Non-canonical Notch Signaling Research Articles

Notch signaling and cardiac repair

Notch signaling is critical for proper heart development and recently has been reported to participate in adult cardiac repair. Notch resides at the cell surface as a single pass transmembrane receptor, transits through the cytoplasm following activation, and acts as a transcription factor upon entering the nucleus. This dynamic and widespread cellular distribution allows for potential interactions with many signaling and binding partners. Notch displays temporal as well as spatial versatility, acting as a strong developmental signal, controlling cell fate determination and lineage commitment, and playing a pivotal role in embryonic and adult stem cell proliferation and differentiation. This review serves as an update of recent literature addressing Notch signaling in the heart, with attention to findings from noncardiac research that provide clues for further interpretation of how the Notch pathway influences cardiac biology. Specific areas of focus include Notch signaling in adult myocardium following pathologic injury, the role of Notch in cardiac progenitor cells with respect to differentiation and cardiac repair, crosstalk between Notch and other cardiac signaling pathways, and emerging aspects of noncanonical Notch signaling in heart.

Non-canonical Notch signaling: emerging role and mechanism

Notch is an ancient transmembrane receptor with crucial roles in cell-fate choices. Although the 'canonical' Notch pathway and its core members are well established - involving ligand-induced cleavage of Notch for transcriptional regulation - it has been unclear whether Notch can also function independently of ligand and transcription ('non-canonically') through a common mechanism. Recent studies suggest that Notch can non-canonically exert its biological functions by post-translationally targeting Wnt/β-catenin signaling, an important cellular and developmental regulator. The non-canonical Notch pathway appears to be highly conserved from flies to mammals. Here, we discuss the emerging conserved mechanism and role of ligand/transcription-independent Notch signaling in cell and developmental biology.

Mamld1 Knockdown Reduces Testosterone Production and Cyp17a1 Expression in Mouse Leydig Tumor Cells

Background MAMLD1 is known to be a causative gene for hypospadias. Although previous studies have indicated that MAMLD1 mutations result in hypospadias primarily because of compromised testosterone production around the critical period for fetal sex development, the underlying mechanism(s) remains to be clarified. Furthermore, although functional studies have indicated a transactivation function of MAMLD1 for the non-canonical Notch target Hes3, its relevance to testosterone production remains unknown. To examine these matters, we performed Mamld1 knockdown experiments.Methodology/Principal Findings Mamld1 knockdown was performed with two siRNAs, using mouse Leydig tumor cells (MLTCs). Mamld1 knockdown did not influence the concentrations of pregnenolone and progesterone but significantly reduced those of 17-OH pregnenolone, 17-OH progesterone, dehydroepiandrosterone, androstenedione, and testosterone in the culture media. Furthermore, Mamld1 knockdown significantly decreased Cyp17a1 expression, but did not affect expressions of other genes involved in testosterone biosynthesis as well as in insulin-like 3 production. Hes3 expression was not significantly altered. In addition, while 47 genes were significantly up-regulated (fold change >2.0×) and 38 genes were significantly down-regulated (fold change <0.5×), none of them was known to be involved in testosterone production. Cell proliferation analysis revealed no evidence for compromised proliferation of siRNA-transfected MLTCs.Conclusions/SignificanceThe results, in conjunction with the previous data, imply that Mamld1 enhances Cyp17a1 expression primarily in Leydig cells and permit to produce a sufficient amount of testosterone for male sex development, independently of the Hes3-related non-canonical Notch signaling.

