Expression Of Notch Target Genes Research Articles

Protein O-fucosyltransferase 1 expression impacts myogenic C2C12 cell commitment via the Notch signaling pathway.

The Notch signaling pathway plays a crucial role in skeletal muscle regeneration in mammals by controlling the transition of satellite cells from quiescence to an activated state, their proliferation, and their commitment toward myotubes or self-renewal. O-fucosylation on Notch receptor epidermal growth factor (EGF)-like repeats is catalyzed by the protein O-fucosyltransferase 1 (Pofut1) and primarily controls Notch interaction with its ligands. To approach the role of O-fucosylation in myogenesis, we analyzed a murine myoblastic C2C12 cell line downregulated for Pofut1 expression by short hairpin RNA (shRNA) inhibition during the time course of differentiation. Knockdown of Pofut1 affected the signaling pathway activation by a reduction of the amount of cleaved Notch intracellular domain and a decrease in downstream Notch target gene expression. Depletion in Pax7(+)/MyoD(-) cells and earlier myogenic program entrance were observed, leading to an increase in myotube quantity with a small number of nuclei, reflecting fusion defects. The rescue of Pofut1 expression in knockdown cells restored Notch signaling activation and a normal course in C2C12 differentiation. Our results establish the critical role of Pofut1 on Notch pathway activation during myogenic differentiation.

Abstract 3294: Notch signaling is regulated by vacuolar ATPase in triple negative breast cancer

Abstract Triple Negative Breast Cancer (TNBC) constitutes 15 % of all diagnosed breast cancers. It is more common among African American and younger women. TNBC cells do not express Estrogen, Progesterone and Her2 receptors. As a result, patients have limited treatment options. To effectively treat TNBC, it is important to identify and target specific signaling pathways that contribute to TNBC tumorigenesis. Notch signaling is an evolutionarily conserved juxtacrine pathway that is important in normal mammary development and has been shown to play a role in TNBC progression. Although there has been considerable research on role of Notch in TNBC, little is known about its regulation. Identifying novel molecules that can regulate Notch can lead to the development of effective combinatorial treatments. The Vacuolar ATPase (V-ATPase) has been shown to regulate Notch Signaling in Drosophila. V-ATPase is an ATP driven proton pump, which is responsible for maintaining pH. Tumor cells increase expression of V-ATPase, which aids in cancer proliferation and metastasis. Here, we report that the a2 isoform of V-ATPase regulates Notch Signaling in breast cancer. Among isoforms a1, a2, a3 and a4, breast cancer cell lines robustly expressed a2 V-ATPase on their surface. a2 V-ATPase is present towards the leading edge of cells and is higher in TNBC cell lines (MDA-MB-231, MDA-MB-468) as compared to normal chemically transformed cell line (184A1) and hormonal positive cell line (MCF7). Expression of Notch ligands, receptors and target genes was also higher in TNBC cell lines as compared to Non-TNBC cell lines. siRNA mediated knockdown of a2 V-ATPase resulted in an increase of Notch Signaling in the Triple Negative MDA-MB-231 cell line. To understand the molecular mechanism behind this regulation, we used Bafilomycin to inhibit V-ATPase activity. Following V-ATPase inhibition, the expression of Jagged1, a Notch ligand was increased. Our results suggest that Vacuolar ATPase can regulate Notch Signaling by affecting ligand internalization through endocytosis. We further assessed the effect of V-ATPase and Notch signaling inhibitors on cell proliferation of the breast cancer cell lines (184A1, MCF7, MDA-MB-231, MDA-MB-468). Combined inhibition of V-ATPase and Notch signaling caused significant cell death in all cell lines tested. Together our results identify a2 V-ATPase as a novel regulator of Notch signaling in Breast Cancer and that targeting both V-ATPase and Notch Signaling can be beneficial for Triple Negative Breast Cancer. Citation Format: Sahithi Pamarthy, Kenneth D. Beaman. Notch signaling is regulated by vacuolar ATPase in triple negative breast cancer. [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 3294. doi:10.1158/1538-7445.AM2014-3294

Abstract 2990: Notch1 monoclonal antibody inhibits tumor growth and modulates angiogenesis

