Target Genes Hey1 Research Articles

RhBMP-2 induces terminal differentiation of human bone marrow mesenchymal stromal cells only by synergizing with other signals

BackgroundRecombinant human bone morphogenetic protein 2 (rhBMP-2) and human bone marrow mesenchymal stromal cells (hBM-MSCs) have been thoroughly studied for research and translational bone regeneration purposes. rhBMP-2 induces bone formation in vivo, and hBM-MSCs are its target, bone-forming cells. In this article, we studied how rhBMP-2 drives the multilineage differentiation of hBM-MSCs both in vivo and in vitro.MethodsrhBMP-2 and hBM-MSCs were tested in an in vivo subcutaneous implantation model to assess their ability to form mature bone and undergo multilineage differentiation. Then, the hBM-MSCs were treated in vitro with rhBMP-2 for short-term or long-term cell-culture periods, alone or in combination with osteogenic, adipogenic or chondrogenic media, aiming to determine the role of rhBMP-2 in these differentiation processes.ResultsThe data indicate that hBM-MSCs respond to rhBMP-2 in the short term but fail to differentiate in long-term culture conditions; these cells overexpress the rhBMP-2 target genes DKK1, HEY-1 and SOST osteogenesis inhibitors. However, in combination with other differentiation signals, rhBMP-2 acts as a potentiator of multilineage differentiation, not only of osteogenesis but also of adipogenesis and chondrogenesis, both in vitro and in vivo.ConclusionsAltogether, our data indicate that rhBMP-2 alone is unable to induce in vitro osteogenic terminal differentiation of hBM-MSCs, but synergizes with other signals to potentiate multiple differentiation phenotypes. Therefore, rhBMP-2 triggers on hBM-MSCs different specific phenotype differentiation depending on the signalling environment.

Polyethylenimine Triggers Dll4 Degradation to Regulate Angiogenesis In Vitro.

The Dll4-Notch signaling pathway plays a crucial role in the regulation of angiogenesis and is a promising therapeutic target for diseases associated with abnormal angiogenesis, such as cancer and ophthalmic diseases. Here, we find that polyethylenimine (PEI), a cationic polymer widely used as nucleic acid transfection reagents, can target the Notch ligand Dll4. By immunostaining and immunoblotting, we demonstrate that PEI significantly induces the clearance of cell-surface Dll4 and facilitates its degradation through the lysosomal pathway. As a result, the activation of Notch signaling in endothelial cells is effectively inhibited by PEI, as evidenced by the observed decrease in the generation of the activated form of Notch and expression of Notch target genes Hes1 and Hey1. Furthermore, through blocking Dll4-mediated Notch signaling, PEI treatment enhances angiogenesis in vitro. Together, our study reveals a novel biological effect of PEI and establishes a foundation for the development of a Dll4-targeted biomaterial for the treatment of angiogenesis-related disease.

Discovery of anticancer compound possessing potential to bind γ-secretase catalytic subunit and inhibit notch promoter activity

Gamma secretase (GS) is an important therapeutic target in anticancer drug discovery. Increased GS activity activates notch signaling pathway which is associated with cancer stemness and drug resistance in cancer cells. A total of 69,075 natural and their derivative compounds were screened to identify the lead compound on the basis of in silico GS catalytic domain binding potential and in vitro selective anticancer efficacy. STOCK1N-23234 showed higher dock score (−11.82) compared to DAPT (−9.2) in molecular docking experiment and formed hydrogen bond with the key amino acid (Asp385) involve in catalysis process. Molecular dynamics (MD) simulation parameters (RMSD, RMSF, Rg, SASA and hydrogen bond formation) revealed that the STOTCK1N-23234 formed structurally and energetically stable complex with the GS catalytic domain with lower binding energy (−22.79 kcal/mol) compared to DAPT (−16.22 kcal/mol). STOCK1N-23234 showed better toxicity (up to 60%) against colon and breast cancer cells (HCT-116 and MDA-MB-453) at 1–70 µM concentration. Interestingly, STOCK1N-23234 did not showed cytotoxicity against human normal breast cells (MCF-10A). STOCK1N-23234 treatment significantly decreased sphere formation, notch promoter activity, and transcription of notch target genes (Hes-1 and Hey-1) in HCT-116 cells derived colonosphere. Confocal microscopy revealed that STOTCK1N-23234 treatment at test concentration induced apoptosis related morphological changes, reduced mitochondria membrane potential and increased reactive oxygen species production in HCT-116 cells compared to non-treated cells. In conclusion, STOCK1N-23234 is a novel lead natural anticancer compound which requires in depth validation in cancer preclinical models. Communicated by Ramaswamy H. Sarma

