Notch Signaling Controls Ciliary Body Morphogenesis and Secretion by Directly Regulating Nectin Protein Expression

The intestinal mucosa undergoes a continual process of proliferation, differentiation, and apoptosis that is regulated by multiple signaling pathways. Previously, we have shown that the nuclear factor of activated T-cells 5 (NFAT5) is involved in the regulation of intestinal enterocyte differentiation. Here we show that treatment with sodium chloride (NaCl), which activates NFAT5 signaling, increased mTORC1 repressor regulated in development and DNA damage response 1 (REDD1) protein expression and inhibited mTOR signaling; these alterations were attenuated by knockdown of NFAT5. Knockdown of NFAT5 activated mammalian target of rapamycin (mTOR) signaling and significantly inhibited REDD1 mRNA expression and protein expression. Consistently, overexpression of NFAT5 increased REDD1 expression. In addition, knockdown of REDD1 activated mTOR and Notch signaling, whereas treatment with mTOR inhibitor rapamycin repressed Notch signaling and increased the expression of the goblet cell differentiation marker mucin 2 (MUC2). Moreover, knockdown of NFAT5 activated Notch signaling and decreased MUC2 expression, while overexpression of NFAT5 inhibited Notch signaling and increased MUC2 expression. Our results demonstrate a role for NFAT5 in the regulation of mTOR signaling in intestinal cells. Importantly, these data suggest that NFAT5 participates in the regulation of intestinal homeostasis via the suppression of mTORC1/Notch signaling pathway.

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Notch signaling in the pigmented epithelium of the anterior eye segment promotes ciliary body development at the expense of iris formation

Pigment Cell & Melanoma Research ◽

10.1111/pcmr.12236 ◽

2014 ◽

Vol 27 (4) ◽

pp. 580-589 ◽

Cited By ~ 3

Author(s):

Bhushan Sarode ◽

Craig S. Nowell ◽

JongEun Ihm ◽

Corinne Kostic ◽

Yvan Arsenijevic ◽

...

Keyword(s):

Notch Signaling ◽

Ciliary Body ◽

Body Development ◽

Anterior Eye Segment ◽

Pigmented Epithelium

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Notch Signaling Pathway Is Activated by Sulfate Reducing Bacteria

Frontiers in Cellular and Infection Microbiology ◽

10.3389/fcimb.2021.695299 ◽

2021 ◽

Vol 11 ◽

Author(s):

Sudha B. Singh ◽

Cristina N. Coffman ◽

Amanda Carroll-Portillo ◽

Matthew G. Varga ◽

Henry C. Lin

Keyword(s):

Protein Expression ◽

Notch Signaling ◽

Signaling Pathway ◽

Notch Pathway ◽

Sulfate Reducing Bacteria ◽

Notch Signaling Pathway ◽

Sulfate Reducing ◽

Inflammatory Conditions ◽

Related Proteins ◽

Reducing Bacteria

Sulfate Reducing Bacteria (SRB), usually rare residents of the gut, are often found in increased numbers (called a SRB bloom) in inflammatory conditions such as Inflammatory Bowel Disease (IBD), pouchitis, and periodontitis. However, the underlying mechanisms of this association remain largely unknown. Notch signaling, a conserved cell-cell communication pathway, is usually involved in tissue development and differentiation. Dysregulated Notch signaling is observed in inflammatory conditions such as IBD. Lipolysaccharide and pathogens also activate Notch pathway in macrophages. In this study, we tested whether Desulfovibrio, the most dominant SRB genus in the gut, may activate Notch signaling. RAW 264.7 macrophages were infected with Desulfovibrio vulgaris (DSV) and analyzed for the expression of Notch signaling pathway-related proteins. We found that DSV induced protein expression of Notch1 receptor, Notch intracellular domain (NICD) and p21, a downstream Notch target, in a dose-and time-dependent manner. DSV also induced the expression of pro-IL1β, a precursor of IL-1β, and SOCS3, a regulator of cytokine signaling. The gamma secretase inhibitor DAPT or Notch siRNA dampened DSV-induced Notch-related protein expression as well the expression of pro-IL1β and SOCS3. Induction of Notch-related proteins by DSV was not affected by TLR4 -IN -C34(C34), a TLR4 receptor antagonist. Additionally, cell-free supernatant of DSV-infected macrophages induced NICD expression in uninfected macrophages. DSV also activated Notch pathway in the human epithelial cell line HCT116 and in mouse small intestine. Thus, our study uncovers a novel mechanism by which SRB interact with host cells by activating Notch signaling pathway. Our study lays a framework for examining whether the Notch pathway induced by SRB contributes to inflammation in conditions associated with SRB bloom and whether it can be targeted as a therapeutic approach to treat these conditions.

