged Probody for the treatment of cancer, we evaluated (i) the heterogeneity of Jagged expression in multiple tumor types, (ii) the activation of the Jagged Probody in tumor biopsies, and (iii) the blockade of Notch signaling using an intracellular pharmacodynamic biomarker. Patient tumor tissues were screened for Jagged-1/-2 IHC across lung, pancreatic and breast cancer_ indications where activation of the Notch pathway is implicated. Overall, more than 75% of patient tumors demonstrated moderate to high Jagged-1/-2 expression. We also developed and validated a technique termed IHZ™ analysis that enables the measurement of proteolytic Probody activation and binding to Jagged ligands ex vivo in human tumor sections. We evaluated 120 patients across the three tumor types and observed ∼99% of samples that were positive for antibody binding were also positive by IHZ analysis, signifying sufficient proteolytic activity to activate the Probody. Finally, we evaluated the accumulation of the Notch Intracellular Domain (NICD), a biomarker of Notch signaling, in the nucleus of tumor cells in animals treated with antibody. Notably, our data showed that Notch signaling blockade as indicated by a decrease in the accumulation of NICD correlated with dose-response in in vivo models. Taken together, our data suggest that the anti-Jagged-1/-2 Probody may have wide therapeutic utility as a tumor-specific inhibitor of Jagged-dependent Notch signaling in oncology. Citation Format: Olga Vasiljeva, Elizabeth Menendez, Jason Sagert, James W. West, Jennifer Richardson, Luc Desnoyers, Shouchun Liu, Judi Ford, Krishna Plou, Henry Lowman. An anti-Jagged-1/-2 Probody demonstrates inhibition of Jagged-dependent Notch signaling and is activated in multiple types of tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2664. doi:10.1158/1538-7445.AM2014-2664

Abstract 1823: Novel regulation of Jagged1 by ErbB2 in breast cancer: implications for anti-ErbB2 therapy

Abstract We have demonstrated that Notch1 is required for trastuzumab resistance in ErbB2 positive breast cancer. This indicates that ErbB2 suppresses Notch1 in breast cancer and therapeutic intervention targeting ErbB2 might have an unintended consequence which is aberrant up regulation of Notch1 which is a breast oncogene. However, the mechanism of action by which ErbB2 restricts Notch1 activation is unknown. In this current study, we investigated the role of cis- and trans-activation of Notch signaling by Notch ligands which are developmentally conversed to tightly regulate Notch activation. To address this hypothesis, we performed co-culture studies using fibroblasts expressing no Notch ligands or over-expressing human Jagged1 or Deltalike1 and ErbB2 positive breast cancer cells. We performed flow cytometry to isolate breast cancer cells after co-culture and extracted RNA to measure expression of Notch gene targets as a measure of Notch activity. The results showed that trastuzumab, Lapatinib, or ErbB2 knockdown increased overall Notch activition. Similarly, Co-culture with Jagged1-expressing fibroblasts increased overall Notch activation. However, Knocked down of Jagged1 in the breast cancer cells had little effect on ligand-induced Notch activation relieving the possibility of cis-inhibition. In contrast, Jagged1 knocked down abrogated trastuzumab-induced Notch activation in the breast cancer cells. These results suggest that ErbB2 might restrict Notch activation by preventing Jagged1-mediated trans activation of Notch and not by promoting cis-inhibition. Confocal immunofluorescence showed that Jagged1 is localized with Notch1 when ErbB2 is hyperactive but is trafficked to the cell surface in response to trastuzumab. K44ADynamin abrogated Jagged1 expression on the cell surface as measured by IF and surface biotinylation studies. Furthermore, K44ADynamin expression abrogated trastuzumab-induced Notch1 activation. Importantly, we measured growth consequences of Jagged1-mediated Notch activation in response to trastuzumab and found that Jagged1 is necessary for survival of ErbB2 positive breast cancer cells and trastuzumab resistance as measured by cell cycle analysis and Annexin V staining. These results taken together indicate that ErbB2 restricts Notch by limiting Jagged1-mediated trans-activation. Citation Format: Clodia Osipo, Kinnari Pandya, Debra Wyatt. Novel regulation of Jagged1 by ErbB2 in breast cancer: implications for anti-ErbB2 therapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1823. doi:10.1158/1538-7445.AM2014-1823