Highlights in DD

“Highlights” calls attention to exciting advances in developmental biology that have recently been reported in Developmental Dynamics. Development is a broad field encompassing many important areas. To reflect this fact, the section spotlights significant discoveries that occur across the entire spectrum of developmental events and problems: from new experimental approaches, to novel interpretations of results, to noteworthy findings utilizing different developmental organisms. Taste test (Epibranchial Placode-Derived Neurons Produce BDNF Required for Early Sensory Neuron Development by Danielle E. Harlow, Hui Yang, Trevor Williams, and Linda A. Barlow, Dev Dyn 240:309–323) In the developing gustatory system, brain-derived neurotrophic factor (BDNF) is best known for its “main course” role supporting the survival of gustatory neurons and proper innervation of peripheral targets. Here, Harlow et al. test whether BDNF is also a key ingredient for an appetizer that sets the stage for the entree. In support of this hypothesis, they demonstrate that BDNF-LacZ expression initiates in a subset of mouse geniculate ganglia (gVII) neurons at embryonic day 9.5, before previously reported BDNF expression. Within gVII, the BDNF receptor, TrkB, is expressed in placode-derived neurons, and BDNF itself in a subset of these neurons. To determine whether this apparent paracrine or autocrine BDNF signaling is important for gustatory system development, phenotypes of mice where bdnf is selectively eliminated from placodal neuroblasts were compared with bdnf knockout mice. In both cases, significant numbers of pre- and postmitotic neurons display a delay in maturation. These findings demonstrate the importance of BDNF in early gustatory neuronal development. Future work will determine how mother nature—a skilled chef—can use the same ingredients throughout the meal, but to different effects. Getting to know noncanonical Notch (Noncanonical Notch Function in Motor Axon Guidance Is Mediated by Rac GTPase and the GEF1 Domain of Trio by Jeong K. Song and Edward Giniger, Dev Dyn 240:324–332) Noncanonical Notch signaling makes the Notch pathway more nimble, enabling cross-talk with diverse regulators. During axon growth and guidance, Drosophila motor neurons rely on noncanonical Notch signaling to interact with key regulators of actin dynamics, the Rho GTPases. Notch associates with and antagonizes components of the Abl tyrosine kinase signaling pathway, including Trio, a guanine nucleotide exchange factor (GEF) for Rho GTPases. Here, Song and Giniger flesh out downstream steps of this context-dependent pathway. First, rescue experiments demonstrate that Trio's Rac-specific GEF1 domain, but not its Rho-specific GEF2 domain, is required for noncanonical Notch signaling. Consistent with the finding, constitutively active and dominant-negative Rac modulates Notch-mediated axon guidance, but manipulation of the Rho GTPases Cdc42 and Rho1 do not. Not for the faint of heart, the experiments demand only slight alterations to Trio or Rac levels, as tipping the balance too far produces combinatorial effects. The authors hypothesize that the Abl/Rac signaling network bridges Notch to actin cytoskeleton rearrangements that push and pull the axonal growth cone to its target. Running dry (An Obesogenic Diet Started Before Puberty Leads to Abnormal Mammary Gland Development During Pregnancy in the Rabbit by Cathy Hue-Beauvais, Pascale Chavatte-Palmer, Etienne Aujean, Michéle Dahirel, Patrice Laigre, Christine Péchoux, Stephan Bouet, Eve Devinoy, and Madia Charlier, Dev Dyn 240:347–356) Health risks to the obese—frequently caused by high fat, high sugar diets during adolescence—are well established. But the impact of obesity on the next generation is just beginning to be understood. Here, Hue-Beauvais and colleagues investigate physiological reasons behind reports that obese women are less likely to breastfeed, by examining mammary gland development in obese rabbits. At mid-pregnancy, rabbits fed an obesogenic (OD) diet before puberty display precocious development of mammary gland alveolar structures. Electron microscopy and Western analysis further show that ducts are prematurely filled with milk proteins and lipids. The data suggest that the observed premature mammary growth and differentiation potentially leads to dysregulation of post-partum lactation. Of interest, the authors also note that pups from mid-gestation OD mothers are significantly underweight, possibly due to decreased placental efficiency. Follow-up work may substantiate these effects on future generations, fueling the need for putting a stop to childhood obesity.