Inhibition of Pi4kb activity causes malformation of vestibular apparatus in zebrafish by downregulating hey1

Phosphatidylinositol 4 kinase-β (PI4KB) plays critical roles in human genetic diseases. In zebrafish, Pi4kb is strongly expressed in hair cells (HCs), which are necessary for detecting sound vibrations, head movements, and water motion. However, the role of PI4KB in HC or semicircular canal development is unclear. Herein, we report that pi4kb morphants exhibit insensitivity to sound stimulation and abnormal morphological vestibular organs, including cilium loss in HCs of the cristae and semicircular canal malformation. As bone morphogenetic protein (BMP) signaling is associated with HC and semicircular canal development, we analyzed the expression of BMP-related genes; the phosphorylated Smad1/5/9 (p-Smad1/5/9) expression was markedly reduced in otic HCs. RNA-sequencing data indicated that the transcriptional levels of BMP membrane receptor 2 (bmpr2a and bmpr2b) and hes-related family of bHLH transcription factors with YRPW motif 1 (hey1), a direct downstream target gene of p-Smad, were significantly reduced in the pi4kb morphants, as verified using quantitative reverse transcription-polymerase chain reaction and in situ hybridization. Co-injection of hey1 mRNA and pi4kb morpholino notably recovered vestibular apparatus development, including the number and length of cilia in HCs of the cristae and semicircular canal formation. Collectively, these results suggest that Pi4kb is involved in vestibular apparatus development in zebrafish by regulating BMP membrane receptor 2 and p-Smad1/5/9 levels, thereby affecting the transcriptional activation of the target gene hey1. This study sheds light on the interaction between Pi4kb and the BMP-Hey1 signaling axis, which is critical for HC and semicircular canal formation.

NOTCH2 sensitizes the chondrocyte to the inflammatory response of tumor necrosis factor α

Notch regulates the immune and inflammatory response and has been associated with the pathogenesis of osteoarthritis in humans and preclinical models of the disease. Notch2tm1.1Ecan mice harbor a NOTCH2 gain-of-function and are sensitized to osteoarthritis, but the mechanisms have not been explored. We examined the effects of tumor necrosis factor α (TNFα) in chondrocytes from Notch2tm1.1Ecan mice and found that NOTCH2 enhanced the effect of TNFα on Il6 and Il1b expression. Similar results were obtained in cells from a conditional model of NOTCH2 gain-of-function, Notch22.1Ecan mice, and following the expression of the NOTCH2 intracellular domain invitro. Recombination signal-binding protein for immunoglobulin Kappa J region partners with the NOTCH2 intracellular domain to activate transcription; in the absence of Notch signaling it inhibits transcription, and Rbpj inactivation in chondrocytes resulted in Il6 induction. Although TNFα induced IL6 to a greater extent in the context of NOTCH2 activation, there was a concomitant inhibition of Notch target genes Hes1, Hey1, Hey2, and Heyl. Electrophoretic mobility shift assay demonstrated displacement of recombination signal-binding protein for immunoglobulin Kappa J region from DNA binding sites by TNFα explaining the increased Il6 expression and the concomitant decrease in Notch target genes. NOTCH2 enhanced the effect of TNFα on NF-κB signaling, and RNA-Seq revealed increased expression of pathways associated with inflammation and the phagosome in NOTCH2 overexpressing cells in the absence and presence of TNFα. Collectively, NOTCH2 has important interactions with TNFα resulting in the enhanced expression of Il6 and inflammatory pathways in chondrocytes.

The effects of propranolol on the biology and Notch signaling pathway of human umbilical vein endothelial cells.

