Target Gene Hes1 Research Articles

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

NOTCH2 promotes osteoclast maturation and metabolism and modulates the transcriptome profile during osteoclastogenesis

Notch signaling plays a key regulatory role in bone remodeling and NOTCH2 enhances osteoclastogenesis, an effect that is mostly mediated by its target gene Hes1. In the present study, we explored mechanisms responsible for the enhanced osteoclastogenesis in bone marrow-derived macrophages (BMM) from Notch2tm1.1Ecan, harboring a NOTCH2 gain-of-function mutation, and control mice. Notch2tm1.1Ecan mice are osteopenic and have enhanced osteoclastogenesis. Bulk RNA-Seq and gene set enrichment analysis of Notch2tm1.1Ecan BMMs cultured in the presence of macrophage colony stimulating factor (M-CSF) and receptor activator of NF-κB ligand revealed enrichment of genes associated with enhanced cell metabolism, aerobic respiration, and mitochondrial function, all associated with osteoclastogenesis. These pathways were not enhanced in the context of a Hes1 inactivation. Analysis of single cell RNA-Seq data of pooled control and Notch2tm1.1Ecan BMMs treated with M-CSF or M-CSF and receptor activator of NF-κB ligand for 3days identified 11 well-defined cellular clusters. Pseudotime trajectory analysis indicated a trajectory of clusters expressing genes associated with osteoclast progenitors, osteoclast precursors, and mature cells. There were an increased number of cells expressing gene markers associated with the osteoclast and with an unknown, albeit related, cluster in Notch2tm1.1Ecan than in control BMMs as well as enhanced expression of genes associated with osteoclast progenitors and precursors in Notch2tm1.1Ecan cells. In conclusion, BMM cultures display cellular heterogeneity, and NOTCH2 enhances osteoclastogenesis, increases mitochondrial and metabolic activity of osteoclasts, and affects cell cluster allocation in BMMs.

SCFAs switch stem cell fate through HDAC inhibition to improve barrier integrity in 3D intestinal organoids from patients with obesity

Stem cells are a keystone of intestinal homeostasis, but their function could be shifted during energy imbalance or by crosstalk with microbial metabolites in the stem cell niche. This study reports the effect of obesity and microbiota-derived short-chain fatty acids (SCFAs) on intestinal stem cell (ISC) fate in human crypt-derived intestinal organoids (enteroids). ISC fate decision was impaired in obesity, resulting in smaller enteroids with less outward protruding crypts. Our key finding is that SCFAs switch ISC commitment to the absorptive enterocytes, resulting in reduced intestinal permeability in obese enteroids. Mechanistically, SCFAs act as HDAC inhibitors in stem cells to enhance Notch signaling, resulting in transcriptional activation of the Notch target gene HES1 to promote enterocyte differentiation. In summary, targeted reprogramming of ISC fate, using HDAC inhibitors, may represent a potential, robust therapeutic strategy to improve gut integrity in obesity.

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.

EOGT enables residual Notch signaling in mouse intestinal cells lacking POFUT1

Notch signaling determines cell fates in mouse intestine. Notch receptors contain multiple epidermal growth factor-like (EGF) repeats modified by O-glycans that regulate Notch signaling. Conditional deletion of protein O-fucosyltransferase 1 (Pofut1) substantially reduces Notch signaling and markedly perturbs lineage development in mouse intestine. However, mice with inactivated Pofut1 are viable, whereas complete elimination of Notch signaling in intestine is lethal. Here we investigate whether residual Notch signaling enabled by EGF-domain-specific O-linked N-acetylglucosamine transferase (Eogt) permits mice conditionally lacking Pofut1 in intestine to survive. Mice globally lacking Eogt alone were grossly unaffected in intestinal development. In contrast, mice lacking both Eogt and Pofut1 died at ~ 28 days after birth with greater loss of body weight, a greater increase in the number of goblet and Paneth cells, and greater downregulation of the Notch target gene Hes1, compared to Pofut1 deletion alone. These data reveal that both O-fucose and O-GlcNAc glycans are fundamental to Notch signaling in the intestine and provide new insights into roles for O-glycans in regulating Notch ligand binding. Finally, EOGT and O-GlcNAc glycans provide residual Notch signaling and support viability in mice lacking Pofut1 in the intestine.

PLS3 promotes papillary thyroid carcinoma progression by activating the Notch signaling pathway.

