Notch Signaling Research Articles

Insulin receptor responsiveness governs TGFβ-induced hepatic stellate cell activation: Insulin resistance instigates liver fibrosis.

The insulin receptor (INSR) has been shown to be hyperactive in hepatic stellate cells (HSCs) in humans and rodents with liver fibrosis. To explore HSC cellular mechanisms that INSR regulates during pro-fibrotic stimulation, we used CRISPR-Cas9 technology. We knocked out a portion of the INSR gene in human LX2 HSC cells (INSRe5-8 KO) that regulates insulin responsiveness but not the insulin-like growth factor (IGF) or transforming growth factor-β (TGFβ) signaling. The INSRe5-8 KO HSCs had significantly higher cell growth, BrdU incorporation, and lower TP53 expression that suppresses growth, and they also exhibited increased migration compared to the Scramble control. We treated the scramble control and INSRe5-8 KO HSCs with insulin or TGFβ and profiled hundreds of kinase activities using the PamGene PamStation kinome technology. Our analysis showed that serine/threonine kinase (STK) activities were reduced, and most of the protein-tyrosine kinase (PTK) activities were increased in the INSRe5-8 KO compared to the Scramble control HSCs. To study gene transcripts altered in activated Scramble control and INSRe5-8 KO HSCs, we treated them with TGFβ for 24 h. We isolated RNA for sequencing and found that the INSRe5-8 KO cells, compared to control HSCs, had altered transcriptional responsiveness to TGFβ stimulation, collagen-activated signaling, smooth muscle cell differentiation pathways, SMAD protein signaling, collagen metabolic process, integrin-mediated cell adhesion, and notch signaling. This study demonstrates that reduced INSR responsiveness enhances HSC growth and selectively mediates TGFβ-induced HSC activation. These findings provide new insights into the development of more effective treatments for liver fibrosis.

Perfluorobutanesulfonate Interfering with the Intestinal Remodeling During Lithobates catesbeiana Metamorphosis via the Hypothalamic-Pituitary-Thyroid Axis.

The intestinal remodeling during amphibian metamorphosis is essential for adapting to various ecological niches of aquatic and terrestrial habitats. However, whether and how the widespread contaminant, perfluorobutanesulfonate (PFBS) affects intestinal remodeling remains unknown. In this study, tadpoles (Lithobates catesbeianus) at the G26 stage were exposed to environmentally relevant concentrations of PFBS (0, 1, 3, and 10 μg/L) until the end of metamorphosis. PFBS exposure resulted in reduced thyroid follicular glia; down-regulation of gene transcripts related to thyroid hormone synthesis; decreased blood hormone (corticotropin-releasing hormone, thyroid-stimulating hormone, and 3,5,3'-triiodothyronine (T3)) and transthyretin concentrations; and up-regulation of gene transcripts related to thyroid hormone degrading enzymes. Moreover, exposure to PFBS induced apoptosis in single-layer columnar epithelial cells, suppressed the proliferation of intestinal stem cells, and hindered their differentiation into adult epithelial cells during intestinal remodeling. The responses of Notch and Wnt signaling pathways regulated by T3 were downregulated, and key gene transcripts (msi, pcna, and lgr5) involved in intestinal remodeling regulated by these two pathways were also downregulated. This is the first report on the effects of PFBS on amphibian metamorphosis. Overall, PFBS reduced thyroid hormone synthesis and transport by interfering with the hypothalamic-pituitary-thyroid axis and transthyretin expression, inhibited downstream Notch and Wnt signaling pathway responses, and ultimately led to incomplete intestinal remodeling to some extent.

Single-Chain Nanobody Inhibition of Notch and Avidity Enhancement Utilizing the β-Pore-Forming Toxin Aerolysin.