Propranolol is the first choice for treating infantile hemangioma (IH). How propranolol works in IH remains unclear. Infantile hemangioma endothelial cells (HemECs) express Notch1, Jagged, Hey1, and other molecules in the Notch pathway, suggesting that Notch pathway-related molecules play an important role in affecting vascular endothelial cell proliferation. Whether propranolol can affect the Notch signaling pathway in IH treatment is unclear. We performed this study to observe the effect of propranolol on the expression of Notch signaling pathway molecules in human umbilical vein endothelial cells (HUVECs) and to explore the therapeutic mechanism of propranolol on IH. HUVECs cultured in vitro were exposed to 60, 120, 240, 360, or 480 µM propranolol. The morphological changes of the HUVECs were observed under an inverted microscope. HUVECs proliferation was detected with Cell Counting Kit-8 (CCK-8). The effects of propranolol on HUVECs apoptosis were detected by flow cytometry. The role of Notch in propranolol inhibition of HUVEC proliferation was analyzed with real-time polymerase chain reaction (PCR) and western blotting. Propranolol reduced HUVECs numbers and altered their morphology. The inhibitory effect of propranolol on cell proliferation was dependent on the reaction time and drug concentration. Propranolol upregulated Jagged1, Notch1, and Hey1 expression and downregulated delta-like ligand4 (DLL4) expression. Propranolol may play a role in IH treatment by increasing Jagged1 expression in endothelial cells, activating the Notch pathway and inducing the upregulation of the downstream target gene HEY1.

Gastrodin protects porcine sertoli cells from zearalenone-induced abnormal secretion of glial cell line-derived neurotrophic factor through the NOTCH signaling pathway

Zearalenone (ZEA) is a prevalent mycotoxin found in moldy diets and is associated with reproductive dysfunction. However, the molecular underpinning of ZEA in impairment of spermatogenesis remains largely unknown. To unveil the toxic mechanism of ZEA, we established a co-culture model using porcine Sertoli cells and porcine spermatogonial stem cells (pSSCs) to investigate the impact of ZEA on these cell types and their associated signaling pathways. Our findings showed that low concentration of ZEA inhibited cell apoptosis, while high concentration induced cell apoptosis. Furthermore, the expression levels of Wilms’ tumor 1 (WT1), proliferating cell nuclear antigen (PCNA) and glial cell line-derived neurotrophic factor (GDNF) were significantly decreased in ZEA treatment group, while concurrently upregulating the transcriptional levels of the NOTCH signaling pathway target genes HES1 and HEY1. The addition of the NOTCH signaling pathway inhibitor DAPT (GSI-IX) alleviated the damage to porcine Sertoli cells caused by ZEA. Gastrodin (GAS) significantly increased the expression levels of WT1, PCNA and GDNF, and inhibited the transcription of HES1 and HEY1. GAS also efficiently restored the decreased expression levels of DDX4, PCNA and PGP9.5 in co-cultured pSSCs suggesting its potential in ameliorating the damage caused by ZEA to Sertoli cells and pSSCs. In conclusion, the present study demonstrates that ZEA disrupts pSSCs self-renewal by affecting the function of porcine Sertoli cell, and highlights the protective mechanism of GAS through the regulation of the NOTCH signaling pathway. These findings may offer a novel strategy for alleviating ZEA-induced male reproductive dysfunction in animal production.

Rutin Potentially Binds the Gamma Secretase Catalytic Site, Down Regulates the Notch Signaling Pathway and Reduces Sphere Formation in Colonospheres.

Rutin, a natural flavonol, can modulate molecular signaling pathways and has considerable potential in cancer treatment. However, little is known about the effect of rutin on the notch signaling pathway (NSP) in cancer and cancer stem-like cells. In this study, we explored the effect of rutin on gamma secretase (GS, a putative notch signaling target) inhibition mediated NICD (Notch Intracellular Domain) production in colon cancer cells. Molecular docking, MM-GBSA, and Molecular dynamics (MD) simulation experiments were performed to check rutin’s GS catalytic site binding potential. The HCT-116 colon cancer and cancer stem-like cells (colonospheres) were utilized to validate the in silico findings. The NICD production, notch promoter assay, expression of notch target genes, and cancer stemness/self-renewal markers were studied at molecular levels. The results were compared with the Notch-1 siRNA transfected test cells. The in silico study revealed GS catalytic site binding potential in rutin. The in vitro results showed a decreased NICD formation, an altered notch target gene (E-cad, Hes-1, and Hey-1) expression, and a reduction in stemness/self-renewal markers (CD44, c-Myc, Nanog, and Sox2) in test cells in a time and dose-dependent manner. In conclusion, rutin inhibits the notch signaling pathway and reduces the stemness/self-renewal property in colon cancer cells and the colonospheres by targeting gamma secretase. The clinical efficacy of rutin in combination therapy in colon cancer may be studied in the future.