Construction and expression of fusion protein TRX-hJagged1 in E.coli BL21

This study was purposed to construct prokaryotic expression vector and to investigate the expression of Notch ligand Jagged1 in E.coli. An expression vector pET-hJagged1 was constructed, which can be inserted in Jagged1 with different lengths, but the DSL domain of human Jagged1 should be contained. Then the recombinant plasmids were transformed into the competent cell of E.coli BL21, and the expression of the fusion protein was induced by IPTG. Fusion protein was purified from the supernatant of cell lysates via the Nickel affinity chromatography. The results showed that prokaryotic expression vectors pET-hJagged1 (Bgl II), pET-hJagged1 (Hind I) and pET-hJagged1 (Stu I) were successfully constructed, but only pET-hJagged1 (Stu I) could express the soluble TRX-hJagged1. The purified TRX-Jagged1 protein could be obtained via the Nickel affinity chromatography, and then confirmed by Western Blot. It is concluded that prokaryotic expression vector pET-hJagged1 is successfully constructed, but only pET-hJagged1 (Stu I) can express the soluble TRX-hJagged1 and the TRX-Jagged1 fusion protein is obtained through the prokaryotic expression system, which laid a solid foundation for further to explore the effects of Jagged1 in hematopoietic and lymphoid system.

Activation of Notch signaling by short-term treatment with Jagged-1 enhances store-operated Ca2+ entry in human pulmonary arterial smooth muscle cells

Notch signaling plays a critical role in controlling proliferation and differentiation of pulmonary arterial smooth muscle cells (PASMC). Upregulated Notch ligands and Notch3 receptors in PASMC have been reported to promote the development of pulmonary vascular remodeling in patients with pulmonary arterial hypertension (PAH) and in animals with experimental pulmonary hypertension. Activation of Notch receptors by their ligands leads to the cleavage of the Notch intracellular domain (NICD) to the cytosol by γ-secretase; NICD then translocates into the nucleus to regulate gene transcription. In this study, we examined whether short-term activation of Notch functionally regulates store-operated Ca(2+) entry (SOCE) in human PASMC. Treatment of PASMC with the active fragment of human Jagged-1 protein (Jag-1) for 15-60 min significantly increased the amplitude of SOCE induced by passive deletion of Ca(2+) from the intracellular stores, the sarcoplasmic reticulum (SR). The Jag-1-induced enhancement of SOCE was time dependent: the amplitude was maximized at 30 min of treatment with Jag-1, which was closely correlated with the time course of Jag-1-mediated increase in NICD protein level. The scrambled peptide of Jag-1 active fragment had no effect on SOCE. Inhibition of γ-secretase by N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester (DAPT) significantly attenuated the Jag-1-induced augmentation of SOCE. In addition to the short-term effect, prolonged treatment of PASMC with Jag-1 for 48 h also markedly enhanced the amplitude of SOCE. These data demonstrate that short-term activation of Notch signaling enhances SOCE in PASMC; the NICD-mediated functional interaction with store-operated Ca(2+) channels (SOC) may be involved in the Jag-1-mediated enhancement of SOCE in human PASMC.

Effective expansion of engrafted human hematopoietic stem cells in bone marrow of mice expressing human Jagged1

The human immune system can be reconstituted in experimental animals by transplanting human hematopoietic stem cells (hHSCs) into immunodeficient mice. To generate such humanized mice, further improvements are required, particularly to ensure that transplanted hHSCs are maintained in mice and proliferate long enough to follow prolonged immune responses to chronic diseases or monitor therapeutic effects. To prepare the relatively human bone marrow environment in mice, we generated nonobese diabetic/severe combined immunodeficiency/interleukin-2 receptor gamma chain null (NOG) mice expressing human Jagged1 (hJ1) in an osteoblast-specific manner (hJ1-NOG mice) to examine whether Notch signaling induced by hJ1 mediates hHSC proliferation and/or maintenance in mice. The established hJ1-NOG mice possess relatively larger bone marrow space and thinner cortical bone compared with nontransgenic littermates, but the number of c-kit(+) Sca-1(+) lineage(-) cells was not significantly different between hJ1-NOG and nontransgenic littermates. In the transplantation experiments of CD34(+) cells obtained from human cord blood, CD34(+)CD38(-) cells (hHSCs) were more increased in hJ1-NOG recipient mice than in nontransgenic littermates in mouse bone marrow environment. In contrast, the transplanted mouse c-kit(+) Sca-1(+) lineage(-) cells did not show significant increase in the same hJ1-NOG mice. These results suggest that hJ1-NOG mice could contribute to the growth of transplanted human CD34(+) cells in a human-specific manner and be useful to study the in vivo behavior and/or development of human stem cells, including cancer stem cells and immune cells.