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Growth Arrest-Specific 2 Promotes the Growth of T-Cell Acute Lymphoblastic Leukemia Cells Via Its Regulation of CXCR4

Blood ◽

10.1182/blood-2018-99-114354 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4081-4081

Author(s):

Wenjuan Ma ◽

Yan Wan ◽

Haixia Zhou ◽

Li Zhu ◽

Yun Zhao

Keyword(s):

Acute Lymphoblastic Leukemia ◽

T Cell ◽

Protein Expression ◽

Notch Signaling ◽

Lymphoblastic Leukemia ◽

Growth Arrest ◽

Jurkat Cells ◽

Control Group ◽

Migration Ability ◽

Cell Acute Lymphoblastic Leukemia

Abstract Growth arrest-specific 2 (GAS2) has multiple functions including the regulation of cell morphology, cell cycle, apoptosis and calpain activity.GAS2 has a dual function in cancer cells, however its expression and underlying mechanism in human T-cell acute lymphoblastic leukemia (T-ALL) remain unclear. In the present study, qRT-PCR analysis showed that GAS2 has significantly higher expression (155.5-fold, P=0.0048) in CD3+ cells from T-ALL patients (n=25) than healthy donors (n=13). GAS2 was present in Jurkat cells, while absent in MOLT-4 or HPB-ALL cells. A tiny CpG island of GAS2 was almost fully methylated in both MOLT-4 (100%) and HPB-ALL cells (80%), while 40% methylation in Jurkat cells; suggesting that DNA methylation played a subtle role in regulating GAS2 expression. Two independent shRNA sequences were delivered into Jurkat cells with lentiviral vector. GAS2 silencing inhibited the growth and colony-forming cell (CFC) production significantly. Conversely, GAS2 overexpression enhanced the growth and CFC production of both MOLT-4 and HPB-ALL cells. In addition, GAS2 overexpression promoted HPB-ALL cell induced leukemia in a xenoengraftment model (5 mice in each control group). In GAS2 expressed group, the disease latency was shortened, the splenomegaly was more severe than control group (0.35±0.04g vs. 0.27±0.05g), and more leukemic cells were present in bone marrow (85±3% vs. 45±7%). To obtain the molecular insights of how GAS2 acts, RNA-seq data comparing GAS2 silenced Jurkat cells with control cells were generated. Several Notch signaling molecules were inhibited, including NOTCH1, HES1 and HES4. Despite the differential expression of these transcripts was validated in Jurkat cells, GAS2 overexpression did not elevated the expression of these transcripts in MOLT-4 or HPB-ALL cells, suggesting GAS2 did not have a consistent impact on Notch signaling. However, we found that GAS2 silencing reduced CXCR4 protein expression in Jurkat cells and GAS2 overexpression enhanced CXCR4 protein expression in MOLT-4 cells, while CXCR4 transcript was not altered upon GAS2 manipulation. Consequently, GAS2 silencing significantly reduced migration ability of Jurkat cells and GAS2 overexpression enhanced migration ability of MOLT-4 cells. Overexpression of CXCR4 "rescued" the inhibited CFC production and migration upon GAS2 silencing. A truncated GAS2 (Δ171-313) coined as GAS2DN (dominant negative form of GAS2) has been known to inhibit normal function of GAS2. Herein, we showed that GAS2DN inhibited the growth of Jurkat cells and the expression of CXCR4. To delineate the role of calpain1 and calpain2 in GAS2 function, shRNA sequences against calpain1 and calpain2 was delivered into GAS2DN expressed Jurkat cells respectively, the results showed that calpain2 but not calpain1 silencing was able to enhance the cell growth and CXCR4 expression. Taken together, the present study has demonstrated that GAS2 is aberrantly expressed in human T-ALL cells, which promotes the growth of T-ALL cells partially via its post-transcriptional regulation of CXCR4 depending on calpain2 activity. These data provide new insights of the pathogenesis of T-ALL and possibly new clues to improve the management of the disease. Disclosures No relevant conflicts of interest to declare.

