Notch1 Signaling Contributed To TLR4-Triggered NF-kB Activation In Macrophages

Abstract Background: Macrophages substantially shape the development, progression, and complications of inflammation-driven diseases. Although numerous researches support a critical role for Notch signaling in most inflammatory diseases, there is limited data on the role of Notch signaling in TLR4-induced macrophage activation and the interaction of Notch signaling with other signaling pathways(e.g., the NF-kB pathway during inflammation) in macrophages. Methods and Results: In this study, we confirmed that stimulation with TLR4 ligand LPS up-regulates Notch1 expression in RAW264.7 monocyte/macrophage-like cell line. LPS also induced the expression of Notch target genes Notch1 and Hes1 mRNA in macrophages, suggesting that TLR4 signaling enhances Notch pathway activation. The upregulation of Notch1, NICD, and Hes1 protein by LPS treatment was inhibited by the Notch1 inhibitor of DAPT. The increase of TNF-a, IL-6, and IL-1b induced by LPS was inhibited by DAPT while jagged1, the Notch1 ligand, rescued them. Furthermore, the suppression of Notch signaling by DAPT up-regulated CYLD expression but down-regulated TRAF6, IKKa/bphosphorylation, and subsequently phosphorylation and degradation of IκB-α, indicating the inhibition of TLR4-triggered NF-kB activation by DAPT. Interestingly, DAPT showed no inhibitory effect on the increase of MyD88 expression induced by LPS in our study. Conclusions: Our study shows that the stimulation of macrophages via the TLR4 signaling cascade triggers the activation of Notch1 signaling, which regulates the expression patterns of genes involved in pro-inflammatory responses through by activating NF-kB. It may be dependent on the CYLD-TRAF6-IKK pathway. The Notch1 signaling may be considered as a potential therapeutic target against infectious and inflammatory driven diseases.

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Challenges and Opportunities for Effective NOTCH1 Targeting in T-ALL.

Blood ◽

10.1182/blood.v112.11.sci-30.sci-30 ◽

2008 ◽

Vol 112 (11) ◽

pp. sci-30-sci-30

Author(s):

Adolfo A. Ferrando

Keyword(s):

T Cell ◽

Cell Growth ◽

Notch Signaling ◽

Signaling Pathway ◽

Target Genes ◽

Cell Transformation ◽

Notch Pathway ◽

Gastrointestinal Toxicity ◽

Thymocyte Development ◽

Notch1 Signaling

The NOTCH pathway is an evolutionarily conserved mechanism responsible for the direct transduction of developmental signals at the cell surface into changes in gene expression in the nucleus. In the hematopoietic system, the NOTCH1 signaling pathway is required for the commitment of multipotent hematopoietic progenitors to the T cell lineage and, later on, to support cell growth, proliferation, and survival at multiple stages of thymocyte development. These physiological functions are disrupted by activating mutations in the NOTCH1 gene in over 50% of T-cell acute lymphoblastic leukemias (T-ALL). NOTCH1 mutations result in ligand-independent activation or increased protein stability, leading to deregulated cell proliferation and survival in T cell progenitors and T cell transformation. Importantly, small molecule inhibitors of the γ-secretase complex (GSIs) effectively abrogate NOTCH1 signaling, making NOTCH1 a promising therapeutic target for the treatment of T-ALL. However, the clinical development of GSIs has been hampered by our incomplete understanding of the effector pathways controlled by NOTCH1, the lack of clinical responses to GSI therapy, and the development of gastrointestinal toxicity secondary to inhibition of NOTCH signaling in the gut. Recent progress in the identification of the transcriptional regulatory networks that control T cell transformation downstream of NOTCH1 has shown a close relationship between oncogenic NOTCH1 signaling and the transcriptional control of cell growth and metabolism. Thus, NOTCH1 directly controls multiple genes involved in anabolic pathways and further promotes cell growth via direct transcriptional upregulation of MYC and indirect upregulation of the PI3K-AKT signaling pathway via HES1-mediated downregulation of PTEN. Moreover, oncogenic NOTCH1 signaling promotes activation of the NFKB pathway and interacts with the function of p53. Although the specific mechanisms mediating some of these interactions have not been elucidated yet, these results suggest that combinations of GSIs with chemotherapy or drugs targeting the PI3K-AKT and NFKB pathways may have a synergistic effect in the treatment of T-ALL. Moreover, inhibitory antibodies capable of selectively blocking the activation of NOTCH1 and inhibitors of the nuclear transcriptional complex mediating the activation of NOTCH1 target genes offer alternative approaches for the inhibition of oncogenic NOTCH1 signaling. Each of these strategies offers the promise to induce potent antileukemic effects and/or to ameliorate the gastrointestinal toxicity associated with systemic inhibition of NOTCH signaling and may ultimately lead to the development of rationally-designed and highly effective anti-NOTCH1 therapies for T-ALL.