Notch signaling and new therapeutic options in liver disease

Notch signaling is a crucial determinant of cell fate decision during development and disease in several organs. Notch effects are strictly dependent on the cellular context in which it is activated. In the liver, Notch signaling is involved in biliary tree development and tubulogenesis. Recent advances have shed light on Notch as a critical player in liver regeneration and repair, as well as in liver metabolism and inflammation and cancer. Notch signaling is finely regulated at several levels. The complexity of the pathway provides several possible targets for development of therapeutic agents able to inhibit Notch. Recent reports have shown that persistent activation of Notch signaling is associated with liver malignancies, particularly hepatocellular with stem cell features and cholangiocarcinoma. These novel findings suggest that interfering with the aberrant activation of the Notch pathway may have therapeutic relevance. However, further studies are needed to clarify the mechanisms regulating physiologic and pathologic Notch activation in the adult liver, to better understand the mechanistic role(s) of Notch in liver diseases and to develop safe and specific therapeutic agents.

Abstract C158: Tumor-specific inhibition of jagged-dependent notch signaling using a Probody™ therapeutic.

Abstract Probodies represent a unique class of antibody-based therapeutics that specifically target activity to diseased tissues including cancer. Probodies are designed to be inactive in circulation and in healthy tissue and activated only within the tumor microenvironment by dysregulated protease activity. The Probody approach enables promising therapeutic targets not druggable with conventional antibodies due to toxicity. Inhibition of Notch signaling has revealed the therapeutic potential of targeting this pathway in cancer; however, systemic inhibition results in severe toxicities (e.g. gastrointestinal and cardiac) in preclinical and clinical studies limiting the development of Notch-targeting therapeutics. We have developed a fully human IgG1 monoclonal antibody that binds and inhibits the activity of both human and mouse Jagged1 and Jagged2 (Jagged1/2). In vitro cell based assays and in vivo ectopic xenograft mouse tumor models (BxPC3 and H292) show dose-dependent inhibition of Notch signaling and anti-tumor activity, respectively. However, the antibody also shows profound toxicities in mice, including weight loss, alopecia, hyperkeratosis, and athymia, due to on-target inhibition of Jagged1/2 in healthy tissue. To address these toxicities, we developed a fully recombinant anti-Jagged1/2 Probody, CTX-033, comprised of the same antibody but with a unique peptide mask that blocks the antigen binding site linked to the light chain of the antibody by a linker containing a substrate that is cleavable by one or more proteases upregulated in cancer. CTX-033 had similar anti-tumor activity in the mouse xenograft models as compared to the antibody. However, despite a two-fold higher systemic exposure to CTX-033 as compared to the antibody, Probody treated mice did not exhibit the toxicities associated with the antibody treatment. Furthermore, CTX-033 shows additive anti-tumor efficacy when combined with gemcitabine in the BxPC3 model. Notably, the Probody plus gemcitabine combination lacks the significant toxicity associated with the antibody plus gemcitabine combination treatment. To validate both Jagged expression and protease activity in patient tumor samples, we used a novel in situ assay, immunofluorescent Probody zymography. The results reveal broad expression of the Jagged ligands and specific activation and binding of CTX-033 in patient pancreatic adenocarcinoma tissue samples. The data described here with the anti-Jagged Probody, CTX-033, demonstrates (1) the potential of the Probody platform to enable the safe targeting of two key ligands in the Notch signaling pathway not possible with a traditional antibody format and (2) the presence of both Jagged1/2 and proteases capable of activating CTX-033 in patient tumor samples. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C158. Citation Format: Jason Sagert, James West, Olga Vasiljeva, Jennifer Richardson, Luc Desnoyers, Shouchun Liu, Annie Yang, Chihunt Wong, Elizabeth Menendez, Krishna Polu, Henry Lowman. Tumor-specific inhibition of jagged-dependent notch signaling using a Probody™ therapeutic. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C158.