Abstract AVEO Oncology has developed a series of inducible mouse models of cancer, which through the preservation of critical tumor/stromal interactions, facilitate identification of cell-surface and secreted proteins that represent viable targets for therapeutic antibodies and other biologics. Functional genetic screens performed in vivo in these models identified the Notch pathway as a critical regulator of tumor maintenance. This finding was consistent with emerging evidence that activation of Notch signaling via receptor point mutation, receptor amplification, and elevated receptor and ligand expression, plays a key role in various human cancers. Moreover, the Notch pathway controls diverse aspects of tumorigenesis and tumor maintenance, regulating tumor autonomous processes and interactions with the microenvironment, including angiogenesis. To further understand the role of the Notch pathway in tumor maintenance, and to assess the therapeutic potential of targeting the Notch pathway in cancer, we have generated monoclonal antibodies that inhibit the Notch1 receptor. Characterization of monoclonal antibodies targeting Notch1 through cell-based and biochemical studies demonstrated that the Notch1 monoclonal antibody bound with high affinity and high specificity to the Notch1 receptor, prevented ligand-mediated activation of the target receptor, and specifically repressed Notch-dependent signaling with high potency. Significantly, specific inhibition by the Notch1 monoclonal antibody did not result in the dose-limiting gut toxicity observed with Notch1 mAb resulted in tumor inhibition that was accompanied by increased vascular density, inhibition of Notch target gene expression, and increased expression of angiogenesis-related genes, consistent with expected changes resulting from Notch1/Dll4 pathway inhibition. In addition, several tumor models treated in combination with Notch1 mAb and an investigational VEGF pathway inhibitor, tivozanib, demonstrated increased tumor inhibition compared to either agent alone. Interestingly, combination treatment resulted in decreased vascular density and downregulated expression of angiogenesis genes, similar to treatment with VEGF inhibitor alone. Collectively, our work suggests that combination therapy of Notch1 antibodies with VEGF inhibitors should be evaluated as a potential viable therapeutic option for VEGF-resistant tumors. Citation Format: Heidi Okamura, Theresa Proia, Alisa Bell, Qing Liu, Zakir Siddiquee, Jie Lin, Jeno Gyuris. Notch1 monoclonal antibody inhibits tumor growth and modulates angiogenesis. [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 2990. doi:10.1158/1538-7445.AM2014-2990

Osteoblasts as leukemia-initiating cells.

Osteoblasts as leukemia-initiating cells.

Adropin Is a Brain Membrane-bound Protein Regulating Physical Activity via the NB-3/Notch Signaling Pathway in Mice

Adropin is a highly conserved polypeptide that has been suggested to act as an endocrine factor that plays important roles in metabolic regulation, insulin sensitivity, and endothelial functions. However, in this study, we provide evidence demonstrating that adropin is a plasma membrane protein expressed abundantly in the brain. Using a yeast two-hybrid screening approach, we identified NB-3/Contactin 6, a brain-specific, non-canonical, membrane-tethered Notch1 ligand, as an interaction partner of adropin. Furthermore, this interaction promotes NB3-induced activation of Notch signaling and the expression of Notch target genes. We also generated and characterized adropin knockout mice to explore the role of adropin in vivo. Adropin knockout mice exhibited decreased locomotor activity and impaired motor coordination coupled with defective synapse formation, a phenotype similar to NB-3 knockout mice. Taken together, our data suggest that adropin is a membrane-bound protein that interacts with the brain-specific Notch1 ligand NB3. It regulates physical activity and motor coordination via the NB-3/Notch signaling pathway and plays an important role in cerebellum development in mice.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

CRTC1/MAML2 gain-of-function interactions with MYC create a gene signature predictive of cancers with CREB-MYC involvement.

Chimeric oncoproteins created by chromosomal translocations are among the most common genetic mutations associated with tumorigenesis. Malignant mucoepidermoid salivary gland tumors, as well as a growing number of solid epithelial-derived tumors, can arise from a recurrent t (11, 19)(q21;p13.1) translocation that generates an unusual chimeric cAMP response element binding protein (CREB)-regulated transcriptional coactivator 1 (CRTC1)/mastermind-like 2 (MAML2) (C1/M2) oncoprotein comprised of two transcriptional coactivators, the CRTC1 and the NOTCH/RBPJ coactivator MAML2. Accordingly, the C1/M2 oncoprotein induces aberrant expression of CREB and NOTCH target genes. Surprisingly, here we report a gain-of-function activity of the C1/M2 oncoprotein that directs its interactions with myelocytomatosis oncogene (MYC) proteins and the activation of MYC transcription targets, including those involved in cell growth and metabolism, survival, and tumorigenesis. These results were validated in human mucoepidermoid tumor cells that harbor the t (11, 19)(q21;p13.1) translocation and express the C1/M2 oncoprotein. Notably, the C1/M2-MYC interaction is necessary for C1/M2-driven cell transformation, and the C1/M2 transcriptional signature predicts other human malignancies having combined involvement of MYC and CREB. These findings suggest that such gain-of-function properties may also be manifest in other oncoprotein fusions found in human cancer and that agents targeting the C1/M2-MYC interface represent an attractive strategy for the development of effective and safe anticancer therapeutics in tumors harboring the t (11, 19) translocation.

