Notch-Hes1 Pathway Research Articles

HES1 deficiency impairs development of human intestinal mesenchyme by suppressing WNT5A expression

Serious intestinal side-effects that target the NOTCH-HES1 pathway in human cancer differentiation therapy make it necessary to understand the pathway at the human organ level. Herein, we endogenously introduced HES1−/− mutations into human embryonic stem cells (hESCs) and differentiated them into human intestinal organoids (HIO). The HES1−/− hESCs retained ES cell properties and showed gene expression patterns similar to those of wild-type hESCs when they differentiated into definitive endoderm and hindgut. During the formation of the HES1−/− lumen we noted an impaired development of mesenchymal cells in addition to the increased differentiation of secretory epithelium. RNA-Seq revealed that inhibited development of the mesenchymal cells may have been due to a downregulation of WNT5A signaling. Overexpression of HES1 and silencing of WNT5A in the intestinal fibroblast cell line CCD-18Co indicated that HES1 was involved in the activation of WNT5A-induced fibroblast growth and migration, suggesting the likelihood of the Notch pathway in epithelial-mesenchymal crosstalk. Our results facilitated the identification of more precise underlying molecular mechanisms displaying distinct roles in HES1 signaling in stromal and epithelial development in human intestinal mucosa.

Somatostatin stimulates colonic MUC2 expression through SSTR5-Notch-Hes1 signaling pathway

Colonic mucus barrier is regarded as the first defense line against bacteria and antigens from directly attaching to the epithelium, which would further lead to intestinal inflammation activation and pathological conditions. As MUC2 mucin is the predominant component of the mucus, understanding the regulatory mechanisms of MUC2 is important for mucus barrier protection. Somatostatin (SST) has been found to play a role in colon protection through various manners. However, whether SST involves in colonic mucus barrier regulation is still unclear. The aim of this study is to investigate the effects and potential mechanisms of SST on colonic MUC2 expression and mucus secretion. In vivo study, exogenous somatostatin (octreotide) administration effectively stimulated mice colonic MUC2 expression and mucus secretion. In human goblet-like cell LS174T cells, SST exposure also significantly stimulated MUC2 expression and mucus secretion. Further studies indicated that SST receptor 5 (SSTR5) was significantly activated by SST, whereas specific SSTR5 siRNA transfection of LS174T cells significantly blocked SST-induced increase in MUC2 expression and mucus secretion. In addition, SSTR5 agonist L817,818 also upregulated MUC2 expression and mucus secretion in LS174T cells. Mechanistic studies further demonstrated that SST/SSTR5-mediated MUC2 upregulation was dependent on Notch-Hes1 pathway suppression by detecting notch intracellular domain (NICD) and Hes1 proteins. Taken together, our findings suggested that SST could participate in colonic mucus barrier regulation through SSTR5-Notch-Hes1-MUC2 signaling pathway. These findings provide a deep insight into the role of SST on colonic mucus regulation under physiological conditions.

Sequential combination of cisplatin with eugenol targets ovarian cancer stem cells through the Notch-Hes1 signalling pathway

BackgroundOvarian carcinomas are the deadliest gynecological malignancies owing to their high rate of recurrence and high resistance to platinum-based chemotherapy. Recent studies have shown platinum-dependent enrichment of ovarian tumors with side population as well as cancer stem cells, which are highly resistant to the treatment. To overcome this treatment-limiting factor, we sought to combine cisplatin with eugenol, a natural substance known to have anti-cancer effects.MethodsThe efficiency of combining cisplatin with eugenol was first tested in vitro on two ovarian cancer cell lines SKOV3 and OV2774 using the WST1 and the flow cytometry techniques. The effect of this combination on ovarian cancer stem cells was determined by the tumorsphere formation assay, while the implication of the Notch pathway was evaluated post-ectopic expression of the Hes1 gene. The resulting changes in the expression of several markers was assessed by immunoblotting, immunofluorescence as well as quantitative RT-PCR. Cell sorting was also used to isolate specific ovarian cancer sub-population of cells. Furthermore, tumor-bearing mouse models were utilized to prove the potential therapeutic value of the cisplatin/eugenol combination treatment in vivo.ResultsWe have shown that adding eugenol to cisplatin-treated ovarian cancer cells synergistically inhibited their growth and survival through induction of apoptosis. Importantly, this sequential inhibition strongly reduced the proportion of side population cells and suppressed cisplatin-dependent enrichment in ovarian cancer stem cells. Additionally, while increase in the level of Hes1 promoted stemness and enhanced resistance to cisplatin, cisplatin/eugenol cotreatment inhibited the Notch-Hes1 pathway and strongly downregulated the drug resistance ABC transporter genes. These findings were confirmed in vivo by showing that cisplatin/eugenol cotherapy inhibited tumor growth in animals, reduced the proportion and self-renewal capacities of cancer stem cells and significantly improved disease-free survival of tumor-bearing animals compared with either therapy alone.ConclusionsThese results indicate that cisplatin/eugenol sequential combination could be of great therapeutic value for ovarian cancer patients through targeting the Notch-Hes1 pathway and the consequent elimination of the resistant cancer stem cells.