Abstract 6308: Antitumor effects of RBPJ Inhibitor-1 on osteosarcoma cells

Abstract Osteosarcoma (OS) is the most common primary malignancy of the bone in adolescents and children with limited treatment options. Drug resistance to current therapy together with severe side effects urgently calls for alternative treatment strategies targeting critical cancer signaling pathways. Recently, our and other studies suggest that hyperactivation of Notch signaling contributes to the pathogenesis of human and murine OS, and its inhibition may be a therapeutic approach for the treatment of OS. Notch pathophysiological activity involves activated Notch (i.e. NICD, the notch intracellular domain) forming a nuclear complex with DNA-binding factors and coactivators such as recombination signal binding protein for immunoglobulin kappa J region (RBPJ). This evolutionary conserved mechanism is involved in the maintenance and survival of cancer stem cells, which can govern various cell activities like metastasis and chemoresistance. This study aims to examine the in vitro effects of targeting the NICD-RBPJ interaction using a small molecular inhibitor of the RBPJ (RIN1) on osteosarcoma cell lines. We found that treatment of 10 µM RIN1 on SJSA-1 (ATCC, CRL-2098), a human osteosarcoma cell line with a high Notch activity, can significantly decrease cell proliferation at 48 and 72 hours (h). We also found that at 10 µM of RIN1 affects SJSA-1 migration using a Boyden chamber-based cell system at 24 h, a time point where we find minimal contribution from an effect on cell proliferation. Treatment with RIN1 can block SJSA-1 clonogenicity, but we showed that it has limited ability to block SJSA-1 cell invasion. Treatment with RIN1 does not initiate or enhance the osteoblast-like differentiation of SJSA-1. Notch target genes HEY1 and CyclinD1 was significantly modulated in SJSA-1 cells upon treatment in a Notch-independent manner. Interestingly, treatment with RIN1 on SJSA-1 decreases SOX2 protein expression, a well-known cancer stem cell marker, and upregulates tumor suppressor RB1 protein expression. Ongoing studies include additional cellular function assays and RNA-seq transcriptomic analysis of RIN1’s effect on two Notch-dependent cancer cell lines including MB 157 (ATCC, CRL-7721) and T29 in its activating context. In summary, RIN1 has been shown to have a significant effect on the different cell behaviors of human osteosarcoma, which may eventually facilitate with the treatment of patients that have metastatic disease. Citation Format: Dakota Callahan, Leetoria Hinojosa, Haydee M. Torres, Jianning Tao. Antitumor effects of RBPJ Inhibitor-1 on osteosarcoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6308.

Notch2 Blockade Mitigates Methotrexate Chemotherapy-Induced Bone Loss and Marrow Adiposity.

Childhood cancer methotrexate (MTX) chemotherapy often causes bone growth impairments, bone loss, and increased risks of fractures during or after treatment, for which the pathobiology is unclear and there is a lack of specific treatment. Our time course analyses of long bones from rats receiving intensive MTX treatment (mimicking a clinical protocol) found decreased trabecular bone volume, increased osteoclast formation and activity, increased adipogenesis in the expense of osteogenesis from the bone marrow stromal cells at days 6 and 9 following the first of five daily MTX doses. For exploring potential mechanisms, PCR array expression of 91 key factors regulating bone homeostasis was screened with the bone samples, which revealed MTX treatment-induced upregulation of Notch receptor NOTCH2, activation of which is known to be critical in skeletal development and bone homeostasis. Consistently, increased Notch2 activation in bones of MTX-treated rats was confirmed, accompanied by increased expression of Notch2 intracellular domain protein and Notch target genes HEY1, HES1 and HEYL. To confirm the roles of Notch2 signalling, a neutralising anti-Notch2 antibody or a control IgG was administered to rats during MTX treatment. Microcomputed tomography analyses demonstrated that trabecular bone volume was preserved by MTX+anti-Notch2 antibody treatment. Anti-Notch2 antibody treatment ameliorated MTX treatment-induced increases in osteoclast density and NFATc1 and RANKL expression, and attenuated MTX-induced bone marrow adiposity via regulating Wnt/β-catenin signalling and PPARγ expression. Thus, Notch2 signalling plays an important role in mediating MTX treatment-induced bone loss and bone marrow adiposity, and targeting Notch2 could be a potential therapeutic option.

Immune cells and Notch1 signaling appear to drive the epithelial to mesenchymal transition in the development of adenomyosis in mice.

