Inhibition Of Notch Signaling Research Articles

Robust reprogramming of glia into neurons by inhibition of Notch signaling and nuclear factor I (NFI) factors in adult mammalian retina.

Generation of neurons through direct reprogramming has emerged as a promising therapeutic approach for treating neurodegenerative diseases. In this study, we present an efficient method for reprogramming retinal glial cells into neurons. By suppressing Notch signaling by disrupting either Rbpj or Notch1/2, we induced mature Müller glial cells to reprogram into bipolar- and amacrine-like neurons. We demonstrate that Rbpj directly activates both Notch effector genes and genes specific to mature Müller glia while indirectly repressing expression of neurogenic basic helix-loop-helix (bHLH) factors. Combined loss of function of Rbpj and Nfia/b/x resulted in conversion of nearly all Müller glia to neurons. Last, inducing Müller glial proliferation by overexpression of dominant-active Yap promotes neurogenesis in both Rbpj- and Nfia/b/x/Rbpj-deficient Müller glia. These findings demonstrate that Notch signaling and NFI factors act in parallel to inhibit neurogenic competence in mammalian Müller glia and help clarify potential strategies for regenerative therapies aimed at treating retinal dystrophies.

Tanshinol ameliorates imiquimod-induced psoriasis by inhibiting M1 macrophage polarization through suppression of the notch signaling pathway.

Psoriasis is a common immune-related chronic inflammatory skin disease, often accompanied by significant itching, and once diseased, the course of the disease lasts for most of the lifetime. Tanshinol (TAN) is an active ingredient of Salvia miltiorrhiza, which possesses pharmacological effects such as anti-inflammatory and antioxidant properties. However, the effects of TAN on psoriasis have not been widely reported. Therefore, the aim of this study was to investigate the therapeutic effects and mechanisms of TAN in psoriasis. An imiquimod (IMQ)-induced psoriasis mouse model was constructed and treated with different doses of TAN to observe the changes in skin lesion phenotype, macrophage polarization, inflammation and Notch signaling pathway in mice. Further removal of macrophages or inhibition or activation of Notch signaling pathway was performed to examine the changes in skin lesion phenotype, macrophage polarization, inflammation and Notch signaling pathway in mice. In addition, in vitro experiments verified that TAN regulates RAW264.7 macrophage polarization and cytokine secretion through the Notch pathway. The results showed that TAN alleviated IMQ-induced skin lesions and pathological phenotypes in psoriasis mice and inhibited Notch signaling pathway and M1-type macrophage polarization. Moreover, macrophage clearance and Notch signaling pathway activation inhibited the effect of TAN on psoriasis. Further in vitro experiments showed that Notch agonists reversed the effects of TAN on macrophage polarization and inflammatory cytokines. Collectively, these findings suggest that TAN may exert a therapeutic effect on psoriasis by inhibiting the Notch signaling pathway and thus M1-type macrophage polarization.

Reduction of specific enterocytes from loss of intestinal LGR4 improves lipid metabolism in mice

Whether intestinal Leucine-rich repeat containing G-protein-coupled receptor 4 (LGR4) impacts nutrition absorption and energy homeostasis remains unknown. Here, we report that deficiency of Lgr4 (Lgr4iKO) in intestinal epithelium decreased the proportion of enterocytes selective for long-chain fatty acid absorption, leading to reduction in lipid absorption and subsequent improvement in lipid and glucose metabolism. Single-cell RNA sequencing demonstrates the heterogeneity of absorptive enterocytes, with a decrease in enterocytes selective for long-chain fatty acid-absorption and an increase in enterocytes selective for carbohydrate absorption in Lgr4iKO mice. Activation of Notch signaling and concurrent inhibition of Wnt signaling are observed in the transgenes. Associated with these alterations is the substantial reduction in lipid absorption. Decrement in lipid absorption renders Lgr4iKO mice resistant to high fat diet-induced obesity relevant to wild type littermates. Our study thus suggests that targeting intestinal LGR4 is a potential strategy for the intervention of obesity and liver steatosis.