Notch plays critical roles in developmental processes and disease pathogenesis, which have led to numerous efforts to modulate its function with small molecules and antibodies. Here we present a nanobody inhibitor of Notch signaling derived from a synthetic phage-display library targeting the Notch negative regulatory region (NRR). The nanobody inhibits Notch signaling in a luciferase reporter assay with an IC50 of about 5 μM and in a Notch-dependent hematopoietic progenitor cell differentiation assay, despite a modest 19 μM affinity for the Notch NRR. We addressed the low affinity by fusion to a mutant varient of the β-pore-forming toxin aerolysin, resulting in a significantly improved IC50 for Notch inhibition. The nanobody-aerolysin fusion inhibits proliferation of T-ALL cell lines with efficacy similar to that of other Notch pathway inhibitors. Overall, this study reports the development of a Notch inhibitory antibody and demonstrates a proof-of-concept for a generalizable strategy to increase the efficacy and potency of low-affinity antibody binders.

Comprehensive Bioinformatics Analysis Reveals Molecular Signatures and Potential Caloric Restriction Mimetics with Neuroprotective Effects: Validation in an In Vitro Stroke Model.

Caloric restriction (CR) is a dietary intervention that reduces calorie intake without inducing malnutrition, demonstrating lifespan-extending effects in preclinical studies and some human trials, along with potential benefits in ameliorating age-related ailments. Caloric restriction mimetics (CRMs) are compounds mimicking CR effects, offering a potential therapeutic avenue for age-related diseases. This study explores the potential protective effects of CR on the brain neocortex (GSE11291) and the identification of CRMs using integrative bioinformatics and systems biology approaches. Our findings indicate that long-term CR activates cellular pathways improving mitochondrial function, enhancing antioxidant capacity, and reducing inflammation, potentially providing neuroprotection. The key signaling pathways enriched in our study include PPAR, mTOR, FoxO, AMPK, and Notch signaling pathways, which are crucial regulators of metabolism, cellular stress response, neuroprotection, and longevity. We identify key signaling molecules and molecular mechanisms associated with CR, including transcription factors, kinase regulators, and microRNAs linked to differentially expressed genes. Furthermore, potential CRMs such as rapamycin, replicating CR-related health benefits, are identified. Additionally, machine learning models were developed to classify small molecules based on their CNS activity and anti-inflammatory properties. As a proof of concept, we have demonstrated the ischemic neuroprotective effects of two top-ranked candidate reference molecules (CRMs) using the oxygen-glucose deprivation (OGD) model, an established in vitro stroke model. However, further investigations are essential to fully elucidate the therapeutic potential of these CRMs. In summary, our study suggests that long-term CR entails protective mechanisms preserving and safeguarding neuronal function, potentially impacting the treatment of age-related neurological diseases. Moreover, our findings contribute to the identification of potential genes and regulatory molecules involved in CR, along with potential CRMs, providing a promising foundation for future research in the field of neurological disorder treatment.

MiR-34a-5p modulation of polycystic ovary syndrome via targeting the NOTCH signaling pathway

PurposePolycystic ovary syndrome (PCOS) is currently recognized as a condition that affects several systems in the body, including the reproductive, endocrine, and cardiovascular systems. Prevalent among teenagers and women of reproductive age. Prior research has demonstrated an elevation of miR-34a-5p within the follicular fluid (FF) of women of PCOS. Despite this, the precise mechanisms through which miR-34a-5p influences granulosa cells (GC) development and function remain poorly characterized.MethodsTherefore, this study investigates the involvement and pathogenic mechanisms of miR-34a-5p within GCs in the context of PCOS. The human granulosa-like tumor cell line (KGN) got transfected at a control, as well as a miR-34a-5p mimic and inhibitor, respectively. Monitor cellular proliferation in each experimental group. The experimental methods included RT-qPCR, CCK8, flow cytometry and western blotting. Also, the interaction between miR-34a-5p and the particular sequence of JAG1 has been verified using the dual luciferase assay. Further investigation of the connection involving miR-34a-5p and the Notch signaling pathway was conducted using bioinformatics analysis and experimental methods.ResultsThe results demonstrated that miR-34a-5p expression was significantly elevated in the serum(p<0. 0001)and FF (p = 0. 0402) of PCOS, whereas its expression in GCs (p = 0. 5522) showed no significant variation. Overexpressing miR-34a-5p caused a decrease in the rate at which KGN cells multiplied and an increase in programmed cell death. Conversely, inhibiting miR-34a-5p resulted in an increase in cell growth and a decrease in programmed cell death. Bioinformatics analysis and experimental results further demonstrated thatmiR-34a-5p interacts with the 3’UTR region of JAG1, leading to a negative regulation of the Jagged1-Notch signaling pathway.ConclusionIn summary, the miR-34a-5p molecule inhibits the growth of GCs as well as triggers programmed cell death by regulating the Jagged1-Notch signaling pathway. Silencing miR-34a-5p prevents dysfunction in GCs. Our analysis implies that miR-34a-5p is a new molecular site to treat PCOS.