Thyroid cancer is the most common endocrine malignancy worldwide. Although significant progress has been made in understanding the genetic and molecular alterations that drive thyroid cancer, the mechanisms underlying thyroid tumor progression remain unclear. In this study, we explored the involvement of Plastin-3 (PLS3) in the progression of papillary thyroid cancer and elucidated the underlying molecular mechanisms. We first analyzed clinical samples from papillary thyroid cancer patients and found that PLS3 expression was significantly upregulated in tumor tissues compared to adjacent normal tissues. Moreover, high PLS3 expression was associated with advanced tumor stage and poor prognosis. Further in vitro and in vivo experiments showed that PLS3 could promote the proliferation, migration, and invasive behavior of papillary thyroid cancer cells, while PLS3 knockdown suppressed these processes. Mechanistically, we found that PLS3 promoted papillary thyroid cancer progression by activating the Notch signaling pathway. Specifically, PLS3 upregulated the expression of Notch receptors (Notch1) and downstream target gene (Hes1) in papillary thyroid cancer cells. In summary, our findings collectively indicate that PLS3 plays a pivotal role in driving the progression of papillary thyroid cancer and holds promise as a viable therapeutic target for the treatment of this disease.

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.

NDRG2 regulates the formation of reactive astrocyte-derived progenitor cells via Notch signaling pathway after brain traumatic injury in rats.

In response to traumatic brain injury, a subpopulation of cortical astrocytes is activated, resulting in acquisition of stem cell properties, known as reactive astrocytes-derived progenitor cells (Rad-PCs). However, the underlying mechanisms remain largely unknown during this process. In this study, we examined the role of N-myc downstream-regulated gene 2 (NDRG2), a differentiation- and stress-associated molecule, in Rad-PCs after cortical stab injury in adult rats. Immunohistochemical analysis showed that in the cerebral cortex of normal adult rats, NDRG2 was exclusively expressed in astrocytes. After liu cortical injury, the expression of NDRG2 was significantly elevated around the wound and most cells expressing NDRG2 also expressed GFAP, a reactive astrocyte marker. Importantly, NDRG2-expressing cells were co-labeled with Nestin, a marker for neural stem cells, some of which also expressed cell proliferation marker Ki67. Overexpression of NDRG2 further increased the number of NDRG2/Nestin double-labeling cells around the lesion. In contrast, shRNA knockdown of NDRG2 decreased the number of NDRG2+/Nestin+ cells. Intracerebroventricular administration of stab-injured rats with a Notch antagonist, DAPT, led to a significant decrease in Nestin+/NDRG2+ cells around the injured boundary, but did not affect NDRG2+ cells. Moreover, overexpression or knockdown of NDRG2 led to up- and down-regulation of the expression of Notch intracellular domain NICD and Notch target gene Hes1, respectively. Taken together, these results suggest that NDRG2 may play a role in controlling the formation of Rad-PCs in the cerebral cortex of adult rats following traumatic injury, and that Notch signaling pathway plays a key role in this process.

The OTUD1-Notch2-ICD Axis Orchestrates Allogeneic T Cell-Mediated Graft-Versus-Host Disease

Disorders of the ubiquitin-proteasome system (UPS) are known to influence the incidence and mortality of various diseases. It remains largely unknown whether and how the UPS affects the onset and progression of acute graft-versus host disease (aGVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). The present study demonstrated that deubiquitinase OTUD1 is an essential regulator of aGVHD. OTUD1 expression level predicts the cumulative incidence and severity of aGVHD patients after allo-HSCT. Donor OTUD1 deficiency markedly prolonged the survival of aGVHD mice, reduced aGVHD scores, ameliorated pathological damage of target organs through inhibiting the differentiation and functions of Th1 and Th17 cells. Activation of CD4+ T cells after allo-HSCT elevated the expression of OTUD1, which in turn interacted with the Notch2-ICD (NICD) protein to cleave the ubiquitin of NICD at the K1770 site, thereby inducing the accumulation of the NICD proteins in T cells. OTUD1-driven NICD signaling promoted the differentiation and functions of Th1 and Th17 cells and amplified the cascade of aGVHD. Additionally, the expression level of OTUD1 is positively correlated with Notch2 target genes (Hes1, Hes5, Hey1, HeyL) in aGVHD patients. Moreover, screening performed in the present study identified dapagliflozin as an inhibitor targeting the OTUD1/NICD axis. Dapagliflozin administration significantly prolonged the survival of aGVHD mice. Taken together, the current work not only characterized a previously undiscovered role of OTUD1 in T cell-mediated allogeneic responses, and identified OTUD1 as a novel deubiquitinase of Notch2, but also revealed a promising therapeutic strategy to target OTUD1 for the alleviation of aGVHD.