Endothelial HIF2α suppresses retinal angiogenesis in neonatal mice by upregulating NOTCH signaling.

Prolyl hydroxylase domain (PHD) proteins are oxygen sensors that use intracellular oxygen as a substrate to hydroxylate hypoxia-inducible factor (HIF) α proteins, routing them for polyubiquitylation and proteasomal degradation. Typically, HIFα accumulation in hypoxic or PHD-deficient tissues leads to upregulated angiogenesis. Here, we report unexpected retinal phenotypes associated with endothelial cell (EC)-specific gene targeting of Phd2 (Egln1) and Hif2alpha (Epas1). EC-specific Phd2 disruption suppressed retinal angiogenesis, despite HIFα accumulation and VEGFA upregulation. Suppressed retinal angiogenesis was observed both in development and in the oxygen-induced retinopathy (OIR) model. On the other hand, EC-specific deletion of Hif1alpha (Hif1a), Hif2alpha, or both did not affect retinal vascular morphogenesis. Strikingly, retinal angiogenesis appeared normal in mice double-deficient for endothelial PHD2 and HIF2α. In PHD2-deficient retinal vasculature, delta-like 4 (DLL4, a NOTCH ligand) and HEY2 (a NOTCH target) were upregulated by HIF2α-dependent mechanisms. Inhibition of NOTCH signaling by a chemical inhibitor or DLL4 antibody partially rescued retinal angiogenesis. Taken together, our data demonstrate that HIF2α accumulation in retinal ECs inhibits rather than stimulates retinal angiogenesis, in part by upregulating DLL4 expression and NOTCH signaling.

Activation of Notch Signaling Pathway is involved in Extracellular Matrix Degradation in human induced pluripotent stem cells chondrocytes induced by HT-2 toxin

Notch signaling regulates cartilage formation and homeostasis. Kashin-Beck Disease (KBD), an endemic osteochondropathy, is characterized by severe cartilage degradation. The etiology of KBD is related to the exposure of HT-2 toxin, a mycotoxin and primary metabolite of T-2 toxin. This study aims to explore the role of HT-2 toxin in the Notch signaling regulation and extracellular matrix (ECM) metabolism of hiPSCs-Chondrocytes. Immunohistochemistry and qRT-PCR were employed to investigate the expression of Notch pathway molecules in KBD articular cartilage and primary chondrocytes. hiPSCs-Chondrocytes, derived from hiPSCs, were treated with 100 ng/mL HT-2 toxin and the γ-secretase inhibitor (DAPT) for 48h, respectively. The markers related to the Notch signaling pathway and ECM were assessed using qRT-PCR and Western blot. Notch pathway dysregulation was prominent in KBD cartilage. HT-2 toxin exposure caused cytotoxicity in hiPSCs-Chondrocytes, and activated Notch signaling by increasing the mRNA and protein levels of NOTCH1 and HES1. HT-2 toxin also upregulated ECM catabolic enzymes and downregulated ECM components (COL2A1 and ACAN), indicating ECM degradation. DAPT-mediated Notch signaling inhibition suppressed the mRNA and protein level of ADAMTS5 expression while enhancing ECM component expression in hiPSCs-Chondrocytes. This study suggests that HT-2 toxin may induce ECM degradation in hiPSCs-Chondrocytes through activating Notch signaling.