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The Immunomodulatory Role of G2013 (α-L-Guluronic Acid) on the Expression of TLR2 and TLR4 in HT29 cell line

Current Drug Discovery Technologies ◽

10.2174/1570163815666180226093711 ◽

2019 ◽

Vol 16 (1) ◽

pp. 91-95 ◽

Cited By ~ 4

Author(s):

Hamid Farhang ◽

Laleh Sharifi ◽

Mohammad Mehdi Soltan Dallal ◽

Mona Moshiri ◽

Zahra Norouzbabaie ◽

...

Keyword(s):

Inflammatory Responses ◽

Inflammatory Diseases ◽

Rna Extraction ◽

Cell Plate ◽

Inhibitory Effect ◽

Control Group ◽

Ht29 Cells ◽

Guluronic Acid ◽

Tlr4 Mrna

Background: The non-steroidal anti-inflammatory drugs (NSAIDs) play crucial role in the controlling of inflammatory diseases. Due to the vast side effects of NSAIDs, its use is limited. G2013 or &#945;-L-Guluronic Acid is a new NSAID with immunomodulatory features. Objectives: Considering the leading role of TLRs in inflammatory responses, in this study, we aimed to evaluate G2013 cytotoxicity and its effect on the expression of TLR2 and TLR4 molecules. Methods: HEK293-TLR2 and HEK293-TLR4 cells were cultured and seeded on 96-well cell plate, and MTT assay was performed for detecting the viability of the cells after treatment with different concentrations of G2013. HT29 cells were grown and treated with low and high doses of G2013. After total RNA extraction and cDNA synthesis, quantitative real-time PCR were performed to assess the TLR2 and TLR4 mRNA synthesis. Results: We found that concentrations of ≤125 &#181;g/ml of G2013 had no apparent cytotoxicity effect on the HEK293-TLR2 and -TLR4 cells. Our results indicated that after G2013 treatment (5 &#181;g/ml) in HT29 cells, TLR2 and TLR4 mRNA expression decreased significantly compared with the untreated control group (p=0.02 and p=0.001 respectively). Conclusion: The results of this study revealed that G2013 can down regulate the TLR2 and TLR4 gene expression and exerts its inhibitory effect. Our findings are parallel to our previous finding which showed G2013 ability to down regulate the signaling pathway of TLRs. However, further studies are needed to identify the molecular mechanism of G2013.<p>

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CSIG-13. TRPM7 INDUCES TUMORIGENESIS AND STEMNESS THROUGH NOTCH ACTIVATION IN GLIOMA

Neuro-Oncology ◽

10.1093/neuonc/noaa215.125 ◽

2020 ◽

Vol 22 (Supplement_2) ◽

pp. ii30-ii30

Author(s):

Jingwei Wan ◽

Alyssa Guo ◽

Mingli Liu

Keyword(s):