Structural Analysis Uncovers Lipid-Binding Properties of Notch Ligands

SummaryThe Notch pathway is a core cell-cell signaling system in metazoan organisms with key roles in cell-fate determination, stem cell maintenance, immune system activation, and angiogenesis. Signals are initiated by extracellular interactions of the Notch receptor with Delta/Serrate/Lag-2 (DSL) ligands, whose structure is highly conserved throughout evolution. To date, no structure or activity has been associated with the extreme N termini of the ligands, even though numerous mutations in this region of Jagged-1 ligand lead to human disease. Here, we demonstrate that the N terminus of human Jagged-1 is a C2 phospholipid recognition domain that binds phospholipid bilayers in a calcium-dependent fashion. Furthermore, we show that this activity is shared by a member of the other class of Notch ligands, human Delta-like-1, and the evolutionary distant Drosophila Serrate. Targeted mutagenesis of Jagged-1 C2 domain residues implicated in calcium-dependent phospholipid binding leaves Notch interactions intact but can reduce Notch activation. These results reveal an important and previously unsuspected role for phospholipid recognition in control of this key signaling system.

Missexpression of Human JAGGED1 Causes Disorganization of The Biliary Tree in Zebrafish Consistent With a Biliary Atresia Phenotype

Biliary atresia (BA) is a progressive, fibro-obliterative cholangiopathy of infants, which if left untreated is fatal. The pathogenesis of BA is not fully understood but may involve dysregulation of hepatic progenitor/stem cells (HPSC) during development of the biliary tree. Recent data indicate that ductular proliferation, a pathologic hallmark of BA, represents massive expansion of HPSC marked with an epithelial stem cell marker GCTM5 (Stamp et al, Stem Cells 2012). Alagille syndrome (AGS), a congenital disorder marked by a paucity of bile ducts, is caused by haploinsufficiency of JAGGED1 (JAG1), a gene that encodes a Notch pathway ligand critical for HPSC proliferation and differentiation at the onset of biliary organogenesis. Kohsaka et al reported that missense mutations in JAG1 occur in a subset of cases of BA and are associated with poorer prognosis (Hepatology 2004). Preliminary in vitro data indicate that these BA-associated JAG1 mutations result in increased Notch activity in contrast to AGS possibly through enhanced subcellular trafficking of ligand. We, therefore, hypothesized that in vivo misexpression of wild-type human JAG1 in transgenic zebrafish would result in increased Notch pathway activation, thus, disrupting biliary organogenesis leading to a disorganized biliary tree.

Disruptions of Global and Jagged1-Mediated Notch Signaling Affect Thyroid Morphogenesis in the Zebrafish

The mechanisms underlying the early steps of thyroid development are largely unknown. In search for novel candidate genes implicated in thyroid function, we performed a gene expression analysis on thyroid cells revealing that TSH regulates the expression of several elements of the Notch pathway, including the ligand Jagged1. Because the Notch pathway is involved in cell-fate determination of several foregut-derived endocrine tissues, we tested its contribution in thyroid development using the zebrafish, a teleost model recapitulating the mammalian molecular events during thyroid development. Perturbing the Notch signaling (e.g. mib mutants, γ-secretase inhibition, or Notch intracellular domain overexpression), we obtained evidence that this pathway has a biological role during the earlier phases of thyroid primordium induction, limiting the number of cells that proceed to a specialized fate and probably involving actions from surrounding tissues. Moreover, we were able to confirm the expression of Jagged1 during different phases of zebrafish thyroid development, as well as in mouse and human thyroid tissues. The two orthologues to the single jagged1 gene (JAG1) in humans, jag1a and jag1b, are expressed with different spatiotemporal patterns in the developing zebrafish thyroid. Both jag1a and jag1b morphants, as well as jag1b mutant fish line, display thyroid hypoplasia and impaired T(4) production; this thyroid phenotype was rescued by coinjection of human JAG1 mRNA. In conclusion, Notch pathway is involved in the early steps of thyroid morphogenesis, and Jagged1-Notch signal is required for zebrafish thyroid development and function. Thus, genetic alterations affecting the Notch pathway may confer susceptibility for thyroid dysgenesis.