Bombyx moriSuppressor of Hairless is involved in the regulation of the silkworm cell cycle and endoreplication of the silk glands

The Notch signaling pathway is important in cell cycle regulation and cell proliferation. The transcriptional repressor Suppressor of Hairless [Su(H)] is a molecular switch for downstream target genes of the Notch signaling pathway but the regulatory mechanism of the Su(H) gene in the cell cycle is unclear. We determined the function of the Notch signaling pathway and Bombyx mori Su(H) [BmSu(H)] in the regulation of the silkworm cell cycle. Inhibition of Notch signaling promoted the replication of DNA in silkworm gland cells and expression of the BmSu(H) gene was significantly reduced. Overexpression of the BmSu(H) gene inhibited DNA replication and cell proliferation of silkworm cells, whereas knockout of the BmSu(H) gene promoted DNA replication and cell proliferation. Knockout of the BmSu(H) in silkworms improved the efficiency of silk gland cell endoreplication and increased important economic traits. We demonstrated that BmSu(H) protein can directly bind to the promoters of BmCyclinA, BmCyclinE and BmCDK1 genes, inhibiting or promoting their transcription at the cell and individual level. This study identified molecular targets for genetic improvement of the silkworm and also provided insights into the regulatory mechanism of the cell cycle.

Differentiation of intestinal stem cells toward goblet cells under systemic iron overload stress are associated with inhibition of Notch signaling pathway and ferroptosis

Iron overload can lead to oxidative stress and intestinal damage and happens frequently during blood transfusions and iron supplementation. However, how iron overload influences intestinal mucosa remains unknown. Here, the aim of current study was to investigate the effects of iron overload on the proliferation and differentiation of intestinal stem cells (ISCs). An iron overload mouse model was established by intraperitoneal injection of 120 mg/kg body weight iron dextran once a fortnight for a duration of 12 weeks, and an iron overload enteroid model was produced by treatment with 3 mM or 10 mM of ferric ammonium citrate for 24 h. We found that iron overload caused damage to intestinal morphology with a 64 % reduction in villus height/crypt depth ratio, and microvilli injury in the duodenum. Iron overload mediated epithelial function by inhibiting the expression of nutrient transporters and enhancing the expression of secretory factors in the duodenum. Meanwhile, iron overload inhibited the proliferation of ISCs and regulated their differentiation into secretory mature cells, such as goblet cells, through inhibiting Notch signaling pathway both in mice and enteroid. Furthermore, iron overload caused oxidative stress and ferroptosis in intestinal epithelial cells. In addition, ferroptosis could also inhibit Notch signaling pathway, and affected the proliferation and differentiation of ISCs. These findings reveal the regulatory role of iron overload on the proliferation and differentiation of ISCs, providing a new insight into the internal mechanism of iron overload affecting intestinal health, and offering important theoretical basis for the scientific application of iron nutrition regulation.

Notch Signaling Inhibition Alleviates Allergies Caused by Antarctic Krill Tropomyosin through Improving Th1/Th2 Imbalance and Modulating Gut Microbiota.

Antarctic krill tropomyosin (AkTM) has been shown in mice to cause IgE-mediated food allergy. The objective of this work was to investigate the role of Notch signaling in AkTM-sensitized mice, as well as to determine the changes in gut microbiota composition and short-chain fatty acids (SCFAs) in the allergic mice. An AkTM-induced food allergy mouse model was built and N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) was used as an γ-secretase inhibitor to inhibit the activation of Notch signaling. Food allergy indices, some key transcription factors, histologic alterations in the small intestine, and changes in gut microbiota composition were examined. The results showed that DAPT inhibited Notch signaling, which reduced AkTM-specific IgE, suppressed mast cell degranulation, decreased IL-4 but increased IFN-γ production, and alleviated allergic symptoms. Quantitative real-time PCR and Western blotting analyses revealed that expressions of Hes-1, Gata3, and IL-4 were down-regulated after DAPT treatment, accompanied by increases in T-bet and IFN-γ, indicating that Notch signaling was active in AkTM-sensitized mice and blocking it could reverse the Th1/Th2 imbalance. Expressions of key transcription factors revealed that Notch signaling could promote Th2 cell differentiation in sensitized mice. Furthermore, 16S rRNA sequencing results revealed that AkTM could alter the diversity and composition of gut microbiota in mice, leading to increases in inflammation-inducing bacteria such as Enterococcus and Escherichia-Shigella. Correlation analysis indicated that reduced SCFA concentrations in AkTM-allergic mice may be related to decreases in certain SCFA-producing bacteria, such as Clostridia_UCG-014. The changes in gut microbiota and SCFAs could be partially restored by DAPT treatment. Our findings showed that inhibiting Notch signaling could alleviate AkTM-induced food allergy by correcting Th1/Th2 imbalance and modulating the gut microbiota.