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Notch Signaling Pathway Is Involved in bFGF-Induced Corneal Lymphangiogenesis and Hemangiogenesis

Journal of Ophthalmology ◽

10.1155/2019/9613923 ◽

2019 ◽

Vol 2019 ◽

pp. 1-13

Author(s):

Fang Xie ◽

Xue Zhang ◽

Wenting Luo ◽

Hongyan Ge ◽

Dawei Sun ◽

...

Keyword(s):

Growth Factor ◽

Protein Expression ◽

Notch Signaling ◽

Signaling Pathway ◽

Notch Signaling Pathway ◽

Expression Level ◽

Control Group ◽

Fluorescence Signal ◽

Mrna And Protein Expression ◽

Corneal Lymphangiogenesis

Background. Notch/Dll4 involvement in cornea neovascularization (CRNV) and lymphangiogenesis is unclear. This study aimed to explore the role of notch signaling in basic fibroblast growth factor- (bFGF-) induced corneal lymphangiogenesis and hemangiogenesis. Methods. Corneal stroma of C57BL/6 mice was implanted with bFGF- or phosphate-buffered saline- (PBS-) soaked pellets. Corneal lymphangiogenesis and neovascularization were evaluated by immunofluorescence. Vascular endothelial growth factor-A (VEGF-A), Delta-like ligand 4 (Dll4), and Notch1 mRNA and protein expression were examined on days 1, 3, 7, and 14 by real-time polymerase chain reaction and western blot. Corneal cells were treated with ranibizumab, dexamethasone, and γ-secretase inhibitor (GSI). Microspheres were used to evaluate corneal hemangiogenesis in vivo. Results. Corneal hemangiogenesis reached its peak on day 7 after bFGF implantation, and corneal lymphangiogenesis was significantly higher on day 7 and 14, compared with PBS. mRNA and protein expression of VEGF-A, Dll4, and Notch1 were higher in bFGF-induced animal models compared with controls. Corneal hemangiogenesis and lymphangiogenesis decreased after 7 days of ranibizumab or dexamethasone treatment. After adding GSI for 24 h in bFGF-induced cells, the expression of Notch1 and Dll4 were downregulated compared with that in the control group whereas the expression level of VEGF-A was upregulated. Fluorescent particle number was higher in the GSI group. Ranibizumab and dexamethasone decreased the fluorescence signal. Conclusion. The notch signaling pathway plays a role in regulating VEGF expression, affecting corneal lymphangiogenesis and hemangiogenesis in mice. The molecular imaging probe technique can visualize the changes in the VEGF-A expression level of corneal limbus hemangiogenesis.

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IL-17A-Mediated Notch Signaling in Multiple Myeloma

Blood ◽

10.1182/blood.v124.21.3434.3434 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3434-3434

Author(s):

Rao H. Prabhala ◽

Teru Hideshima ◽

Mariateresa Fulciniti ◽

Sophia Adamia ◽

Rajya Lakshmi Bandi ◽

...