The epithelial to mesenchymal transition (EMT) has been implicated in the development of adenomyosis, along with dysregulated immune responses. Inflammation potentially induces Notch signaling, which could promote this EMT. The objective of this study was to investigate the involvement of immune cells and Notch1-mediated EMT in the development of adenomyosis. Adenomyosis was induced in 18 CD-1 mice by neonatal oral administration of tamoxifen (TAM group), while 18 neonates received vehicle only (Control group). Their uteri were sampled at 30, 60 or 90 days of age. Immune cell markers (Cd45, Ly6c1, Cd86, Arginine1, Cd19, Cd4, Cd8), Notch1 and its target genes (Hey1, Hey2, Hes1, Hes5) and biomarkers of EMT (E-Cadherin, Vimentin, Tgfb, Snail1, Slug, Snail3) were analyzed by quantitative RT-PCR and immunohistochemistry. Activated-Notch1 protein was measured by western blot. Aberrant expression of immune cell markers was observed in the uteri of mice as they developed adenomyosis. The expression of inflammatory cell markers, notably M1 macrophages and natural killer cells, was increased from Day 30 in the TAM group compared to controls, followed by an increase in the Cd4 marker (T cells) at Day 60. Conversely, expression of the Cd19 marker (B cells) was significantly reduced at all of the stages studied. Notch1 signaling was also highly activated compared to controls at Day 30 and Day 60. Concomitantly, the levels of several markers for EMT were also higher. Therefore, the activation of Notch1 coincides with aberrant expression of immune and EMT markers in the early development of adenomyosis.

Dysregulation of Notch-FGF signaling axis in germ cells results in cystic dilation of the rete testis in mice

Numb (Nb) and Numb-like (Nbl) are functionally redundant adaptor proteins that critically regulate cell fate and morphogenesis in a variety of organs. We selectively deleted Nb and Nbl in testicular germ cells by breeding Nb/Nbl floxed mice with a transgenic mouse line Tex101-Cre. The mutant mice developed unilateral or bilateral cystic dilation in the rete testis (RT). Dye trace indicated partial blockages in the testicular hilum. Morphological and immunohistochemical evaluations revealed that the lining epithelium of the cysts possessed similar characteristics of RT epithelium, suggesting that the cyst originated from dilation of the RT lumen. Spermatogenesis and the efferent ducts were unaffected. In comparisons of isolated germ cells from mutants to control mice, the Notch activity considerably increased and the expression of Notch target gene Hey1 significantly elevated. Further studies identified that germ cell Fgf4 expression negatively correlated the Notch activity and demonstrated that blockade of FGF receptors mediated FGF4 signaling induced enlargement of the RT lumen in vitro. The crucial role of the FGF4 signaling in modulation of RT development was verified by the selective germ cell Fgf4 ablation, which displayed a phenotype similar to that of germ cell Nb/Nbl null mutant males. These findings indicate that aberrant over-activation of the Notch signaling in germ cells due to Nb/Nbl abrogation impairs the RT development, which is through the suppressing germ cell Fgf4 expression. The present study uncovers the presence of a lumicrine signal pathway in which secreted/diffusible protein FGF4 produced by germ cells is essential for normal RT development.

The ErbB3 receptor tyrosine kinase regulates expression of Notch target genes and intestinal stem cell markers.

BackgroundStem cell‐driven regeneration of the intestinal epithelium is important for recovery of damage from insults including inflammation, chemotherapy, and radiation. This process is controlled through tight regulation of intestinal stem cells (ISCs). Our lab has previously reported that intestine‐specific knockout of the receptor tyrosine kinase ErbB3 leads to precocious development of Paneth cells and increased Paneth cell numbers in mice the mouse intestinal epithelium. Since Paneth cells can play a supportive role for ISCs, we tested the hypothesis that altered ErbB3 signaling results in changes in the ISC compartment.MethodsWe crossed ErbB3flox/flox mice with mice expressing villin promoter‐driven Cre to generate an intestinal epithelial‐specific ErbB3 knockout mouse model (ErbB3‐IEKO). Ileal samples were collected from these mice, fixed, and embedded in paraffin for immunostaining. Tissue samples from the ileum were also processed for RNA and protein for qPCR and immunoblots. Samples were probed for Notch target genes and Notch ligands due to the importance of Notch signaling in maintaining the intestinal stem cell compartment. Enteroids were generated from ileum of C57Bl/6 mice and treated with the ErbB3 ligand Nrg‐1β. To activate and inactivate Notch experimentally, enteroids and HT29 cells were treated with valproic acid (VPA) and the γ‐secretase inhibitor DAPT respectively.ResultsqPCR of ErbB3‐IEKO ileum showed increased expression of Lgr5 (rapid‐cycling ISCs) and Bmi1 (facultative ISCs or secretory progenitors capable of regenerating ISCs). Ileal sections also showed increased numbers of Lgr5+ cells by in situ hybridization, and western blots showed increased protein levels of Bmi1 in ErbB3‐IEKO ileum. qPCR of ErbB3‐IEKO ileum showed increased expression of Notch target genes Hey1, Hey2, and Heyl, as well as expression of Notch ligands Dll1, Jag1, and Jag2. Enteroids treated with Nrg‐1β had decreased expression of Hey1, Lgr5, and Bmi1. Enteroids and HT29 cells treated with VPA to activate Notch had decreased expression of ErbB3 RNA; this was abrogated by DAPT treatment.ConclusionErbB3 negatively regulates expression of Lgr5 and Bmi1, markers for fast‐cycling and quiescent ISCs, respectively, indicating a possible role for ErbB3 in the ISC niche. Furthermore, ErbB3 and Notch appear to negatively regulate one another, suggesting a possible mechanism for ErbB3’s effects on ISCs and intestinal cell differentiation.Support or Funding InformationThis work was supported by NIH award R01DK095004.