Tracing the origin of alveolar stem cells in lung repair and regeneration

Alveolar type 2 (AT2) cells are stem cells of the alveolar epithelia. Previous genetic lineage tracing studies reported multiple cellular origins for AT2 cells after injury. However, conventional lineage tracing based on Cre-loxP has the limitation of non-specific labeling. Here, we introduced a dual recombinase-mediated intersectional genetic lineage tracing approach, enabling precise investigation of AT2 cellular origins during lung homeostasis, injury, and repair. We found AT1 cells, being terminally differentiated, did not contribute to AT2 cells after lung injury and repair. Distinctive yet simultaneous labeling of club cells, bronchioalveolar stem cells (BASCs), and existing AT2 cells revealed the exact contribution of each to AT2 cells post-injury. Mechanistically, Notch signaling inhibition promotes BASCs but impairs club cells' ability to generate AT2 cells during lung repair. This intersectional genetic lineage tracing strategy with enhanced precision allowed us to elucidate the physiological role of various epithelial cell types in alveolar regeneration following injury.

The Notch-PDGFRβ axis suppresses brown adipocyte progenitor differentiation in early post-natal mice

De novo brown adipogenesis holds potential in combating the epidemics of obesity and diabetes. However, the identity of brown adipocyte progenitor cells (APCs) and their regulation have not been extensively explored. Here, through invivo lineage tracing and mouse modeling, we observed that platelet-derived growth factor receptor beta (PDGFRβ)+ pericytes give rise to developmental brown adipocytes but not to those in adult homeostasis. By contrast, T-box 18 (TBX18)+ pericytes contribute to brown adipogenesis throughout both developmental and adult stages, though in a depot-specific manner. Mechanistically, Notch inhibition in PDGFRβ+ pericytes promotes brown adipogenesis by downregulating PDGFRβ. Furthermore, inhibition of Notch signaling in PDGFRβ+ pericytes mitigates high-fat, high-sucrose (HFHS)-induced glucose and metabolic impairment in mice during their development and juvenile phases. Collectively, these findings show that the Notch/PDGFRβ axis negatively regulates developmental brown adipogenesis, and its repression promotes brown adipose tissue expansion and improves metabolic health.

Abstract 489: Unlocking immune resistance and metastasis of PDAC via dual acting circularized nanodisc

Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and deadliest forms of cancer. While major progress in immunotherapy has been made in recent years, it remains poorly effective in this malignancy. Two key players stand behind immune resistance of PDAC and inadequate efficacy of immunotherapy to suppress its metastasis. First, the immune-resistant pancreatic cancer stem cells (CSCs) evade immune surveillance acting as a source for tumor relapse and metastasis. This immune resistance stems from low immunogenicity of PDAC cells as well as the inaccessible CSCs that reside deep in the tumor core. Second, pancreatic cancer associated fibroblasts (CAFs) fuel metastasis of PDAC cells by suppressing their immune recognition and establishing pre-metastatic niche at distant organs to help their colonization. Our hypothesis is to overcome immune resistance of PDAC and inhibit early steps of its metastasis via simultaneous immunogenic eradication of immune resistant CSCs and switching immunosuppressive and pro-metastatic CAFs into quiescent ones. Therefore, we engineered ultrasmall circularized nanodiscs (cND) that encapsulate oncolytic peptide LTX-315 and fibroblast remodeling Notch signaling inhibitor. The engineered oncolytic LTX-315 loaded cND successfully induced immunogenic cell death (ICD) of both bulk PANC1 and pancreatic CSCs. Compared to non-treated and free LTX-315 treated cells, LTX-315 cND resulted in enhanced release of DAMPs including ATP and HMGB-1 as well as surface translocation of calreticulin. The ultrasmall size (9 nm) of cND enabled its deep tumor penetration to eradicate resistant CSCs, as revealed using 3D PDAC heterospheroids. In parallel, the Notch blocking cND promoted quiescence of the activated pancreatic stellate cells as denoted by downregulation of SMA, FAP and collagen. In KPC syngeneic PDAC mouse model, the dual oncolytic/Notch blocking cND combined with aPD-L1 therapy significantly suppressed tumor growth and prevented its metastasis to liver compared to vehicle-treated mice. Immunophenotyping study showed marked reduction in the abundance of CSCs and CAFs in the tumor treated with the cNDs. Moreover, the tumor infiltration of CD86+ MHCII+ dendritic cells and cytotoxic CD8+ T cells was enhanced after treatment with the cND. Overall, our dual strategy of targeting pancreatic CSCs and CAFs via oncolytic/Notch blocking cND boosted the anti-tumor and anti-metastatic efficacy of aPD-L1 therapy to eliminate primary PDAC tumor and suppress its metastasis. Citation Format: Ahmed Elzoghby, Hagar Emam, Seungbin Yim, Cuiyan Xin, Mahmoud Nasr. Unlocking immune resistance and metastasis of PDAC via dual acting circularized nanodisc [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 489.

The bone-protective benefits of kaempferol combined with metformin by regulation of osteogenesis-angiogenesis coupling in OVX rats

This study was to investigate the potential mechanisms of treatment with metformin (Met) combined with kaempferol (Kae) against postmenopausal osteoporosis. Experiments were conducted in both ovariectomy (OVX)-induced osteoporosis rats and in vitro using RAW264.7 cells, MC3T3-E1 cells, and HUVECs. Results demonstrated the therapeutic effect of Met combined with Kae on osteoporosis. In vivo, Kae alone and in combination with Met treatments enhanced tibial trabecular microstructure, bone mineral density (BMD), and mechanical properties in OVX rats without causing hepatotoxicity and nephrotoxicity. It also reduced bone resorption markers (CTX-1 and TRAP) and increased the bone formation marker (PINP) level in the serum of OVX rats. The expression of bone resorption marker TRAP was reduced, while bone formation markers Runx2 and ALP were enhanced in the bone tissue of OVX rats. Furthermore, Met combined with Kae also promoted the expression of angiogenesis-related markers CD31 and VEGF in OVX rats. In vitro, MC3T3-E1s cells treated with Met combined with Kae showed higher expression of ALP, Runx2, and VEGF. Interestingly, the treatment did not directly promote HUVECs migration and angiogenesis, but enhanced osteoblast-mediated angiogenesis by upregulating VEGF levels. Additionally, Met combined with Kae treatment promoted VEGF secretion in MC3T3-E1, and activated the Notch intracelluar pathway by upregulating HES1 and HEY1 in HUVECs. Meantime, their stimulation on CD31 expression were inhibited by DAPT, a Notch signaling inhibitor. Overall, this study demonstrates the positive effects of Met combined with Kae on osteoporotic rats by promoting osteogenesis-angiogenesis coupling, suggesting their potential application in postmenopausal osteoporosis.