DeltaC and DeltaD ligands play different roles in the segmentation clock dynamics

The vertebrate segmentation clock drives periodic somite segmentation during embryonic development. Her1 and Her7 clock proteins generate oscillatory expression of their own genes as well as that of deltaC in zebrafish. In turn, DeltaC and DeltaD ligands activate Notch signaling, which then activates transcription of clock genes in neighboring cells. While DeltaC and DeltaD proteins form homo- and heterodimers, only DeltaC-containing oscillatory dimers were expected to be functional. To investigate the contributions of DeltaC and DeltaD proteins on the transcription of her1 and her7 segmentation clock genes, we counted their transcripts by performing single molecule fluorescent in situ hybridization imaging in different genetic backgrounds of zebrafish embryos. Surprisingly, we found that DeltaD homodimers are also functional. We further found that Notch signaling promotes transcription of both deltaC and deltaD genes, thereby creating a previously unnoticed positive feedback loop. Our computational model highlighted the intriguing differential roles of DeltaC and DeltaD dimers on the clock synchronization and transcript numbers, respectively. We anticipate that a mechanistic understanding of the Notch signaling pathway will not only shed light on the mechanism driving robust somite segmentation but also inspire similar quantitative studies in other tissues and organs.

MpECs with high Piezo2 expression promote fracture healing by driving angiogenesis through the Notch signaling pathway

Fractures will impair or disrupt angiogenesis, resulting in delayed union or non-union. Exploring angiogenic signaling molecules and related pathways can promote fracture healing. In this study, the roles of different endothelial cell (EC) subsets in fracture healing were observed using single-cell RNA sequencing (scRNA-seq). It was found that mpECs did affect the repair and regeneration of fracture sites, and could up-regulate genes related to the Notch signaling, angiogenesis, and cell cycle. In addition, in this study, Piezo2 expression was successfully knocked down by transfection of shRNA in human umbilical vein endothelial cells (HUVECs) for in vitro assays. The results suggested that the reduced expression of Piezo2 in HUVECs can suppress cell proliferation and cell cycle and further impair the activation of the Notch signaling pathway, inhibiting angiogenesis. Subsequently, HUVECs were intervened with the Notch pathway inhibitor DAPT and agonist Jagged1. It was found that inhibition of the Notch signaling pathway by Piezo2 knockdown was more significant in the presence of DAPT, whereas Jagged1 reversed the Piezo2 knockdown-caused changes in the downstream protein expression of the Notch pathway. With Jagged1, Piezo2 knockdown-induced decrease in HUVEC tube formation disappeared. Moreover, the tube formation was significantly enhanced, with a marked increase in tube length. Cell counting kit-8 (CCK-8) assay and flow cytometry demonstrated that Jagged1 can promote cell proliferation and trigger cell cycle entry. In conclusion, Piezo2 affects the phenotype of ECs by modulating the Notch signaling pathway and further promotes angiogenesis, thus accelerating fracture healing.

Modes of Notch signalling in development and disease

Modes of Notch signalling in development and disease

The Notch ligand Jagged1 plays a dual role in cochlear hair cell regeneration.

Hair cells (HCs) within the inner ear cochlea are specialized mechanoreceptors required for hearing. Cochlear HCs are not regenerated in mammals, and their loss is a leading cause of deafness in humans. Cochlear supporting cells (SCs) in newborn mice have the capacity to regenerate HCs, but persistent Notch signaling, presumably activated by SC-specific Notch ligand Jagged1 (JAG1), prevents SCs from converting into HCs. Here, employing an organoid platform, we show that while JAG1 participates in HC-fate repression, JAG1's primary function is to preserve the "progenitor-like characteristics" of cochlear SCs. Transcriptomic and mechanistic studies reveal that JAG1/Notch signaling maintains the expression of progenitor and metabolic genes in cochlear SCs and sustains pro-growth pathways, including PI3K-Akt-mTOR signaling, a function that is mediated by Notch1 and Notch2. Finally, we show that JAG1/Notch signaling stimulation with JAG1-Fc peptide enhances the HC-forming capacity of cochlear SCs undergoing maturation in cochlear explants and in vivo .