Alteration of Notch signaling in skeletal development and disease.

Notch signaling is an evolutionarily conserved mechanism for specifying and regulating organogenesis and tissue renewal. Human and mouse genetic studies have demonstrated mutations in many components of the Notch signaling pathway that cause skeletal patterning defects. More recently, the in vivo effects of Notch signaling on osteoblast specification, proliferation, and differentiation have been demonstrated in addition to its regulation of osteoclast activity. However, while our understanding of canonical Notch signaling in skeletal biology is rapidly evolving, the role of noncanonical Notch signaling is still poorly understood. In a pathologic context, aberration of Notch signaling is also associated with osteosarcoma. These studies raise the question of how Notch may interact with other signaling pathways, such as Wnt. Finally, manipulation of Notch signaling for bone-related diseases remains complex because of the temporal and context-dependent nature of Notch signaling during mesenchymal stem cell and osteoblast differentiation.

DNER, an Epigenetically Modulated Gene, Regulates Glioblastoma-Derived Neurosphere Cell Differentiation and Tumor Propagation

Neurospheres derived from glioblastoma (GBM) and other solid malignancies contain neoplastic stem-like cells that efficiently propagate tumor growth and resist cytotoxic therapeutics. The primary objective of this study was to use histone-modifying agents to elucidate mechanisms by which the phenotype and tumor-promoting capacity of GBM-derived neoplastic stem-like cells are regulated. Using established GBM-derived neurosphere lines and low passage primary GBM-derived neurospheres, we show that histone deacetylase (HDAC) inhibitors inhibit growth, induce differentiation, and induce apoptosis of neoplastic neurosphere cells. A specific gene product induced by HDAC inhibition, Delta/Notch-like epidermal growth factor-related receptor (DNER), inhibited the growth of GBM-derived neurospheres, induced their differentiation in vivo and in vitro, and inhibited their engraftment and growth as tumor xenografts. The differentiating and tumor suppressive effects of DNER, a noncanonical Notch ligand, contrast with the previously established tumor-promoting effects of canonical Notch signaling in brain cancer stem-like cells. Our findings are the first to implicate noncanonical Notch signaling in the regulation of neoplastic stem-like cells and suggest novel neoplastic stem cell targeting treatment strategies for GBM and potentially other solid malignancies.

A hierarchical cascade activated by non-canonical Notch signaling and the mTOR–Rictor complex regulates neglect-induced death in mammalian cells

The regulation of cellular metabolism and survival by trophic factors is not completely understood. Here, we describe a signaling cascade activated by the developmental regulator Notch, which inhibits apoptosis triggered by neglect in mammalian cells. In this pathway, the Notch intracellular domain (NIC), which is released after interaction with ligand, converges on the kinase mammalian target of rapamycin (mTOR) and the substrate-defining protein rapamycin independent companion of mTOR (Rictor), culminating in the activation of the kinase Akt/PKB. Biochemical and molecular approaches using site-directed mutants identified AktS473 as a key downstream target in the antiapoptotic pathway activated by NIC. Despite the demonstrated requirement for Notch processing and its predominant nuclear localization, NIC function was independent of CBF1/RBP-J, an essential DNA-binding component required for canonical signaling. In experiments that placed spatial constraints on NIC, enforced nuclear retention abrogated antiapoptotic activity and a membrane-anchored form of NIC-blocked apoptosis through mTOR, Rictor and Akt-dependent signaling. We show that the NIC-mTORC2-Akt cascade blocks the apoptotic response triggered by removal of medium or serum deprivation. Consistently, membrane-tethered NIC, and AktS473 inhibited apoptosis triggered by cytokine deprivation in activated T cells. Thus, this study identifies a non-canonical signaling cascade wherein NIC integrates with multiple pathways to regulate cell survival.