Frequent Activation of Notch Signaling Pathway in Colorectal Cancers and Its Implication in Patient Survival Outcome.

Colorectal cancer is a major health concern as it ranks third in incidence and second major cause of cancer-related deaths worldwide. A leading cause of treatment failure has been attributed to cancer stem cells that can invariably resist existing chemotherapeutic regimens. Notch signaling pathway has been involved in the maintenance of stem cells besides being crucial in cell fate decision and embryonic development. This pathway has also been implicated in several human malignancies including colorectal cancer. We investigated mRNA expression of four Notch receptors (Notch1–4), five ligands (Jag1, Jag2, Dll1, Dll3, and Dll4), and four target genes (Hes1, Hes5, Hey1, and Hey2) using highly specific TaqMan gene expression assays in colorectal adenomas and cancers. Upregulated expression of Notch receptors ranged between 29 and 73% in colorectal cancers and between 11 and 56% in adenomas. Expression of Notch3 and Notch4 receptors was significantly higher in colorectal cancers compared to normal and adenoma tissues. The Jagged and Delta-like ligands were overexpressed between 25 and 52% in colorectal cancers, while in adenomas, it ranged between 0 and 33%. Combining the data for upregulation of receptors and ligands suggests that 86% colorectal cancers and 56% adenomas exhibited overexpression of Notch pathway genes in our cohort. Notch target genes were upregulated between 24 and 33% in colorectal cancers and between 11 and 22% in adenomas. Collating upregulation of Notch receptors and ligands with the target genes showed concordance in 58% colorectal tumors. Additionally, we evaluated expression of Notch receptors, ligands, and target genes with prognosis using the TCGA mRNA expression dataset. Patients overexpressing Notch3, Notch4, and Hey1 had significantly poorer overall survival relative to those having lower levels of these genes. Taken together, Notch signaling components are aberrantly overexpressed in colorectal tumors, and development of therapeutics targeting the Notch pathway may prove to be beneficial in the management of colorectal cancers.

High-Dose Radiation Increases Notch1 in Tumor Vasculature

Purpose:The aim of this study is to characterize the effects of high-dose radiation therapy (HDRT) on Notch signaling components of the tumor vasculature.Methods and Materials:Human umbilical vein endothelial cells monolayers were exposed to different single fraction doses of irradiation; ribonucleic acid RNA was isolated and polymerase chain reaction was performed for Notch signaling components. The vascular response to radiation therapy was examined in a xenograft model of neuroblastoma. Tumors were treated with 0 Gy, 2 Gy, and 12 Gy single fraction doses and analyzed by double immunofluorescence staining for Notch1, Notch ligands Jagged1 and Dll4, and the endothelial cell (EC) marker endomucin. To assess the role of Notch in vivo, NGP xenograft tumors expressing Fc or Notch1-1–24-decoy (a novel Notch inhibitor) were treated with 0 Gy and 12 Gy. Immunofluorescence staining for endomucin and endomucin/αSMA was performed to analyze the effect of combination treatment on tumor EC and endothelial-to-mesenchymal-transition (EndMT), respectively.Results:In human umbilical vein endothelial cells monolayers doses ≥8 Gy increased expression of NOTCH1, JAG1, and Notch target genes HEY1 and HEY2 as early as 6 hours after irradiation. In vivo, 12 Gy significantly increased Notch1 and Jagged1 in tumor ECs compared with 0 Gy or 2 Gy after 72 hours. Combining HDRT with Notch inhibition using the Notch1-1–24-decoy resulted in a greater loss of EC coverage of tumor vessels than HDRT alone at 6 hours and 72 hours post treatment. Notch inhibition reduced EndMT induced by HDRT, as indicated by diminished αSMA staining in ECs.Conclusions:HDRT induced Notch1 expression and increased Notch1 signaling in the endothelial component of tumor vasculature, which was not observed with lower doses. This increase in Notch1 activation might protect tumor vessels from HDRT induced damage and regulate EndMT process.