Differing expression profiles of Notch/enterocyte and Wnt/secretory lineage signallings are associated with morphological diversity of appendiceal tumours

BackgroundTumours of appendix, including classic carcinoid tumour (CCT), goblet cell carcinoid (GCC), low-grade appendiceal mucinous neoplasm, high-grade appendiceal mucinous neoplasm/mucinous carcinoma (MCA) and non-mucinous adenocarcinoma (NMA), show different and sometimes...

Chronic rapamycin treatment on the nutrient utilization and metabolism of juvenile turbot (Psetta maxima).

High dietary protein inclusion is necessary in fish feeds and also represents a major cost in the aquaculture industry, which demands improved dietary conversion into body proteins in fish. In mammals, the target of rapamycin (TOR) is a key nutritionally responsive molecule governing postprandial anabolism. However, its physiological significance in teleosts has not been fully examined. In the present study, we examined the nutritional physiology of turbot after chronic rapamycin inhibition. Our results showed that a 6-week inhibition of TOR using dietary rapamycin inclusion (30 mg/kg diet) reduced growth performance and feed utilization. The rapamycin treatment inhibited TOR signaling and reduced expression of key enzymes in glycolysis, lipogenesis, cholesterol biosynthesis, while increasing the expression of enzymes involved in gluconeogenesis. Furthermore, rapamycin treatment increased intestinal goblet cell number in turbot, while the expressions of Notch and Hes1 were down regulated. It was possible that stimulated goblet cell differentiation by rapamycin was mediated through Notch-Hes1 pathway. Therefore, our results demonstrate the important role of TOR signaling in fish nutritional physiology.

A genome-wide analysis identifies a notch-RBP-Jκ-IL-7Rα axis that controls IL-17-producing γδ T cell homeostasis in mice.

Notch signaling is an important regulator for the development and function of both αβ and γδ T cells, whereas roles of Notch signaling in T cell maintenance remain unclear. We reported previously that the Notch-Hes1 pathway was involved in the intrathymic development of naturally occurring IL-17-producing (IL-17(+)) γδ T cells. To gain insight into additional roles for the Notch axis in the homeostasis of γδ T cells, we performed a genome-wide analysis of Notch target genes and identified the novel promoter site of IL-7Rα driven by the Notch-RBP-Jκ pathway. Constitutive Notch signaling had the potential to induce IL-7Rα expression on γδ T cells in vivo, as well as in vitro, whereas conditional deletion of RBP-Jκ abrogated IL-7Rα expression, but not Hes1 expression, by γδ T cells and selectively reduced the pool size of IL-7Rα(high) IL-17(+) γδ T cells in the periphery. In the absence of IL-7Rα-mediated signaling, IL-17(+) γδ T cells were barely maintained in adult mice. Addition of exogenous IL-7 in vitro selectively expanded IL-17(+) γδ T cells. Thus, our results revealed a novel role for the Notch-RBP-Jκ-IL-7Rα axis that is independent of Hes1 for homeostasis of IL-17(+) γδ T cells.