Injectable bone marrow microniches by co-culture of HSPCs with MSCs in 3D microscaffolds promote hematopoietic reconstitution from acute lethal radiation.

Hematopoietic syndrome of acute radiation syndrome (h-ARS) is an acute illness resulted from the damage of bone marrow (BM) microenvironment after exposure to radiation. Currently, the clinical management of h-ARS is limited to medication-assisted treatment, while there is still no specific therapy for the hematopoietic injury from high-dose radiation exposure. Our study aimed to assemble biomimetic three-dimensional (3D) BM microniches by co-culture of hematopoietic stem and progenitor cells (HSPCs) and mesenchymal stem cells (MSCs) in porous, injectable and viscoelastic microscaffolds in vitro. The biodegradable BM microniches were then transplanted in vivo into the BM cavity for the treatment of h-ARS. We demonstrated that the maintenance of HSPCs was prolonged by co-culture with MSCs in the porous 3D microscaffolds with 84 μm in pore diameter and 11.2 kPa in Young's modulus compared with 2D co-culture system. Besides, the minimal effective dose and therapeutic effects of the BM microniches were investigated on a murine model of h-ARS, which showed that the intramedullary cavity-injected BM microniches could adequately promote hematopoietic reconstitution and mitigate death from acute lethal radiation with a dose as low as 1000 HSPCs. Furthermore, the mRNA expression of Notch1 and its downstream target gene Hes1 of HSPCs were increased when co-cultured with MSCs, while the Jagged1 expression of the co-cultured MSCs was upregulated, indicating the significance of Notch signaling pathway in maintenance of HSPCs. Collectively, our findings provide evidence that biomimetic and injectable 3D BM microniches could maintain HSPCs, promote hematopoiesis regeneration and alleviate post-radiation injury, which provides a promising approach to renovate conventional HSPCs transplantation for clinical treatment of blood and immune disorders.

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.

Phase 1B/2A safety, pharmacokinetics, and pharmacodynamics study of fosciclopirox alone and in combination with cytarabine in patients with relapsed/refractory acute myeloid leukemia.

TPS7069 Background: Fosciclopirox (F) is a γ-secretase inhibitor being developed for the treatment of acute myeloid leukemia (AML). Following intravenous (IV) administration, F is rapidly and completely metabolized to its active metabolite, ciclopirox (CPX). CPX binds to γ-secretase complex proteins Presenilin 1 and Nicastrin, which are essential for Notch activation. In HL60 cells, CPX inhibits Notch 1 and Notch 2 expression, reduces levels of γ-secretase complex proteins Presenilin 1 and Nicastrin, and decreases expression of the downstream Notch target gene Hes-1. Utilizing Notable Labs predictive precision medicine platform, bone marrow (BM) and peripheral blood (PB) samples obtained from 10 AML patients treated with CPX demonstrated significant blast count reductions. Methods: Study CPX-POM-003 (NCT04956042) is an open-label Phase 1B/2A, trial designed to characterize the efficacy, safety, and PK/PD of F alone and in combination with cytarabine (ara-C) in patients with relapsed/refractory AML (R/R AML). Eligible patients must be 18 years of age or older with relapsed AML after complete remission or with primary refractory AML refractory to at least two cycles of induction therapy. There will be up to three cohorts of patients, approximately 42 R/R AML patients, evaluated. If disease response to F alone (Cohort 1a) is observed in at least 4 of 14 patients, an additional 14 patients will be enrolled in Cohort 1b. If disease response is not observed following F alone, the study may be terminated or a second cohort, Cohort 2a, may be initiated to evaluate the combination of F + ara-C. If disease response to F + ara-C is observed in at least 4 of 14 patients, an additional 14 patients will be enrolled in Cohort 2b. If response to F + ara-C is not observed in at least 4 of 14 patients, the study will be stopped for futility. F is being administered as 900 mg/m2 once daily as a 20-minute IV infusion on Days 1 to 5 of each 21-day treatment cycle. Ara-C is administered as 1 gm/m2 once daily on Days 1 to 5 of each cycle. BM and PB samples are collected prior to and during Cycles 1 (C1) and 2 (C2) for disease response assessment and blast count determination. Additional BM and PB samples are obtained after every two cycles beyond C2 for patients continuing treatment. Disease response is determined based on Döhner et al, Blood 2017;129(4)424-447. Next Generation Sequencing (NGS) profiles will be determined prior to and at the end of C1, and thereafter as clinically indicated. Immunohistochemistry will be performed on BM samples to elucidate drug mechanism. Ex vivo Drug Sensitivity Screening (DSS) will be performed on BM and PB samples obtained prior to treatment as well as on C1 Days 8 and 21. The steady-state plasma pharmacokinetics of F are being characterized during C1. Enrollment began in October 2022 with four patients enrolled to date. Clinical trial information: NCT04956042.