Notch Signaling ◽

Glioma Cell ◽

Glioma Cells ◽

Inhibitory Effect ◽

Notch Signaling Pathway ◽

Normal Brain ◽

Diffuse Astrocytoma ◽

Notch1 Signaling ◽

Signaling Activation ◽

Proliferation And Invasion

Abstract Our group found that the inhibitory effect of TRPM7 on proliferation and invasion of human glioma cell is mediated by multiple mechanisms. TRPM7 regulates miR-28-5p expression, which suppresses cell proliferation and invasion in glioma cells by targeting Ras-related protein Rap1b. In particular, our group found that TRPM7 channels regulate glioma stem cell (GSC) growth/proliferation through STAT3 and Notch signaling. However, which Notch component(s) is crucial for its activity regulated by TRPM7, and its relationship with other GSC markers, such as CD133 and ALDH1, remain unclear. In the current project, we elucidate the mechanisms of TRMP7’s regulation of Notch signaling pathway that contribute to the development and progression of glioma and maintenance of self-renewal and tumorigenicity of GSC using multiple glioma cell lines (GC) with different molecular subtypes and GSCs derived from the GC lines. 1) We first analyzed TRPM7 expression using the Oncomine database (https://www.oncomine.org) and found that the TRPM7 mRNA expression is significantly increased in anaplastic astrocytoma, diffuse astrocytoma, and GBM patients compared to that in normal brain tissue controls. 2) TRPM7 is expressed in GBM, and its channel activity is correlated with Notch1 activation. Inhibition of TRPM7 downregulates Notch1 signaling, while upregulation of TRPM7 upregulates Notch1 signaling. 3) GSC markers, CD133 and ALDH1, are correlated with TRPM7 in GBM. 4) Targeting TRPM7 suppresses the growth and proliferation of glioma cells through G1/S arrests and apoptosis of glioma cells. 5) Targeting Notch1 suppresses the TRPM7-induced growth and proliferation of glioma cells, as well as the expression of GSC markers CD133 and ALDH1. In summary, TRPM7 is responsible for sustained Notch signaling activation, enhanced expression of GSC markers, and regulation of glioma stemness, which contribute to malignant glioma cell growth and invasion. Notch1 and ligand DII4 are key components that contribute GSC stemness.

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Notch restricts lymphatic vessel sprouting induced by vascular endothelial growth factor

Blood ◽

10.1182/blood-2010-11-317800 ◽

2011 ◽

Vol 118 (4) ◽

pp. 1154-1162 ◽

Cited By ~ 73

Author(s):

Wei Zheng ◽

Tuomas Tammela ◽

Masahiro Yamamoto ◽

Andrey Anisimov ◽

Tanja Holopainen ◽

...

Keyword(s):

Notch Signaling ◽

Cell Fate ◽

Target Genes ◽

Notch Pathway ◽

Lymphatic Vessels ◽

Soluble Form ◽

Fibrin Gel ◽

3 Dimensional ◽

Endothelial Growth Factor

Abstract Notch signaling plays a central role in cell-fate determination, and its role in lateral inhibition in angiogenic sprouting is well established. However, the role of Notch signaling in lymphangiogenesis, the growth of lymphatic vessels, is poorly understood. Here we demonstrate Notch pathway activity in lymphatic endothelial cells (LECs), as well as induction of delta-like ligand 4 (Dll4) and Notch target genes on stimulation with VEGF or VEGF-C. Suppression of Notch signaling by a soluble form of Dll4 (Dll4-Fc) synergized with VEGF in inducing LEC sprouting in 3-dimensional (3D) fibrin gel assays. Expression of Dll4-Fc in adult mouse ears promoted lymphangiogenesis, which was augmented by coexpressing VEGF. Lymphangiogenesis triggered by Notch inhibition was suppressed by a monoclonal VEGFR-2 Ab as well as soluble VEGF and VEGF-C/VEGF-D ligand traps. LECs transduced with Dll4 preferentially adopted the tip cell position over nontransduced cells in 3D sprouting assays, suggesting an analogous role for Dll4/Notch in lymphatic and blood vessel sprouting. These results indicate that the Notch pathway controls lymphatic endothelial quiescence, and explain why LECs are poorly responsive to VEGF compared with VEGF-C. Understanding the role of the Notch pathway in lymphangiogenesis provides further insight for the therapeutic manipulation of the lymphatic vessels.

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A modifier in the 129S2/SvPasCrl genome is responsible for the viability of Notch1[12f/12f] mice

BMC Developmental Biology ◽

10.1186/s12861-019-0199-3 ◽

2019 ◽

Vol 19 (1) ◽

Cited By ~ 6

Author(s):

Shweta Varshney ◽

Hua-Xing Wei ◽

Frank Batista ◽

Mohd Nauman ◽

Subha Sundaram ◽

...