Advanced Glycation End Product-Induced Astrocytic Differentiation of Cultured Neurospheres through Inhibition of Notch-Hes1 Pathway-Mediated Neurogenesis

This study aims to investigate the roles of the Notch-Hes1 pathway in the advanced glycation end product (AGE)-mediated differentiation of neural stem cells (NSCs). We prepared pLentiLox3.7 lentiviral vectors that express short hairpin RNA (shRNA) against Notch1 and transfected it into NSCs. Cell differentiation was analyzed under confocal laser-scanning microscopy. The percentage of neurons and astrocytes was quantified by normalizing the total number of TUJ1+ (Neuron-specific class III β-tubulin) and GFAP+ (Glial fibrillary acidic protein) cells to the total number of Hoechst 33342-labeled cell nuclei. The protein and gene expression of Notch-Hes1 pathway components was examined via western blot analysis and real-time PCR. After 1 week of incubation, we found that AGE-bovine serum albumin (BSA) (400 μg/mL) induced the astrocytic differentiation of cultured neurospheres and inhibited neuronal formation. The expression of Notch-Hes1 pathway components was upregulated in the cells in the AGE-BSA culture medium. Immunoblot analysis indicated that shRNA silencing of Notch1 expression in NSCs significantly increases neurogenesis and suppresses astrocytic differentiation in NSCs incubated with AGE-BSA. AGEs promote the astrocytic differentiation of cultured neurospheres by inhibiting neurogenesis through the Notch-Hes1 pathway, providing a potential therapeutic target for hyperglycemia-related cognitive deficits.

Hes1 in the somatic cells of the murine ovary is necessary for oocyte survival and maturation

The Notch pathway plays an important role in ovary development in invertebrates like Drosophila. However its role for the mammalian ovary is unclear. Mammalian Hes genes encode transcriptional factors that mediate many of the activities of the Notch pathway. Here, we have studied the function of Hes1 during embryonic development of the mouse ovary. We find that Hes1 protein is present in somatic cells and oocyte cytoplasm and decreases between E15.5 and P0. Conventional Hes1 knock-out (KO), Hes1 conditional KO in the ovarian somatic, and chemical inhibition of Notch signaling decrease the total number, size and maturation of oocytes and increase the number of pregranulosa cells at P0. These defects correlate with abnormal proliferation and enhanced apoptosis. Expression of the proapoptotic gene Inhbb is increased, while the levels of the antiapoptotic and oocyte maturation marker Kit are decreased in the Hes1 KO ovaries. Conversely, overactivation of the Notch pathway in ovarian somatic cells increases the number of mature oocytes and decreases the number of pregranulosa cells. Fertility is also reduced by either Hes1 deletion or Notch pathway overactivation. In conclusion, our data suggest that the Notch–Hes1 pathway regulates ovarian somatic cell development, which is necessary for oocyte survival and maturation.

TNFα enhances cancer stem cell-like phenotype via Notch-Hes1 activation in oral squamous cell carcinoma cells

Cancer stem-like cell (CSC; also known as tumor initiating cell) is defined as a small subpopulation of cancer cells within a tumor and isolated from various primary tumors and cancer cell lines. CSCs are highly tumorigenic and resistant to anticancer treatments. In this study, we found that prolonged exposure to tumor necrosis factor alpha (TNFα), a major proinflammatory cytokine, enhances CSC phenotype of oral squamous cell carcinoma (OSCC) cells, such as an increase in tumor sphere-forming ability, stem cell-associated genes expression, chemo-radioresistance, and tumorigenicity. Moreover, activation of Notch1 signaling was detected in the TNFα-exposed cells, and suppression of Notch1 signaling inhibited CSC phenotype. Furthermore, we demonstrated that inhibition of a Notch downstream target, Hes1, led to suppression of CSC phenotype in the TNFα-exposed cells. We also found that Hes1 expression is commonly upregulated in OSCC lesions compared to precancerous dysplastic lesions, suggesting the possible involvement of Hes1 in OSCC progression and CSC in vivo. In conclusion, inflammatory cytokine exposure may enhance CSC phenotype of OSCC, in part by activating the Notch-Hes1 pathway.