FRI0363 Deletion of RBP-J in A Murine Model of Inflammatory Arthritis Reveals Pro-Inflammatory Cytokine Expression and Immunophenotypic Differences

BackgroundRBP-J, a DNA-binding protein, serves as the central transcriptional regulator of the Notch signaling pathway. Prior in vitro work has demonstrated that RBP-J plays a critical role in macrophage activation,...

Endothelial Krüppel-like Factor 4 Regulates Angiogenesis and the Notch Signaling Pathway

Regulation of endothelial cell biology by the Notch signaling pathway (Notch) is essential to vascular development, homeostasis, and sprouting angiogenesis. Although Notch determines cell fate and differentiation in a wide variety of cells, the molecular basis of upstream regulation of Notch remains poorly understood. Our group and others have implicated the Krüppel-like factor family of transcription factors as critical regulators of endothelial function. Here, we show that Krüppel-like factor 4 (KLF4) is a central regulator of sprouting angiogenesis via regulating Notch. Using a murine model in which KLF4 is overexpressed exclusively in the endothelium, we found that sustained expression of KLF4 promotes ineffective angiogenesis leading to diminished tumor growth independent of endothelial cell proliferation or cell cycling effects. These tumors feature increased vessel density yet are hypoperfused, leading to tumor hypoxia. Mechanistically, we show that KLF4 differentially regulates expression of Notch receptors, ligands, and target genes. We also demonstrate that KLF4 limits cleavage-mediated activation of Notch1. Finally, we rescue Notch target gene expression and the KLF4 sprouting angiogenesis phenotype by supplementation of DLL4 recombinant protein. Identification of this hitherto undiscovered role of KLF4 implicates this transcription factor as a critical regulator of Notch, tumor angiogenesis, and sprouting angiogenesis.

Notch Signaling in Mesenchymal Stem Cells Harvested From Geriatric Mice

Morbidity associated with geriatric fractures may be attributed, in part, to compromised mesenchymal stem cell (MSC) function within the fracture callus. The Notch signaling pathway is important for the healing of nonskeletal tissues in an age-dependent manner, but the effect of Notch on age-dependent fracture healing and MSC dysfunction has not been substantiated. The objective of this study was to examine Notch signaling in MSCs obtained from young and geriatric mice. Marrow-derived MSCs were harvested from the femora of 5- and 25-month-old C57BL/6 mice. We assessed in vivo MSC number using CFU-F, proliferation using an Alamar Blue assay, osteoblast differentiation by Alizarin Red S staining, and adipogenic differentiation using Oil Red O staining. Notch receptor and ligand expression was assessed using quantitative PCR, and Notch signaling was assessed by evaluating Notch target gene expression (Hey and HES) under basal conditions and when cells were plated to Jagged-1 ligand. MSC from geriatric mice exhibit reduced MSC number (CFU-F), proliferation, adipogenesis, and inconsistent osteogenesis. The highest expressed Notch receptor is Notch 2, and the highest expressed ligand is Jagged-1, but there were no differences in ligand and receptor gene expression between young and old MSCs. Interestingly, geriatric MSCs show decreased basal Notch signaling activity but are fully responsive to Jagged-1 stimulation. These data suggest that therapeutic targeting of Notch signaling should be explored in clinical therapies to improve geriatric fracture healing.