LY450139 Inhibited Ti-Particle-Induced Bone Dissolution via Suppressing Notch and NF-κB Signaling Pathways

Aseptic loosening of the prosthesis caused by wear-particle-induced osteolysis is a long-term complication and one of the most common reasons for the failure of joint implants. The primary cause of aseptic loosening of the prosthesis is overactive bone resorption caused by wear-particle-activated osteoclasts in both direct and indirect ways. Therefore, drugs that can inhibit differentiation and bone resorption of osteoclasts need investigation as a potential therapeutic strategy to prevent and treat peri-prosthetic osteolysis and thereby prolong the service life of the prosthesis. This study has verified the potential inhibitory effect of LY450139 on inflammatory osteolysis induced by titanium particles in a mice skull model. In addition, we found that LY450139 inhibited receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis, bone resorption, and podosomal actin belt formation in a dose-dependent manner without evidence of cytotoxicity in vitro. In addition, LY450139 significantly decreased the expression of osteoclast-specific markers, including TRAP, CTSK, V-ATPase d2, CTR, DC-STAMP, NFATc1, and the downstream target gene Hes1 in Notch signaling pathway. Further investigation of the molecular mechanism demonstrated that LY450139 inhibited the formation of osteoclasts via inhibition of the NF-κB and Notch signaling pathways. In summary, LY450139 inhibited the formation of RANKL-mediated osteoclasts via NF-κB and Notch signaling and inhibited Ti particle-induced inflammatory osteolysis in vivo. LY450139 is a potential targeted drug for the treatment of peri-prosthetic osteolysis and other osteolytic disease associated with overactive osteoclasts.

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.

Cross-talk between NOTCH2 and BMP4/SMAD signaling pathways in bovine follicular granulosa cells

NOTCH and bone morphogenetic protein (BMP)/SMAD signaling play key regulatory roles in mammalian ovarian development. The study aimed to investigate interregulatory mechanisms between NOTCH2 and BMP4/SMAD signaling pathways in bovine follicular granulosa cells (GCs). The results showed that NOTCH2 silence reduced the mRNA expression of SMAD1, SMAD5, SMAD8 (also known as SMAD9) and Mg2+/Mn2+- dependent Protein Phosphatase 1A (PPM1A), which are effectors of BMP/SMAD signaling pathway (P < 0.01). Overexpressing NOTCH2 intracellular sequence increased the mRNA expression of BMPR1A, SMAD1, SMAD4, SMAD5, SMAD8 and PPM1A (P < 0.01). Meanwhile, treating GCs with BMP4 inhibited the mRNA expression of its downstream gene SMAD1 and steroidogenesis genes STAR and CYP11A1 in the presence of follicular stimulating hormone (FSH) (P < 0.01). Moreover, BMP4 inhibited the mRNA expression of NOTCH signaling pathway target gene HES1 (P < 0.05), while the increase in NOTCH2 may be due to negative feedback of HES1. By and large, these results indicated that NOTCH2 up-regulated key genes of BMP/SMAD signaling in bovine follicle GCs, while BMP4 inhibited its downstream signaling factors and NOTCH signaling pathway target gene HES1. This study suggests there are complex synergistic and antagonistic effects between the two signaling pathways, which jointly participate in regulating bovine follicular development through regulating follicular GCs.

Notch1 signaling contributes to TLR4-triggered NF-κB activation in macrophages

Macrophages substantially influence the development, progression, and complications of inflammation-driven diseases. Although numerous studies support the critical role of Notch signaling in most inflammatory diseases, there is limited data on the role of Notch signaling in TLR4-induced macrophage activation and interaction of Notch signaling with other signaling pathways in macrophages during inflammation, such as the NF-κB pathway. This study confirmed that stimulation with lipopolysaccharide (LPS), a TLR4 ligand, upregulated Notch1 expression in monocyte/macrophage-like RAW264.7 cells and bone marrow-derived macrophages (BMDMs). LPS also induced increased mRNA expression of Notch target genes Notch1 and Hes1 in macrophages, suggesting that TLR4 signaling enhances activation of the Notch pathway. The upregulation of Notch1, Notch1 intracellular domain (NICD), and Hes1 proteins by LPS treatment was inhibited by DAPT, a Notch1 inhibitor. Additionally, the increased TNF-α, IL-6, and IL-1β expression induced by LPS was inhibited by DAPT and rescued by jagged1, a Notch1 ligand. Furthermore, suppression of Notch signaling by DAPT upregulated Cylindromatosis (CYLD) expression but downregulated TRAF6 expression, IκB kinase (IKK) α/β phosphorylation, and subsequently, phosphorylation and degradation of IκB-α, indicating that DAPT inhibited NF-κB activation triggered by TLR-4. Interestingly, DAPT did not inhibit the increased MyD88 expression induced by LPS. Our study findings demonstrate that macrophage stimulation via the TLR4 signaling cascade triggers activation of Notch1 signaling, which regulates the expression patterns of genes involved in pro-inflammatory responses by activating NF-κB. This effect may be dependent on the CYLD-TRAF6-IKK pathway. Thus, Notch1 signaling may provide a therapeutic target against infectious and inflammation-driven diseases.