Keyword(s):

Multiple Myeloma ◽

Monoclonal Antibody ◽

Cell Growth ◽

Protein Expression ◽

Notch Signaling ◽

Cellular Protein ◽

Full Length ◽

Growth And Survival ◽

Cell Growth And Survival

Abstract Multiple myeloma (MM) is a plasma cell malignancy, however, significant abnormalities in T cell function are considered to provide help in uncontrolled growth and survival of MM cells. We have previously reported that IL-17A-producing Th17 cells are elevated in MM, that MM cells express IL-17 receptor, and IL-17A promotes MM cell growth and survival. We have reported that MM cells themselves produce IL-17A as confirmed by RT-PCR, Western blotting and immunostaining providing a possibility of both autocrine and paracrine role for IL-17A in MM. As Notch activation has been implicated in Th17 cell differentiation and IL17A production, we have here investigated the role of Notch pathway activation in IL-17A-mediated MM cell growth within the BM microenvironment. Notch consists of 4 proteins (1-4) and has 5 ligands (DLL-1,3,4 and jagged-1, 2). We analyzed RNA-Seq data from 117 newly-diagnosed MM patients and 18 normal plasma cells and observed high expression of Notch 1, and 2 and Notch target genes Hes-1 and Hey-1 but not Notch 3 and 4 in MM. For Notch 2, isoform 2 was highly predominant. Notch expression on MM cells was further confirmed by flow cytometric analysis (Notch1-84%, Notch2-86% and Notch3-3%). Evaluating functional role of Notch in MM, when MM cells were co-cultured with Notch ligand jagged 2-expressing 3T3 cells, IL-17A was able to further induce Notch target gene Hes-1 by 45%. Interestingly, increase in the expression of Notch 2 was also observed during this interaction (increased full-length protein by 65% and active intra-cellular protein by 145%). We next evaluated effect of both anti-IL-17 antibody and Notch inhibitors on MM cells. Anti-IL-17A monoclonal antibody inhibited full-length Notch2 protein expression by 54% and active intra-cellular protein by 85%, as determined by western blot analysis. The antibody inhibitory activity was confirmed with quantitative PCR. Importantly, IL-17A mAb inhibited Hes-1 protein expression by 83%. With the observed impact of Notch signaling in MM, we next evaluated notch inhibitors MRK003, and compound E, a γ-secretase inhibitors, to determine their impact on MM cell growth and survival. We observe that Notch inhibitors affect MM cell growth (inhibition by 43%%, N=5) and IL-6 production (inhibition by 60%, N=3) in co-culture with bone marrow stromal cells. These preclinical data establish the role of IL-17 as well as Notch signaling in myeloma and provides the rationale to evaluate anti-MM activity of anti-IL-17A monoclonal antibody and Notch inhibitors in MM. Disclosures No relevant conflicts of interest to declare.

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Notch signaling in mouse blastocyst development and hatching

10.21203/rs.3.rs-22442/v2 ◽

2020 ◽

Author(s):

Mariana Batista ◽

Patrícia Diniz ◽

Ana Torres ◽

Daniel Murta ◽

Luís Lopes-da-Costa ◽

...

Keyword(s):