The MEK/ERK Pathway Promotes NOTCH Signalling in Pancreatic Cancer Cells

Activation of the NOTCH receptors relies on their intracellular proteolysis by the gamma-secretase complex. This cleavage liberates the NOTCH intracellular domain (NIC) thereby allowing the translocation of NIC towards the nucleus to assemble into a transcriptional platform. Little information is available regarding the regulatory steps operating on NIC following its release from the transmembrane receptor up to its association with transcriptional partners. Interfering with these regulatory steps might potentially influences the nuclear outcome of NOTCH signalling. Herein, we exploited a reliable model to study the molecular events occurring subsequent to NOTCH1 cleavage. In pancreatic cancer cells, pulse of NOTCH1 activation led to increased expression of NOTCH target genes namely HES1 and c-MYC. We uncovered that, upon its release, the NOTCH1 intracellular domain, NIC1, undergoes a series of post-translational modifications that include phosphorylation. Most interestingly, we found that activation of the MEK/ERK pathway promotes HES1 expression. Inhibition of the gamma-secretase complex prevented the MEK/ERK-induced HES1 expression suggesting a NOTCH-dependent mechanism. Finally, higher levels of NIC1 were found associated with its transcriptional partners [CBF1, Su(H) and LAG-1] (CSL) and MASTERMIND-LIKE 1 (MAML1) upon MEK/ERK activation providing a potential mechanism whereby the MEK/ERK pathway promotes expression of NOTCH target genes. For the first time, our data exposed a signalling pathway, namely the MEK/ERK pathway that positively impacts on NOTCH nuclear outcome.

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.

Normal Development in Mice Over-Expressing the Intracellular Domain of DLL1 Argues against Reverse Signaling by DLL1 In Vivo

The Notch signaling pathway mediates the direct communication between adjacent cells and regulates multiple developmental processes. Interaction of the Notch receptor with its ligands induces the liberation of the intracellular portion of Notch (NICD) referred to as regulated intramembraneous proteolysis (RIP). NICD translocates to the nucleus, and by complexing with the DNA binding protein RBPjκ and other cofactors activates transcription of bHLH genes. RIP-like processing of various mammalian Notch ligands (DLL1, JAG1 and JAG2) and the translocation of their intracellular domains (ICDs) to the nucleus has also been observed. These observations together with effects of over-expressed ligand ICDs in cultured cells on cell proliferation, differentiation, and Notch activity and target gene expression have led to the idea that the intracellular domains of Notch ligands have signaling functions. To test this hypothesis in vivo we have generated ES cells and transgenic mice that constitutively express various versions of the intracellular domain of mouse DLL1. In contrast to other cell lines, expression of DICDs in ES cells did not block proliferation or stimulate neuronal differentiation. Embryos with ubiquitous DICD expression developed to term without any apparent phenotype and grew up to viable and fertile adults. Early Notch-dependent processes or expression of selected Notch target genes were unaltered in transgenic embryos. In addition, we show that mouse DICD enters the nucleus inefficiently. Collectively, our results argue against a signaling activity of the intracellular domain of DLL1 in mouse embryos in vivo.

Analysis of gene expression in wild-type and Notch1 mutant retinal cells by single cell profiling.

The vertebrate retina comprises sensory neurons, the photoreceptors, as well as many other types of neurons and one type of glial cell. These cells are generated by multipotent and restricted retinal progenitor cells (RPCs), which express Notch1. Loss of Notch1 in RPCs late during retinal development results in the overproduction of rod photoreceptors at the expense of interneurons and glia. To examine the molecular underpinnings of this observation, microarray analysis of single retinal cells from wild-type or Notch1 conditional knockout retinas was performed. In situ hybridization was carried out to validate some of the findings. The majority of Notch1-mutant cells lost expression of known Notch target genes. These cells also had low levels of RPC and cell cycle genes, and robustly up-regulated rod precursor genes. In addition, single wild-type cells, in which cell cycle marker genes were down-regulated, expressed markers of both rod photoreceptors and interneurons.

Notch signaling induces EMT in OSCC cell lines in a hypoxic environment

Epithelial-mesenchymal transition (EMT) is an early step in the acquisition of invasiveness by malignant tumors. It has been clarified that the tumor microenvironment affects malignancy in a number of different carcinomas, in particular, that a hypoxic environment induces EMT. Activation of Notch signaling induces EMT, but it remains unclear how the Notch pathway is involved in oral squamous cell carcinoma (OSCC) under hypoxia. Three OSCC cell lines were cultured for examination under hypoxic (1% O2) and normoxic (21% O2) conditions. Expression of E-cadherin was investigated as a hallmark of EMT by immunohistochemical examination. Cell motility and invasion were examined by wound-healing and invasion assays, respectively. The expression of Notch pathway molecules was analyzed by qPCR. Hypoxia increased the mRNA expression of Notch receptors, ligands and target genes, and Snail. Hypoxia decreased the expression of E-cadherin, and increased the motility and invasiveness of OSCC cell lines. γ-secretase inhibitor, a Notch-specific inhibitor, prevented these effects caused by h-ypoxia. These findings suggest that hypoxia induces EMT in OSCC cell lines via activation of Notch signaling, and inhibition of the Notch signaling pathway to suppress EMT may be a useful approach for the treatment of OSCC.