CD146 regulates the stemness and chemoresistance of hepatocellular carcinoma via JAG2-NOTCH signaling

CD146 plays a key role in cancer progression and metastasis. Cancer stem cells (CSCs) are responsible for tumor initiation, drug resistance, metastasis, and recurrence. In this study, we explored the role of CD146 in the regulation of liver CSCs. Here, we demonstrated that CD146 was highly expressed in liver CSCs. CD146 overexpression promoted the self-renewal ability and chemoresistance of Hepatocellular Carcinoma (HCC) cells in vitro and tumorigenicity in vivo. Inversely, knockdown of CD146 restrained these abilities. Mechanistically, CD146 activated the NF-κB signaling to up-regulate JAG2 expression and activated the Notch signaling, which resulted in increased stemness of HCC. Furthermore, JAG2 overexpression restored the Notch signaling activity, the stemness, and chemotherapeutic resistance caused by CD146 knockdown. These results demonstrated that CD146 positively regulates HCC stemness by activating the JAG2-NOTCH signaling. Combined targeting of CD146 and JAG2 may represent a novel therapeutic strategy for HCC treatment.

The role of senescence-related genes in major depressive disorder: insights from machine learning and single cell analysis

BackgroundEvidence indicates that patients with Major Depressive Disorder (MDD) exhibit a senescence phenotype or an increased susceptibility to premature senescence. However, the relationship between senescence-related genes (SRGs) and MDD remains underexplored.MethodsWe analyzed 144 MDD samples and 72 healthy controls from the GEO database to compare SRGs expression. Using Random Forest (RF) and Support Vector Machine-Recursive Feature Elimination (SVM-RFE), we identified five hub SRGs to construct a logistic regression model. Consensus cluster analysis, based on SRGs expression patterns, identified subclusters of MDD patients. Weighted Gene Co-expression Network Analysis (WGCNA) identified gene modules strongly linked to each cluster. Single-cell RNA sequencing was used to analyze MDD SRGs functions.ResultsThe five hub SRGs: ALOX15B, TNFSF13, MARCH 15, UBTD1, and MAPK14 showed differential expression between MDD patients and controls. Diagnostics models based on these hub genes demonstrated high accuracy. The hub SRGs correlated positively with neutrophils and negatively with T lymphocytes. SRGs expression pattern revealed two distinct MDD subclusters. WGCNA identified significant gene modules within these subclusters. Additionally, individual endothelial cells with high senescence scores were found to interact with astrocytes via the Notch signaling pathway, suggesting a specific role in MDD pathogenesis.ConclusionThis comprehensive study elucidates the significant role of SRGs in MDD, highlighting the importance of the Notch signaling pathway in mediating senescence effects.

Developing zebrafish models of Notch-related CNS pathologies.

Developing zebrafish models of Notch-related CNS pathologies.

Novel determinants of NOTCH1 trafficking and signaling in breast epithelial cells.

The evolutionarily conserved Notch signaling pathway controls cell-cell communication, enacting cell fate decisions during development and tissue homeostasis. Its dysregulation is associated with a wide range of diseases, including congenital disorders and cancers. Signaling outputs depend on maturation of Notch receptors and trafficking to the plasma membrane, endocytic uptake and sorting, lysosomal and proteasomal degradation, and ligand-dependent and independent proteolytic cleavages. We devised assays to follow quantitatively the trafficking and signaling of endogenous human NOTCH1 receptor in breast epithelial cells in culture. Based on such analyses, we executed a high-content screen of 2,749 human genes to identify new regulators of Notch that might be amenable to pharmacologic intervention. We uncovered 39 new NOTCH1 modulators for NOTCH1 trafficking and signaling. Among them, we find that PTPN23 and HCN2 act as positive NOTCH1 regulators by promoting endocytic trafficking and NOTCH1 maturation in the Golgi apparatus, respectively, whereas SGK3 serves as a negative regulator that can be modulated by pharmacologic inhibition. Our findings might be relevant in the search of new strategies to counteract pathologic Notch signaling.