Keyword(s):

Target Genes ◽

Notch Pathway ◽

X Ray ◽

X Ray Crystallography ◽

Notch1 Signaling ◽

Notch Receptors ◽

F1 Progeny ◽

Epidermal Growth ◽

Notch Ligands

Abstract Background Mouse NOTCH1 carries a highly conserved O-fucose glycan at Thr466 in epidermal growth factor-like repeat 12 (EGF12) of the extracellular domain. O-Fucose at this site has been shown by X-ray crystallography to be recognized by both DLL4 and JAG1 Notch ligands. We previously showed that a Notch1 Thr466Ala mutant exhibits very little ligand-induced NOTCH1 signaling in a reporter assay, whereas a Thr466Ser mutation enables the transfer of O-fucose and reverts the NOTCH1 signaling defect. We subsequently generated a mutant mouse with the Thr466Ala mutation termed Notch1[12f](Notch1tm2Pst). Surprisingly, homozygous Notch1[12f/12f] mutants on a mixed background were viable and fertile. Results We now report that after backcrossing to C57BL/6 J mice for 11–15 generations, few homozygous Notch1[12f/12f] embryos were born. Timed mating showed that embryonic lethality occurred by embryonic day (E) ~E11.5, somewhat delayed compared to mice lacking Notch1 or Pofut1 (the O-fucosyltransferase that adds O-fucose to Notch receptors), which die at ~E9.5. The phenotype of C57BL/6 J Notch1[12f/12f] embryos was milder than mutants affected by loss of a canonical Notch pathway member, but disorganized vasculogenesis in the yolk sac, delayed somitogenesis and development were characteristic. In situ hybridization of Notch target genes Uncx4.1 and Dll3 or western blot analysis of NOTCH1 cleavage did not reveal significant differences at E9.5. However, qRT-PCR of head cDNA showed increased expression of Dll3, Uncx4.1 and Notch1 in E9.5 Notch1[12f/12f] embryos. Sequencing of cDNA from Notch1[12f/12f] embryo heads and Southern analysis showed that the Notch1[12f] locus was intact following backcrossing. We therefore looked for evidence of modifying gene(s) by crossing C57BL/6 J Notch1 [12f/+] mice to 129S2/SvPasCrl mice. Intercrosses of the F1 progeny gave viable F2 Notch1[12f/12f] mice. Conclusion We conclude that the 129S2/SvPasCrl genome contains a dominant modifying gene that rescues the functions of NOTCH1[12f] in signaling. Identification of the modifying gene has the potential to illuminate novel factor(s) that promote Notch signaling when an O-fucose glycan is absent from EGF12 of NOTCH1.

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Impaired angiogenesis and altered Notch signaling in mice overexpressing endothelial Egfl7

Blood ◽

10.1182/blood-2010-03-274860 ◽

2010 ◽

Vol 116 (26) ◽

pp. 6133-6143 ◽

Cited By ~ 71

Author(s):

Donna Nichol ◽

Carrie Shawber ◽

Michael J. Fitch ◽

Kathryn Bambino ◽

Anshula Sharma ◽

...

Keyword(s):

Endothelial Cells ◽

Notch Signaling ◽

Target Genes ◽

Critical Role ◽

Endothelial Cell Proliferation ◽

Vascular Patterning ◽

Cardiac Morphogenesis ◽

Epidermal Growth ◽

And Migration

Abstract Epidermal growth factor-like domain 7 (Egfl7) is important for regulating tubulogenesis in zebrafish, but its role in mammals remains unresolved. We show here that endothelial overexpression of Egfl7 in transgenic mice leads to partial lethality, hemorrhaging, and altered cardiac morphogenesis. These defects are accompanied by abnormal vascular patterning and remodeling in both the embryonic and postnatal vasculature. Egfl7 overexpression in the neonatal retina results in a hyperangiogenic response, and EGFL7 knockdown in human primary endothelial cells suppresses endothelial cell proliferation, sprouting, and migration. These phenotypes are reminiscent of Notch inhibition. In addition, our results show that EGFL7 and endothelial-specific NOTCH physically interact in vivo and strongly suggest that Egfl7 antagonizes Notch in both the postnatal retina and in primary endothelial cells. Specifically, Egfl7 inhibits Notch reporter activity and down-regulates the level of Notch target genes when overexpressed. In conclusion, we have uncovered a critical role for Egfl7 in vascular development and have shown that some of these functions are mediated through modulation of Notch signaling.