CAF-1 promotes Notch signaling through epigenetic control of target gene expression during Drosophila development

The histone chaperone CAF-1 is known for its role in DNA replication-coupled histone deposition. However, loss of function causes lethality only in higher multicellular organisms such as mice and flies, but not in unicellular organisms such as yeasts, suggesting that CAF-1 has other important functions than histone deposition during animal development. Emerging evidence indicates that CAF-1 also has a role in higher order chromatin organization and heterochromatin-mediated gene expression; it remains unclear whether CAF-1 has a role in specific signaling cascades to promote gene expression during development. Here, we report that knockdown of one of the subunits of Drosophila CAF-1, dCAF-1-p105 (Caf1-105), results in phenotypes that resemble those of, and are augmented synergistically by, mutations of Notch positive regulatory pathway components. Depletion of dCAF-1-p105 leads to abrogation of cut expression and to downregulation of other Notch target genes in wing imaginal discs. dCAF-1-p105 is associated with Suppressor of Hairless [Su(H)] and regulates its binding to the enhancer region of E(spl)mβ. The association of dCAF-1-p105 with Su(H) on chromatin establishes an active local chromatin status for transcription by maintaining a high level of histone H4 acetylation. In response to induced Notch activation, dCAF-1 associates with the Notch intracellular domain to activate the expression of Notch target genes in cultured S2 cells, manifesting the role of dCAF-1 in Notch signaling. Together, our results reveal a novel epigenetic function of dCAF-1 in promoting Notch pathway activity that regulates normal Drosophila development.

Notch1 Receptor Regulates AKT Protein Activation Loop (Thr308) Dephosphorylation through Modulation of the PP2A Phosphatase in Phosphatase and Tensin Homolog (PTEN)-null T-cell Acute Lymphoblastic Leukemia Cells

Notch1 activating mutations occur in more than 50% of T-cell acute lymphoblastic leukemia (T-ALL) cases and increase expression of Notch1 target genes, some of which activate AKT. HES1 transcriptionally silences phosphatase and tensin homolog (PTEN), resulting in AKT activation, which is reversed by Notch1 inhibition with γ-secretase inhibitors (GSIs). Mutational loss of PTEN is frequent in T-ALL and promotes resistance to GSIs due to AKT activation. GSI treatments increased AKT-Thr(308) phosphorylation and signaling in PTEN-deficient, GSI-resistant T-ALL cell lines (Jurkat, CCRF-CEM, and MOLT3), suggesting that Notch1 represses AKT independent of its PTEN transcriptional effects. AKT-Thr(308) phosphorylation and downstream signaling were also increased by knocking down Notch1 in Jurkat (N1KD) cells. This was blocked by treatment with the AKT inhibitor perifosine. The PI3K inhibitor wortmannin and the protein phosphatase type 2A (PP2A) inhibitor okadaic acid both impacted AKT-Thr(308) phosphorylation to a greater extent in nontargeted control than N1KD cells, suggesting decreased dephosphorylation of AKT-Thr(308) by PP2A in the latter. Phosphorylations of AMP-activated protein kinaseα (AMPKα)-Thr(172) and p70S6K-Thr(389), both PP2A substrates, were also increased in both N1KD and GSI-treated cells and responded to okadaic acid treatment. A transcriptional regulatory mechanism was implied because ectopic expression of dominant-negative mastermind-like protein 1 increased and wild-type HES1 decreased phosphorylation of these PP2A targets. This was independent of changes in PP2A subunit levels or in vitro PP2A activity, but was accompanied by decreased association of PP2A with AKT in N1KD cells. These results suggest that Notch1 can regulate PP2A dephosphorylation of critical cellular regulators including AKT, AMPKα, and p70S6K.