Protein Expression ◽

Notch Signaling ◽

Gene Transcription ◽

Embryo Culture ◽

Expression Patterns ◽

Mouse Blastocyst ◽

Blastocyst Development ◽

Gene Markers ◽

Signaling Activity ◽

Blastocyst Hatching

Abstract Background Mammalian early embryo development requires a well-orchestrated interplay of cell signaling pathways. Notch is a major regulatory pathway involved in cell-fate determination in embryonic and adult scenarios. However, the role of Notch in embryonic pre-implantation development is controversial. In particular, Notch role on blastocyst development and hatching remains elusive, and a complete picture of the transcription and expression patterns of Notch components during this time-period is not available.Results This study provided a comprehensive view on the dynamics of individual embryo gene transcription and protein expression patterns of Notch components (receptors Notch1-4; ligands Dll1 and Dll4, Jagged1-2; and effectors Hes1-2), and their relationship with transcription of gene markers of pluripotency and differentiation (Sox2, Oct4, Klf4, Cdx2) during mouse blastocyst development and hatching. Transcription of Notch1-2, Jagged1-2 and Hes1 was highly prevalent and dynamic along stages of development, whereas transcription of Notch3-4, Dll4 and Hes2 had a low prevalence among embryos. Transcription levels of Notch1, Notch2, Jagged2 and Hes1 correlated with each other and with those of pluripotency and differentiation genes. Gene transcription was associated to protein expression, except for Jagged2, where high transcription levels in all embryos were not translated into protein. Presence of Notch signaling activity was confirmed through nuclear NICD and Hes1 detection, and downregulation of Hes1 transcription following canonical signaling blockade with DAPT. In vitro embryo culture supplementation with Jagged1 had no effect on embryo developmental kinetics. In contrast, supplementation with Jagged2 abolished Jagged1 transcription, downregulated Cdx2 transcription and inhibited blastocyst hatching. Notch signaling blockade by DAPT downregulated transcription of Sox2, and retarded embryo hatching. Conclusion Transcription of Notch genes showed a dynamic pattern along blastocyst development and hatching. Data confirmed Notch signaling activity, and lead to the suggestion that Notch canonical signaling may be operating through Notch1, Notch3, Jagged1 and Hes1. Embryo culture supplementation with Jagged1 and Jagged2 unveiled a possible regulatory effect between Jagged1, Cdx2 and blastocyst hatching. Overall, results indicate that a deregulation in Notch signaling, either by its over or under-activation, affects blastocyst development and hatching.

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Effects of topical pilocarpine on ocular growth and refractive development in rabbits

European Journal of Ophthalmology ◽

10.1177/1120672120934962 ◽

2020 ◽

pp. 112067212093496

Author(s):

Liyang Tong ◽

Dongmei Cui ◽

Junwen Zeng

Keyword(s):

Protein Expression ◽

Muscarinic Receptors ◽

Ciliary Body ◽

Anterior Chamber Depth ◽

Receptor Expression ◽

Corneal Curvature ◽

Expression Levels ◽

Ocular Growth ◽

Refractive Development ◽

Mrna And Protein Expression

Purpose: This study aimed to investigate whether topical pilocarpine affects ocular growth and refractive development as well as the underlying biochemical processes in early eye development in rabbits. Methods: Twenty three-week-old New Zealand white rabbits were treated with 0.5% pilocarpine in the right eye for 6 weeks. The left eyes served as contralateral controls. The effects of pilocarpine on refractive error, corneal curvature and ocular biometrics were assessed using streak retinoscopy, keratometry, and A-scan ultrasonography, respectively. Eyeballs were enucleated for histological analysis. The ciliary body and sclera were homogenized to determine the mRNA and protein expression levels of five subtypes of muscarinic receptors. Results: Compared to control eyes, pilocarpine-treated eyes exhibited approximately −1.63 ± 0.54 D myopia accompanied by a 0.11 ± 0.04 mm increase in axial length (AL) ( p < 0.001, respectively). The anterior chamber depth (ACD) was reduced, whereas the lens thickness (LT) and vitreous chamber depth (VCD) increased ( p < 0.001, respectively). Corneal curvature decreased over time but was not significantly different between treated and control eyes. The mRNA and protein expression levels of five subtypes of muscarinic receptors were upregulated in the ciliary body and downregulated in the sclera. Conclusions: Based on these results, pilocarpine can induce myopic shift, increase LT, elongate VCD and AL, and reduce muscarinic receptor expression in the sclera early in development. These changes raise the possibility that pilocarpine may promote axial elongation in ocular development and facilitate the emmetropization of hyperopic eyes.

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