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The Pivotal Role of TBK1 in Inflammatory Responses Mediated by Macrophages

Mediators of Inflammation ◽

10.1155/2012/979105 ◽

2012 ◽

Vol 2012 ◽

pp. 1-8 ◽

Cited By ~ 95

Author(s):

Tao Yu ◽

Young-Su Yi ◽

Yanyan Yang ◽

Jueun Oh ◽

Deok Jeong ◽

...

Keyword(s):

Rheumatoid Arthritis ◽

Inflammatory Responses ◽

Inflammatory Diseases ◽

Cell Damage ◽

Critical Role ◽

Biological Response ◽

Antiviral Defense ◽

Functional Roles ◽

Virus Interaction

Inflammation is a complex biological response of tissues to harmful stimuli such as pathogens, cell damage, or irritants. Inflammation is considered to be a major cause of most chronic diseases, especially in more than 100 types of inflammatory diseases which include Alzheimer's disease, rheumatoid arthritis, asthma, atherosclerosis, Crohn's disease, colitis, dermatitis, hepatitis, and Parkinson's disease. Recently, an increasing number of studies have focused on inflammatory diseases. TBK1 is a serine/threonine-protein kinase which regulates antiviral defense, host-virus interaction, and immunity. It is ubiquitously expressed in mouse stomach, colon, thymus, and liver. Interestingly, high levels of active TBK1 have also been found to be associated with inflammatory diseases, indicating that TBK1 is closely related to inflammatory responses. Even though relatively few studies have addressed the functional roles of TBK1 relating to inflammation, this paper discusses some recent findings that support the critical role of TBK1 in inflammatory diseases and underlie the necessity of trials to develop useful remedies or therapeutics that target TBK1 for the treatment of inflammatory diseases.

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Regulation of Notch 1 signaling in THP-1 cells enhances M2 macrophage differentiation

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00896.2013 ◽

2014 ◽

Vol 307 (11) ◽

pp. H1634-H1642 ◽

Cited By ~ 24

Author(s):

Reetu D. Singla ◽

Jing Wang ◽

Dinender K. Singla

Keyword(s):

Notch Signaling ◽

Proinflammatory Cytokine ◽

Inflammatory Responses ◽

Inflammatory Diseases ◽

Macrophage Polarization ◽

Cellular Stress ◽

Culture Conditions ◽

M2 Macrophage ◽

Macrophage Differentiation ◽

Anti Inflammatory

Macrophage polarization is emerging as an important area of research for the development of novel therapeutics to treat inflammatory diseases. Within the current study, the role of Notch1R in macrophage differentiation was investigated as well as downstream effects in THP-1 monocytes cultured in “inflammation mimicry” media. Interference of Notch signaling was achieved using either the pharmaceutical inhibitor DAPT or Notch1R small interfering RNA (siRNA), and Notch1R expression, macrophage phenotypes, and anti- and proinflammatory cytokine expression were evaluated. Data presented show that Notch1R expression on M1 macrophages as well as M1 macrophage differentiation is significantly elevated during cellular stress ( P < 0.05). However, under identical culture conditions, interference to Notch signaling via Notch1R inhibition mitigated these results as well as promoted M2 macrophage differentiation. Moreover, when subjected to cellular stress, macrophage secretion of proinflammatory cytokines was significantly heightened ( P < 0.05). Importantly, Notch1R inhibition not only diminished proinflammatory cytokine secretion but also enhanced anti-inflammatory protein release ( P < 0.05). Our data suggest that Notch1R plays a pivotal role in M1 macrophage differentiation and heightened inflammatory responses. Therefore, we conclude that inhibition of Notch1R and subsequent downstream signaling enhances monocyte to M2 polarized macrophage outcomes and promotes anti-inflammatory mediation during cellular stress.