Regulatory Crosstalk between Physiological Low O2 Concentration and Notch Pathway in Early Erythropoiesis.

Physiological low oxygen (O2) concentration (<5%) favors erythroid development ex vivo. It is known that low O2 concentration, via the stabilization of hypoxia-induced transcription factors (HIFs), intervenes with Notch signaling in the control of cell fate. In addition, Notch activation is implicated in the regulation of erythroid differentiation. We test here if the favorable effects of a physiological O2 concentration (3%) on the amplification of erythroid progenitors implies a cooperation between HIFs and the Notch pathway. To this end, we utilized a model of early erythropoiesis ex vivo generated from cord blood CD34+ cells transduced with shHIF1α and shHIF2α at 3% O2 and 20% O2 in the presence or absence of the Notch pathway inhibitor. We observed that Notch signalization was activated by Notch2R–Jagged1 ligand interaction among progenitors. The inhibition of the Notch pathway provoked a modest reduction in erythroid cell expansion and promoted erythroid differentiation. ShHIF1α and particularly shHIF2α strongly impaired erythroid progenitors’ amplification and differentiation. Additionally, HIF/NOTCH signaling intersects at the level of multipotent progenitor erythroid commitment and amplification of BFU-E. In that, both HIFs contribute to the expression of Notch2R and Notch target gene HES1. Our study shows that HIF, particularly HIF2, has a determining role in the early erythroid development program, which includes Notch signaling.

Asymmetric Notch activity by differential inheritance of lysosomes in human neural stem cells.

Symmetric and asymmetric cell divisions are conserved strategies for stem cell expansion and the generation of more committed progeny, respectively. Here, we demonstrate that in human neural stem cells (NSCs), lysosomes are asymmetrically inherited during mitosis. We show that lysosomes contain Notch receptors and that Notch activation occurs the acidic lysosome environment. The lysosome asymmetry correlates with the expression of the Notch target gene HES1 and the activity of Notch signaling in the daughter cells. Furthermore, an asymmetry of lysosomes and Notch receptors was also observed in a human organoid model of brain development with mitotic figures showing preferential inheritance of lysosomes and Notch receptor in that daughter cell remaining attached to the apical membrane. Thus, this study suggests a previously unknown function of lysosomes as a signaling hub to establish a bias in Notch signaling activity between daughter cells after an asymmetric cell division of human NSCs.

Effect of Nano Herbal Andaliman (Zanthoxylum acanthopodium) Fruits in NOTCH1 and Hes1 Expressions to Human Placental Trophoblasts.

Andaliman fruit (Zanthoxylum acanthopodium) is a well-known spice antioxidant in Northern Sumatera (Indonesia). The cellular activity requires antioxidants in counteracting free radicals. The cellular proteins that play a role in development, proliferation, differentiation and embryonic processes in the human placenta are NOTCH1 and Hes1. The aim of this research was to analyze the expression of NOTCH1 and Hes1 genes after administering nano herbal andaliman to the trophoblast cells of the human placenta. HTR8 trophoblast cells were divided into two groups, namely, control and treatment (nano herbal andaliman). RNA isolation, reverse transcription and RT-PCR (real-time polymerase chain reaction) were performed to analyze the NOTCH1 and Notch target gene (Hes1) expressions. The NOTCH1 and Hes1 gene expressions were quantified using the CT method (2-ΔΔCT) and normalized with Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene expressions. Nanoherbal andaliman reduced the expression of NOTCH1 genes in the human placental trophoblast. However, it increased the expression of Hes1 when the incubation time was 16 hrs. Nanoherbal andaliman decreases the expression of genes that are crucial in hypoxia and free radicals in the placenta, namely, NOTCH1 and Hes1 increased after incubation for 16 hrs. Therefore, this herb needs to be evaluated further.