Icariin promotes osteogenic differentiation by suppressing Notch signaling

Osteoporosis is a bone disease characterized by microarchitectural deterioration, low bone mass, and increased risk of fractures. Icariin (ICA), an active flavonoid glucoside isolated from Herba epimedii (HEF), is a potent stimulator of osteogenic differentiation and has potential applications for preventing bone loss in postmenopausal women. However, the molecular mechanism underlying the osteogenic effect of ICA has not yet been fully elucidated. In this study, we report that ICA treatment significantly elevated gene expression of osteogenic markers and increased alkaline phosphatase (ALP) activity in MC3T3-E1 and C3H10T1/2 cells. RNA sequencing revealed that the expression of several genes involved in the Notch pathway was decreased following ICA treatment. Real-time PCR further demonstrated that the mRNA levels of Notch ligands Jagged-1 (Jag1), lunatic fringe (Lfng), and Notch signaling downstream target gene Hey-1 were significantly decreased following ICA treatment. In addition, we found that constitutive activation of Notch signaling through overexpression of the intracellular domain of Notch (NICD) fully blocked ICA-induced osteoblast differentiation. Moreover, inhibiting Notch signaling with DAPT markedly enhanced osteogenic differentiation following ICA treatment. We found that the mRNA levels of Notch pathway molecules (Lfng, Notch1, Rbpjk and Nfatc1) were increased in ovariectomized (OVX) mice, and administration of ICA significantly decreased the expression of these genes. Our results suggest that ICA promotes osteogenic differentiation in vitro and alleviates osteoporosis in vivo through inhibition of the Notch signaling pathway.

P4484Notch-dependent endothelial dysfunction in patients with aortic valve calcification

Abstract Background Aortic valve calcification associated with either bicuspid or tricuspid aortic valve remains among the most dangerous cardiovascular diseases but cellular and molecular reasons of this pathology are not fully understood. It has been shown that NOTCH1 haploinsufficiency in endothelial cells derived from iPS and also inhibition of NOTCH1 in aortic valve endothelial cells gives rise to the activation of proosteogenic pathways. Endothelial cells regulate underlying interstitial cells and govern them to differentiation. Thus, elucidation of molecular mechanisms of endothelial dysfunction could shed light on valve calcification pathogenesis. Purpose To elucidate key genes responsible for valve endothelial dysfunction in aortic valve calcification. Methods Primary human aortic valve endothelial cells (VEC) were isolated from 45 patients (BAV, n=21; TAV, n=26) with calcific aortic valve disease and from 8 healthy donors. Expression of corresponding genes was estimated by qPCR. Results We checked expression of about 30 genes related to Notch/Wnt/Bmp pathways and shear stress response. Endothelial cells of patients with calcification had significantly lower mRNA levels of Notch component genes NOTCH2, NOTCH3, DLL4 and JAG1 as well as of Notch target genes HEY1, SNAIL and SLUG. Expression of Wnt-related genes such as DKK1, GREM1 and TCF4 was altered in VEC from calcified valves. Interestingly, mRNA level of ROBO4, recently suggested to be involved in endothelial dysfunctions, was significantly higher in VEC of patients with calcification. Moreover, endothelium from calcified valves had impaired expression of matrix gla protein (MGP), which is known to sequester calcium and inhibit soft tissue calcification in the vasculature and aortic valve. Conclusions Our data show that Notch pathway genes are downregulated in valve endothelial cells of the patients with aortic valve calcification. Our results suggest also implication of shear stress responsive genes and genes of endothelial performance in changing the properties of valve endothelial population of calcified valves and thus presumably to the pathogenesis of aortic valve calcification. Acknowledgement/Funding Russian Foundation For Basic Research 18-34-00277