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Modeling Inflammation in Zebrafish for the Development of Anti-inflammatory Drugs

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2020.620984 ◽

2021 ◽

Vol 8 ◽

Author(s):

Yufei Xie ◽

Annemarie H. Meijer ◽

Marcel J. M. Schaaf

Keyword(s):

Immune System ◽

Inflammatory Response ◽

Molecular Mechanisms ◽

Inflammatory Responses ◽

Inflammatory Diseases ◽

Critical Role ◽

Model Systems ◽

Trinitrobenzene Sulfonic Acid ◽

Inflammatory Drugs ◽

Anti Inflammatory

Dysregulation of the inflammatory response in humans can lead to various inflammatory diseases, like asthma and rheumatoid arthritis. The innate branch of the immune system, including macrophage and neutrophil functions, plays a critical role in all inflammatory diseases. This part of the immune system is well-conserved between humans and the zebrafish, which has emerged as a powerful animal model for inflammation, because it offers the possibility to image and study inflammatory responses in vivo at the early life stages. This review focuses on different inflammation models established in zebrafish, and how they are being used for the development of novel anti-inflammatory drugs. The most commonly used model is the tail fin amputation model, in which part of the tail fin of a zebrafish larva is clipped. This model has been used to study fundamental aspects of the inflammatory response, like the role of specific signaling pathways, the migration of leukocytes, and the interaction between different immune cells, and has also been used to screen libraries of natural compounds, approved drugs, and well-characterized pathway inhibitors. In other models the inflammation is induced by chemical treatment, such as lipopolysaccharide (LPS), leukotriene B4 (LTB4), and copper, and some chemical-induced models, such as treatment with trinitrobenzene sulfonic acid (TNBS), specifically model inflammation in the gastro-intestinal tract. Two mutant zebrafish lines, carrying a mutation in the hepatocyte growth factor activator inhibitor 1a gene (hai1a) and the cdp-diacylglycerolinositol 3-phosphatidyltransferase (cdipt) gene, show an inflammatory phenotype, and they provide interesting model systems for studying inflammation. These zebrafish inflammation models are often used to study the anti-inflammatory effects of glucocorticoids, to increase our understanding of the mechanism of action of this class of drugs and to develop novel glucocorticoid drugs. In this review, an overview is provided of the available inflammation models in zebrafish, and how they are used to unravel molecular mechanisms underlying the inflammatory response and to screen for novel anti-inflammatory drugs.

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Structural and functional analysis of the repressor complex in the Notch signaling pathway ofDrosophila melanogaster

Molecular Biology of the Cell ◽

10.1091/mbc.e11-05-0420 ◽

2011 ◽

Vol 22 (17) ◽

pp. 3242-3252 ◽

Cited By ~ 31

Author(s):

Dieter Maier ◽

Patricia Kurth ◽

Adriana Schulz ◽

Andrew Russell ◽

Zhenyu Yuan ◽

...

Keyword(s):

Cell Culture ◽

Notch Signaling ◽

Target Genes ◽

Notch Pathway ◽

Structural Similarity ◽

Receptor Activation ◽

Notch Signaling Pathway ◽

Repressor Complex

In metazoans, the highly conserved Notch pathway drives cellular specification. On receptor activation, the intracellular domain of Notch assembles a transcriptional activator complex that includes the DNA-binding protein CSL, a composite of human C-promoter binding factor 1, Suppressor of Hairless of Drosophila melanogaster [Su(H)], and lin-12 and Glp-1 phenotype of Caenorhabditis elegans. In the absence of ligand, CSL represses Notch target genes. However, despite the structural similarity of CSL orthologues, repression appears largely diverse between organisms. Here we analyze the Notch repressor complex in Drosophila, consisting of the fly CSL protein, Su(H), and the corepressor Hairless, which recruits general repressor proteins. We show that the C-terminal domain of Su(H) is necessary and sufficient for forming a high-affinity complex with Hairless. Mutations in Su(H) that affect interactions with Notch and Mastermind have no effect on Hairless binding. Nonetheless, we demonstrate that Notch and Hairless compete for CSL in vitro and in cell culture. In addition, we identify a site in Hairless that is crucial for binding Su(H) and subsequently show that this Hairless mutant is strongly impaired, failing to properly assemble the repressor complex in vivo. Finally, we demonstrate Hairless-mediated inhibition of Notch signaling in a cell culture assay, which hints at a potentially similar repression mechanism in mammals that might be exploited for therapeutic purposes.

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