Enhanced cell growth and tumorigenicity of rat glioma cells by stable expression of human CD133 through multiple molecular actions

CD133 (Prominin-1/AC133) is generally treated as a cell surface marker found on multipotent stem cells and tumor stem-like cells, and its biological function remains debated. Genetically modified rat glioma cell lines were generated by lentiviral gene delivery of human CD133 into rat C6 glioma cells (hCD133(+) -C6) or by infection of C6 cells with control lentivirus (mock-C6). Stable hCD133 expression promoted the self-renewal ability of C6-formed spheres with an increase in the expression of the stemness markers, Bmi-1 and SOX2. Akt phosphorylation, Notch-1 activation, and Notch-1 target gene expression (Hes-1, Hey1 and Hey2) were increased in hCD133(+) -C6 when compared to mock-C6. The inhibition of Akt phosphorylation, Notch-1 activation, and Hes-1 in hCD133(+) -C6 cells effectively suppressed their clonogenic ability, indicating that these factors are involved in expanding the growth of hCD133(+) -C6. An elevated expression of GTPase-activating protein 27 (Arhgap27) was detected in hCD133(+) -C6. A decline in the invasion of hCD133(+) -C6 by knockdown of Arhgap27 expression indicated the critical role of Arhgap27 in promoting cell migration of hCD133(+) -C6. In vivo study further showed that hCD133(+) -C6 formed aggressive tumors in vivo compared to mock-C6. Exposure of hCD133(+) -C6 to arsenic trioxide not only reduced Akt phosphorylation, Notch-1 activation and Hes-1 expression in vitro, but also inhibited their tumorigenicity in vivo. The results show that C6 glioma cells with stable hCD133 expression enhanced their stemness properties with increased Notch-1/Hes-1 signaling, Akt activation, and Arhgap27 action, which contribute to increased cell proliferation and migration of hCD133(+) -C6 in vitro, as well as progressive tumor formation in vivo.

Jagged1 immobilization to an osteoconductive polymer activates the Notch signaling pathway and induces osteogenesis

Treatment of nonunion fractures is a significant problem. Common therapeutics, including autologous bone grafts and bone morphogenetic proteins, show well-established limitations. Therefore, a need persists for the identification of novel clinical therapies to promote healing. The Notch signaling pathway regulates bone development. Clinically, loss-of-function mutations to the Notch ligand Jagged1 decrease bone mass and increase fracture risk. Jagged1 is also the most highly upregulated ligand during fracture repair, identifying it as a potential target to promote bone formation. Therefore, the objective of this study was to develop a clinically translatable construct comprised of Jagged1 and an osteoconductive scaffold, and characterize its activity in human mesenchymal stem cells (hMSC). We first evaluated the effects of Jagged1 directly immobilized to a novel poly(β-amino ester) relative to indirect coupling via antibody. Direct was more effective at activating hMSC Notch target gene expression and osteogenic activity. We then found that directly immobilized Jagged1 constructs induced osteoblast differentiation. This is the first study to demonstrate that Jagged1 delivery transiently activates Notch signaling and increases osteogenesis. A positive correlation was found between Jagged1-induced Notch and osteogenic expression. Collectively, these results indicate that Jagged1 coupled to an osteogenic biomaterial could promote bone tissue formation during fracture healing.

Flt-1 (Vascular Endothelial Growth Factor Receptor-1) Is Essential for the Vascular Endothelial Growth Factor–Notch Feedback Loop During Angiogenesis

Vascular endothelial growth factor (VEGF) signaling induces Notch signaling during angiogenesis. Flt-1/VEGF receptor-1 negatively modulates VEGF signaling. Therefore, we tested the hypothesis that disrupted Flt-1 regulation of VEGF signaling causes Notch pathway defects that contribute to dysmorphogenesis of Flt-1 mutant vessels. Wild-type and flt-1(-/-) mouse embryonic stem cell-derived vessels were exposed to pharmacological and protein-based Notch inhibitors with and without added VEGF. Vessel morphology, endothelial cell proliferation, and Notch target gene expression levels were assessed. Similar pathway manipulations were performed in developing vessels of zebrafish embryos. Notch inhibition reduced flt-1(-/-) embryonic stem cell-derived vessel branching dysmorphogenesis and endothelial hyperproliferation, and rescue of flt-1(-/-) vessels was accompanied by a reduction in elevated Notch targets. Surprisingly, wild-type vessel morphogenesis and proliferation were unaffected by Notch suppression, Notch targets in wild-type endothelium were unchanged, and Notch suppression perturbed zebrafish intersegmental vessels but not caudal vein plexuses. In contrast, exogenous VEGF caused wild-type embryonic stem cell-derived vessel and zebrafish intersegmental vessel dysmorphogenesis that was rescued by Notch blockade. Elevated Notch signaling downstream of perturbed VEGF signaling contributes to aberrant flt-1(-/-) blood vessel formation. Notch signaling may be dispensable for blood vessel formation when VEGF signaling is below a critical threshold.