Role of integrin β1 as a biomarker of stemness in head and neck squamous cell carcinoma

ObjectivesSignaling between cancer stem cells (CSC) and their extracellular matrix has a crucial role in CSC progression and maintenance. However, mediators of this signaling pathway in head and neck squamous cell carcinoma (HNSCC) are largely unknown. Here, we explored whether integrin β1, which is one of the key regulators of the communication between cells and their microenvironment, affected the stemness of HNSCC cells. Materials and methodsWe examined self-renewal capacity, chemoresistance, and xenograft tumorigenicity after knockdown of integrin β1 in primary HNSCC cells. In addition, we studied the role of focal adhesion kinase (FAK), an intracellular downstream molecule of integrin signaling, in influencing stemness of HNSCC. The relevance of Notch1 and integrin β1 interactions in HNSCC cells was also examined. Finally, immunohistochemical analysis was carried out to test whether the coexpression of integrin β1 and Notch1 in the samples from HNSCC patients correlated with their survival. ResultsTargeting integrin β1 in HNSCC cells inhibited self-renewal, chemoresistance, and in vivo tumor-forming capacity. Treatment with an inhibitor of FAK decreased self-renewal capacities and expression of various putative stem cell markers (Oct4, Sox2, and Nanog) in a dose-dependent manner. Moreover, knockdown of integrin β1 decreased the expression of Notch1 and its target genes (Hey1 and Hes1). Notably, HNSCC patients demonstrating simultaneous expression of integrin β1 and Notch1 in their tissue samples had significantly worse survival rate. ConclusionIntegrin β1/Notch1 axis has a significant role in the regulation of stemness in HNSCC.

Identification of Pyrvinium, an Anthelmintic Drug, as a Novel Anti-Adipogenic Compound Based on the Gene Expression Microarray and Connectivity Map.

Obesity is a serious health problem, while the current anti-obesity drugs are not very effective. The Connectivity Map (C-Map), an in-silico drug screening approach based on gene expression profiles, has recently been indicated as a promising strategy for drug repositioning. In this study, we performed mRNA expression profile analysis using microarray technology and identified 435 differentially expressed genes (DEG) during adipogenesis in both C3H10T1/2 and 3T3-L1 cells. Then, DEG signature was uploaded into C-Map, and using pattern-matching methods we discovered that pyrvinium, a classical anthelminthic, is a novel anti-adipogenic differentiation agent. Pyrvinium suppressed adipogenic differentiation in a dose-dependent manner, as evidenced by Oil Red O staining and the mRNA levels of adipogenic markers. Furthermore, we identified that the inhibitory effect of pyrvinium was resulted primarily from the early stage of adipogenesis. Molecular studies showed that pyrvinium downregulated the expression of key transcription factors C/EBPa and PPARγ. The mRNA levels of notch target genes Hes1 and Hey1 were obviously reduced after pyrvinium treatment. Taken together, this study identified many differentially expressed genes involved in adipogenesis and demonstrated for the first time that pyrvinium is a novel anti-adipogenic compound for obesity therapy. Meanwhile, we provided a new strategy to explore potential anti-obesity drugs.

The Notch system during pubertal development of the bovine mammary gland

The development of the mammary gland of cows during pre-weaning and puberty will condition its future productive capacity and warrants special study. In this respect, Notch signaling regulates tissue development and fate by modifying cell proliferation and differentiation and has been involved in stem cell maintenance, but has not been extensively studied in the developing mammary glands in cows. We therefore investigated Notch receptor expression and localization, as well as the expression of Notch ligands and target genes in the mammary gland of Holstein heifers in pre- and post-pubertal stages. Notch receptors 1 to 4 were detected by immunohistochemistry in the parenchyma and stroma of the developing gland. The subcellular localization of the four receptors was predominantly cytoplasmic except for NOTCH4, which was mostly nuclear. The membrane and the active intracellular domains of NOTCH paralogues were identified by western blot. NOTCH1 and NOTCH2 active domains increased during pubertal stages while NOTCH3 and NOTCH4 active domains decreased, suggesting strikingly different involvement of NOTCH paralogues in bovine mammary gland development and differentiation. The mRNA expression levels of the target genes HEY1 and HEY2 increased during peri-puberty whereas no variation of HES1 mRNA levels was observed. The mRNA levels of the Notch ligands JAGGED1 and DELTA1 also increased gradually during development. In conclusion, Notch signaling system dynamically varies throughout the development of the mammary gland during puberty pointing to specific time involvement of each component.