Notch signaling mediates G1/S cell-cycle progression in T cells via cyclin D3 and its dependent kinases

AbstractNotch signaling plays a role in normal lymphocyte development and function. Activating Notch1-mutations, leading to aberrant downstream signaling, have been identified in human T-cell acute lymphoblastic leukemia (T-ALL). While this highlights the contribution of Notch signaling to T-ALL pathogenesis, the mechanisms by which Notch regulates proliferation and survival in normal and leukemic T cells are not fully understood. Our findings identify a role for Notch signaling in G1-S progression of cell cycle in T cells. Here we show that expression of the G1 proteins, cyclin D3, CDK4, and CDK6, is Notch-dependent both in vitro and in vivo, and we outline a possible mechanism for the regulated expression of cyclin D3 in activated T cells via CSL (CBF-1, mammals; suppressor of hairless, Drosophila melanogaster; Lag-1, Caenorhabditis elegans), as well as a noncanonical Notch signaling pathway. While cyclin D3 expression contributes to cell-cycle progression in Notch-dependent human T-ALL cell lines, ectopic expression of CDK4 or CDK6 together with cyclin D3 shows partial rescue from γ-secretase inhibitor (GSI)-induced G1 arrest in these cell lines. Importantly, cyclin D3 and CDK4 are highly overexpressed in Notch-dependent T-cell lymphomas, justifying the combined use of cell-cycle inhibitors and GSI in treating human T-cell malignancies.

Soluble Forms of the Notch Ligands Delta1 and Jagged1 Promote in Vivo Tumorigenicity in NIH3T3 Fibroblasts with Distinct Phenotypes

We previously found that soluble forms of the Notch ligands Jagged1 and Delta1 induced fibroblast growth factor receptor-dependent cell transformation in NIH3T3 fibroblasts. However, the phenotypes of these lines differed, indicating distinct functional differences among these Notch ligands. In the present study, we used allografts to test the hypothesis that NIH3T3 fibroblasts that express soluble forms of Delta1 and Jagged1 accelerate tumorigenicity in vivo. With the exception of the full-length Jagged1 transfectant, all other cell lines, including the control, generated tumors when injected subcutaneously in athymic mice. Suppression of Notch signaling by the soluble ligands significantly increased tumor onset and growth, whereas full-length Jagged1 completely suppressed tumor development. In addition, there were striking differences in tumor pathology with respect to growth kinetics, vascularization, collagen content, size and number of necrotic foci, and invasiveness into the underlying tissue. Further, the production of angiogenic factors, including vascular endothelial growth factor, also differed among the tumor types. Lastly, both Jagged1- and Delta1-derived tumors contained phenotypically distinct populations of lipid-filled cells that corresponded with increased expression of adipocyte markers. The divergence of tumor phenotype may be attributed to ligand-specific alterations in Notch receptor responses in exogenous and endogenous cell populations within the allographs. Our findings demonstrate distinct functional properties for these Notch ligands in the promotion of tumorigenicity in vivo.

Differentiation of the Drosophila serotonergic lineage depends on the regulation of Zfh-1 by Notch and Eagle

Elucidating mechanisms that differentiate motor neurons from interneurons are fundamental to understanding CNS development. Here we demonstrate that within the Drosophila NB 7-3/serotonergic lineage, different levels of Zfh-1 are required to specify unique properties of both motor neurons and interneurons. We present evidence that Zfh-1 is induced by Notch signaling and suppressed by the transcription factor Eagle. The antagonistic regulation of zfh-1 by Notch and Eagle results in Zfh-1 being expressed at low levels in the NB 7-3 interneurons and at higher levels in the NB 7-3 motor neurons. Furthermore, we present evidence that the induction of Zfh-1 by Notch occurs independently from canonical Notch signaling. We present a model where the differentiation of cell fates within the NB 7-3 lineage requires both canonical and non-canonical Notch signaling. Our observations on the regulation of Zfh-1 provide a new approach for examining the function of Zfh-1 in motor neurons and larval locomotion.