A phase I/IIa safety and efficacy trial of intratympanic gamma-secretase inhibitor as a regenerative drug treatment for sensorineural hearing loss

Inhibition of Notch signalling with a gamma-secretase inhibitor (GSI) induces mammalian hair cell regeneration and partial hearing restoration. In this proof-of-concept Phase I/IIa multiple-ascending dose open-label trial (ISRCTN59733689), adults with mild-moderate sensorineural hearing loss received 3 intratympanic injections of GSI LY3056480, in 1 ear over 2 weeks. Phase I primary outcome was safety and tolerability. Phase lla primary outcome was change from baseline to 12 weeks in average pure-tone air conduction threshold across 2,4,8 kHz. Secondary outcomes included this outcome at 6 weeks and change from baseline to 6 and 12 weeks in pure-tone thresholds at individual frequencies, speech reception thresholds (SRTs), Distortion Product Otoacoustic Emissions (DPOAE) amplitudes, Signal to Noise Ratios (SNRs) and distribution of categories normal, present-abnormal, absent and Hearing Handicap Inventory for Adults/Elderly (HHIA/E). In Phase I (N = 15, 1 site) there were no severe nor serious adverse events. In Phase IIa (N = 44, 3 sites) the average pure-tone threshold across 2,4,8 kHz did not change from baseline to 6 and 12 weeks (estimated change −0.87 dB; 95% CI −2.37 to 0.63; P = 0.252 and −0.46 dB; 95% CI −1.94 to 1.03; P = 0.545, respectively), nor did the means of secondary measures. DPOAE amplitudes, SNRs and distribution of categories did not change from baseline to 6 and 12 weeks, nor did SRTs and HHIA/E scores. Intratympanic delivery of LY3056480 is safe and well-tolerated; the trial’s primary endpoint was not met.

CD151 expression marks atrial- and ventricular- differentiation from human induced pluripotent stem cells

Current differentiation protocols for human induced pluripotent stem cells (hiPSCs) produce heterogeneous cardiomyocytes (CMs). Although chamber-specific CM selection using cell surface antigens enhances biomedical applications, a cell surface marker that accurately distinguishes between hiPSC-derived atrial CMs (ACMs) and ventricular CMs (VCMs) has not yet been identified. We have developed an approach for obtaining functional hiPSC-ACMs and -VCMs based on CD151 expression. For ACM differentiation, we found that ACMs are enriched in the CD151low population and that CD151 expression is correlated with the expression of Notch4 and its ligands. Furthermore, Notch signaling inhibition followed by selecting the CD151low population during atrial differentiation leads to the highly efficient generation of ACMs as evidenced by gene expression and electrophysiology. In contrast, for VCM differentiation, VCMs exhibiting a ventricular-related gene signature and uniform action potentials are enriched in the CD151high population. Our findings enable the production of high-quality ACMs and VCMs appropriate for hiPSC-derived chamber-specific disease models and other applications.

Dang-Gui-Bu-Xue decoction improves wound healing in diabetic rats by the activation of Notch signaling

Diabetes serves as a severe chronic disease that severely affects the normal life of human beings. Diabetes causes the complication of diabetic wound dysfunction, which is characterized by sustained inflammation, altered angiogenesis, delayed epithelialization and abnormal secretion of protease. Dang-Gui-Bu-Xue decoction (DBD) is a Chinese traditional medicine that comprises Radix Astragali and Radix Angelicae sinensis and is widely applied in treatment of multiple diseases owing to its functions against inflammation, lipid peroxidation and oxidative stress. Nevertheless, the impact of DBD on diabetic wound healing remains elusive. In this study, we aimed to explore the function of DBD in the regulation of wound healing. We observed that the gavage administration of DBD reduced the wound area, inflammatory infiltration, inflammatory factor levesl, and enhanced granulation tissue formation, wound extracellular matrix (ECM) production, and CD31 accumulation in the diabetic rat wound model, and the co-treatment of gavage administration and the external administration of gauze containing DBD further improved the wound healing effect, while the combination of Notch signaling inhibitor DAPT ((N- [N- (3, 5-difluorophenacetyl)-l-alanyl]-s-phenylglycinet-butyl ester)) could attenuate the improvement. Regarding to the mechanism, the expression levels of Notch1, Delta-like canonical Notch ligand 4 (Dll4), Jagged1, and Hairy Enhancer of Split-1 (Hes1) were increased by DBD, while the treatment of DAPT impaired the effect in the rats. Furthermore, we found that the high glucose (HG)-inhibited viability and tube formation were induced by DBD in human umbilical vein endothelial cells (HUVECs), in which DAPT could reverse this effect. Therefore, we concluded that DBD contributed to wound healing by the activation of Notch signaling. Our finding provides new insight into the potential role of DBD in promoting diabetic wound healing.