Notch signaling pathway in keloid disease: Enhanced fibroblast activity in a Jagged‐1 peptide‐dependent manner in lesional vs. extralesional fibroblasts

Keloid disease (KD) is a fibroproliferative disorder of unknown etiopathogenesis with ill-defined treatment. There is increasing evidence to suggest that aberrant Notch signaling may contribute directly to skin pathogenesis and altered expression of Notch receptors identified in KD. Therefore, the aim of this study was to investigate the Notch signaling pathway in KD compared to normal skin (NS). In this study, we employed in vitro primary cell culture models to elucidate the role of Notch signaling in 44 tissue samples from patients with KD split into keloid and extralesional (EL) samples (internal control) from the same patients, and six NS tissue samples (external control). We show the presence of a significant (p < 0.05) up-regulation of Notch receptors and ligand Jagged-1 (JAG-1) in KD compared to EL and NS tissue samples. Cell spreading, attachment, and proliferation were significantly (p < 0.05) reduced in JAG-1 antisense-treated primary dermal fibroblasts isolated from KD and treated with γ-secretase inhibitor (blocks proteolytic cleavage and activation of Notch), evaluated by real-time cell analyzer (RTCA) on a microelectronic sensory array. In contrast, extralesional skin fibroblasts (ELF) treated with recombinant human JAG-1 (rh-JAG-1) peptide showed significant (p < 0.05) enhancement of cell spreading, attachment, and proliferation in RTCA. Activation/inhibition of JAG-1 and Notch signaling significantly (p < 0.05) altered the behavior of primary keloid fibroblasts and ELF, in cell migration (using a scratch wound assay), invasion (using a 3D invasion assay), and angiogenesis (in vitro coculture tube formation assay). In conclusion, this is the first study to demonstrate a potential role for the Notch signaling pathway in KD progression and that targeting this pathway may provide a novel strategy for treatment of KD.

Flexibility of the PDZ-binding motif in the micelle-bound form of Jagged-1 cytoplasmic tail

Human Jagged-1, one of the ligands of Notch receptors, is a transmembrane protein composed of a large extracellular region and a 125-residue cytoplasmic tail which bears a C-terminal PDZ recognition motif. To investigate the interaction between Jagged-1 cytoplasmic tail and the inner leaflet of the plasma membrane we determined, by solution NMR, the secondary structure and dynamics of the recombinant protein corresponding to the intracellular region of Jagged-1, J1_tmic, bound to negatively charged lysophospholipid micelles. NMR showed that the PDZ binding motif is preceded by four α-helical segments and that, despite the extensive interaction between J1_tmic and the micelle, the PDZ binding motif remains highly flexible. Binding of J1_tmic to negatively charged, but not to zwitterionic vesicles, was confirmed by surface plasmon resonance. To study the PDZ binding region in more detail, we prepared a peptide corresponding to the last 24 residues of Jagged-1, J1C24, and different phosphorylated variants of it. J1C24 displays a marked helical propensity and undergoes a coil–helix transition in the presence of negatively charged, but not zwitterionic, lysophospholipid micelles. Phosphorylation at different positions drastically decreases the helical propensity of the peptides and abolishes the coil–helix transition triggered by lysophospholipid micelles. We propose that phosphorylation of residues upstream of the PDZ binding motif may shift the equilibrium from an ordered, membrane-bound, interfacial form of Jagged-1 C-terminal region to a more disordered form with an increased accessibility of the PDZ recognition motif, thus playing an indirect role in the interaction between Jagged-1 and the PDZ-containing target protein.

Notch Initiates the Endothelial-to-Mesenchymal Transition in the Atrioventricular Canal through Autocrine Activation of Soluble Guanylyl Cyclase

The heart is the most common site of congenital defects, and valvuloseptal defects are the most common of the cardiac anomalies seen in the newborn. The process of endothelial-to-mesenchymal transition (EndMT) in the cardiac cushions is a required step during early valve development, and Notch signaling is required for this process. Here we show that Notch activation induces the transcription of both subunits of the soluble guanylyl cyclase (sGC) heterodimer, GUCY1A3 and GUCY1B3, which form the nitric oxide receptor. In parallel, Notch also promotes nitric oxide (NO) production by inducing Activin A, thereby activating a PI3-kinase/Akt pathway to phosphorylate eNOS. We thus show that the activation of sGC by NO through a Notch-dependent autocrine loop is necessary to drive early EndMT in the developing atrioventricular canal (AVC).