Decoding a Cell's Fate: How Notch and receptor tyrosine kinase signals specify the Drosophila R7 photoreceptor.

Decoding a Cell's Fate: How Notch and receptor tyrosine kinase signals specify the Drosophila R7 photoreceptor.

Segetalin B promotes bone formation in ovariectomized mice by activating PLD1/SIRT1 signaling to inhibit γ-secretase-mediated Notch1 overactivation.

Segetalin B promotes bone formation in ovariectomized mice by activating PLD1/SIRT1 signaling to inhibit γ-secretase-mediated Notch1 overactivation.

Cannabidiol alleviates LPS-inhibited odonto/osteogenic differentiation in human dental pulp stem cells invitro.

Cannabidiol (CBD), derived from the Cannabis sativa plant, exhibits benefits in potentially alleviating a number of oral and dental pathoses, including pulpitis and periodontal diseases. This study aimed to explore the impact of CBD on several traits of human dental pulp stem cells (hDPSC), such as their proliferation, apoptosis, migration and odonto/osteogenic differentiation. hDPSCs were harvested from human dental pulp tissues. The cells were treated with CBD at concentrations of 1.25, 2.5, 5, 10, 25 and 50 μg/mL. Cell responses in terms of cell proliferation, colony-forming unit, cell cycle progression, cell migration, apoptosis and odonto/osteogenic differentiation of hDPSCs were assessed in the normal culture condition and P. gingivalis lipopolysaccharide (LPS)-induced 'inflammatory' milieus. RNA sequencing and proteomic analysis were performed to predict target pathways impacted by CBD. CBD minimally affects hDPSCs' behaviour under normal culture growth milieu in normal conditions. However, an optimal concentration of 1.25 μg/mL CBD significantly countered the harmful effects of LPS, indicated by the promoting cell proliferation and restoring the odonto/osteogenic differentiation potential of hDPSCs under LPS-treated conditions. The proteomic analysis demonstrated that several proteins involved in cell proliferation and differentiation were upregulated following CBD exposure, including CCL8, CDC42 and KFL5. RNA sequencing data indicated that CBD upregulated the Notch signalling pathway. In an inhibitory experiment, DAPT, a Notch inhibitor, reduced the effect of CBD-rescued LPS-attenuated mineralization in hDPSCs, suggesting that CBD potentially mediates Notch activation to exert its impact on odonto/osteogenic differentiation of hDPSCs. CBD recovers the proliferation and survival of hDPSCs following exposure to LPS. Additionally, we report that CBD-mediated Notch activation effectively restores the odonto/osteogenic differentiation ability of hDPSCs under inflamed conditions. These results underscore the potential role of CBD as a therapeutic option to enhance dentine regeneration.

Buyang Huanwu decoction promotes gray and white matter remyelination by inhibiting Notch signaling activation in the astrocyte and microglia after ischemic stroke.

Buyang Huanwu decoction promotes gray and white matter remyelination by inhibiting Notch signaling activation in the astrocyte and microglia after ischemic stroke.

NOTCH1, 2, and 3 receptors enhance osteoblastogenesis of mesenchymal C3H10T1/2 cells and inhibit this process in preosteoblastic MC3T3-E1 cells.

NOTCH1, 2, and 3 receptors enhance osteoblastogenesis of mesenchymal C3H10T1/2 cells and inhibit this process in preosteoblastic MC3T3-E1 cells.

NUMB alternative splicing and isoform-specific functions in development and disease.

NUMB alternative splicing and isoform-specific functions in development and disease.

MYG1 interacts with HSP90 to promote breast cancer progression through Wnt/β-catenin and Notch signaling pathways.

MYG1 interacts with HSP90 to promote breast cancer progression through Wnt/β-catenin and Notch signaling pathways.