Resveratrol Inhibits T-acute Lymphoblastic Leukemia in Mice by Regulating Notch1 Signaling Pathway

To observe the effect of resveratrol (Res) on T-acute lymphoblastic leukemia (T-ALL) mice, and further explore its mechanism on Notch1 signaling pathway. Twenty-five 6-8 weeks old female C57BL/6 mice were randomly divided into control group, T-ALL group and Res group. Res group was further divided into low-Res, middle-Res and high-Res group. The percentage of leukemia cells in peripheral blood and spleen cell suspension were detected by flow cytometry and Wright-Giemsa staining, pathological morphology of spleen and bone marrow tissues were observed by HE staining, the expression levels of Notch1, Hes-1, c-Myc, miR-19b and PTEN mRNA in spleen tissue were detected by RT-qPCR, and the protein levels of Notch1, Hes-1, c-Myc, p-PTEN and PTEN were detected by Western blot. Compared with control group, the leukemia cells in peripheral blood of mice in T-ALL group were markedly increased, accompanied by diffuse infiltration of leukemia cells in spleen and bone marrow tissues, the mRNA levels of Notch1, Hes-1, c-Myc, miR-19b and the protein levels of Notch1, Hes-1, c-Myc were increased (P <0.01), while the expression of PTEN mRNA and protein were significantly decreased in the spleen tissue of T-ALL mice (P <0.01). The above indicators in the H-Res group were reversed compared with T-ALL group after administration of resveratrol. Resveratrol may play a role in anti T-ALL by inhibiting Notch1 signaling pathway in mice.

Paneth-like cells produced from OLFM4+ stem cells support OLFM4+ stem cell growth in advanced colorectal cancer

Tumor tissues consist of heterogeneous cells that originate from stem cells; however, their cell fate determination program remains incompletely understood. Using patient-derived organoids established from patients with advanced colorectal cancer (CRC), we evaluated the potential of olfactomedin 4 (OLFM4)+ stem cells to produce a bifurcated lineage of progenies with absorptive and secretory properties. In the early phases of organoid reconstruction, OLFM4+ cells preferentially gave rise to secretory cells. Additionally, we found that Paneth-like cells, which do not exist in the normal colon, were induced in response to Notch signaling inhibition. Video recordings of single OLFM4+ cells revealed that organoids containing Paneth-like cells were effectively propagated and that their selective ablation led to organoid collapse. In tumor tissues, Paneth-like cells were identified only in the region where tumor cells lost cell adhesion. These findings indicate that Paneth-like cells are directly produced by OLFM4+ stem cells and that their interaction contributes to tumor formation by providing niche factors. This study reveals the importance of the cell fate specification program for building a complete tumor cellular ecosystem, which might be targeted with novel therapeutics.

Notch signaling pathway induces expression of type IV collagen in angiogenesis.

Mural cell adhesion is important for the localization of basement membrane components during angiogenesis, and cell-cell interactions are thought to be critical for basement membrane formation. Type IV collagen, a component of the basement membrane, and non-triple helical type IV collagen α1 chain (NTH α1(IV)) co-localize in the basement membrane of neovascular vessels. However, it remains unclear how type IV collagen and NTH α1(IV) are produced around the basement membrane. In the present study, we developed a de novo angiogenesis model using human umbilical vein endothelial cell spheroids and TIG-1 fibroblast cells and demonstrated that NTH α1(IV), probably with α1(IV) chain before forming triple helix molecule, was localized in the fibroblasts in contact with vascular endothelial cells. This localization was disrupted by DAPT, a Notch signaling inhibitor. DAPT treatment also reduced type IV collagen and NTH α1(IV) secretion in TIG-1 fibroblasts, along with diminished COL4A1 and COL4A2 gene expression. Downregulation of Notch3 in TIG-1 fibroblasts decreased the secretion of type IV collagen and NTH α1(IV). Taken together, these findings suggest that heterogeneous and homogeneous intercellular Notch signaling via Notch3 induces type IV collagen and NTH α1(IV) expression in fibroblasts and contributes to basement membrane formation in neovascular vessels.

Exosome-mediated Delivery of miR-519e-5p Promotes Malignant Tumor Phenotype and CD8+ T-Cell Exhaustion in Metastatic PTC.

Distant metastases are the primary cause of therapy failure and mortality in patients with papillary thyroid carcinomas (PTCs). However, the underlying mechanism responsible for the initiation of tumor cell dissemination and metastasis in PTCs has rarely been investigated. The aim of this study was to investigate effects and underlying molecular mechanisms of circulating exosomal microRNAs (miRNAs) in distant metastatic PTCs. The most relevant circulating exosomal miRNA to distant metastatic PTCs were verified between distant metastatic PTCs and nondistant metastatic PTCs by miRNA microarray, quantitative real-time polymerase chain reaction (qRT-PCR) assays and receiver operating characteristic (ROC) curves. The parental and recipient cells of that circulating exosomal miRNA were then explored. In vitro and in vivo experiments were further performed to elucidate the function and potential mechanisms of circulating exosomal miRNAs that contribute to the development of distant metastases. We determined that PTC-derived exosomal miR-519e-5p was significantly upregulated in the circulatory system in distant metastatic PTCs. Further tests demonstrated that PTC cells can acquire a more malignant phenotype via hnRNPA2B1-mediated sorting of tumor suppressor miR-519e-5p into exosomes to activate Wnt signaling pathway via upregulating PLAGL2. Furthermore, miR-519e-5p included in PTC-derived exosomes can be transferred to recipient CD8+ T cells and aid in tumor immune escape in distant organs through inhibiting Notch signaling pathway by downregulating NOTCH2. Our findings highlight the dual role of PTC-derived exosomal miR-519e-5p in distant metastasis, which may improve our understanding of exosome-mediated distant metastatic mechanisms.

Effect of Notch Signal Pathway on Steroid Synthesis Enzymes in TM3 Cells.

Studies have indicated that the conservative Notch pathway contributes to steroid hormone synthesis in the ovaries; however, its role in hormone synthesis of the testis remains unclear. We have previously reported Notch 1, 2, and 3 to be expressed in murine Leydig cells and that inhibition of Notch signaling caused G0/G1 arrest in TM3 Leydig cells. In this study, we have further explored the effect of different Notch signal pathways on key steroidogenic enzymes in murine Leydig cells. TM3 cells were treated with Notch signaling pathway inhibitor MK-0752, and different Notch receptors were also overexpressed in TM3 cells. We evaluated the expression of key enzymes of steroid synthesis, including p450 cholesterol side-chain cleavage enzyme (P450Scc), 3β-hydroxysteroid dehydrogenase (3β-HSD) and steroidogenic acute regulatory protein (StAR), and key transcriptional factors for steroid synthesis, including steroidogenic factor 1 (SF1), GATA-binding protein 4 (GATA4) and GATA6. We found the level of P450Scc, 3β-HSD, StAR and SF1 to be decreased after treatment with MK-0752, while overexpression of Notch1 up-regulated the expression of 3β-HSD, P450Scc, StAR and SF1. MK-0752 and overexpression of different Notch members had no influence on the expression of GATA4 and GATA6. In conclusion, Notch1 signaling may contribute to the steroid synthesis in Leydig cells through regulating SF1 and downstream steroidogenic enzymes (3β-HSD, StAR and P450Scc).