Notch-Hes1 pathway is required for the development of IL-17–producing γδ T cells

AbstractUnlike conventional T cells, which are exported from the thymus as naive cells and acquire effector functions upon antigen encounter in the periphery, a subset of γδ T cells differentiates into effectors that produce IL-17 within the fetal thymus. We demonstrate here that intrathymic development of the naturally occurring IL-17–producing γδ T cells is independent of STAT3 and partly dependent on RORγt. Comparative gene-expression analysis identified Hes1, one of the basic helix-loop-helix proteins involved in Notch signaling, as a factor specifically expressed in IL-17–producing γδ T cells. Hes1 is critically involved in the development of IL-17–producing γδ T cells, as evidenced by their severe decrease in the thymi of Hes1-deficient fetal mice. Delta-like 4 (Dll4)–expressing stromal cells support the development of IL-17–producing γδ T cells in vitro. In addition, conditional Hes1 ablation in peripheral γδ T cells decreases their IL-17 production but not their IFN-γ production. These results reveal a unique differentiation pathway of IL-17–producing γδ T cells.

Notch-Hes1 Pathway and TNF-α Synergistically up-Regulates OLFM4 Expression in the Inflamed Mucosa of the Human Intestine

Background & Aims: Recent studies have shown that OLFM4 is a robust marker for intestinal epithelial stem cells. However, the precise distribution of OLFM4-expressing cells within the inflamed human intestinal mucosa has never been described. Also, the molecular mechanism regulating its expression within intestinal epithelial cells (IECs), with regard to the inflammatory environment, remains largely unknown. Our previous studies have shown that NotchHes1 pathway is constitutively activated in IECs of the inflamed intestinal mucosa, and function as a key pathway for lineage-specific gene expression. Thus, we planned the present study to identify the distribution of OLFM4-expressing IECs, and also to reveal the underlying molecular mechanism regulating expression of OLFM4, under such an inflammatory environment. Methods: Immunostaining using human intestinal tissues were performed to determine the distribution of OLFM4-expressing cells. The expression level of OLFM4 by IECs in response to various inflammatory cytokines was analyzed by RT-PCR or western blot. Also, tetracycline inducible over-expression of Notch1 intracellular domain (NICD1), or Hes1, was employed to examine the involvement of Notch-Hes1 pathway upon OLFM4 expression. Finally, a series of promoter assay was performed to analyze the transcriptional regulation of the human OLFM4 gene. Results: Immunostaining of normal small intestinal and colonic tissues clearly showed the distribution of OLFM4-positive IECs at the lowest part of the crypt, including the crypt base columnar cells. However, in inflamed intestinal tissues of inflammatory bowel disease patients, increased number of OLFM4-expressing IECs was observed, expanding its distribution to the upper part of the crypt. In LS174T cells, stimulation by TNF-α, but not by IL1-β and IFN-γ, significantly up-regulated the mRNA expression of OLFM4. Also, forced expression of both NICD1 and Hes1 significantly up-regulated mRNA and protein expression of OLFM4. Surprisingly, combination of NICD1 or Hes1 over-expression with TNF-α stimulation had synergistic effect upon up-regulation of OLFM4 mRNA expression, reaching up to 2500 fold increase in LS174T cells. Promoter assays using 5' flanking region of the human OLFM4 gene revealed that such a synergistic effect of TNF-α and Notch-Hes1 pathway is mediated through transcriptional regulation, depending on the proximal NF-κB binding site. Consistently, TNF-αmediated up-regulation of NF-κB-dependent transcriptional activity was significantly enhanced by both NICD1 and Hes1 over-expression in LS174T cells. Conclusion: In the inflamed human intestinal mucosa, TNF-α and Notch-Hes1 pathway synergistically up-regulate expression of OLFM4 in IECs. Such an up-regulation of a stem-cell specific genemight be required to promote the regenerative response of the inflamed intestinal environment.

Slco1b1*1b Haplotype is Associated With Risk of Aspirin Induced Peptic Ulcer and Bleeding

Background & Aims: Recent studies have shown that OLFM4 is a robust marker for intestinal epithelial stem cells. However, the precise distribution of OLFM4-expressing cells within the inflamed human intestinal mucosa has never been described. Also, the molecular mechanism regulating its expression within intestinal epithelial cells (IECs), with regard to the inflammatory environment, remains largely unknown. Our previous studies have shown that NotchHes1 pathway is constitutively activated in IECs of the inflamed intestinal mucosa, and function as a key pathway for lineage-specific gene expression. Thus, we planned the present study to identify the distribution of OLFM4-expressing IECs, and also to reveal the underlying molecular mechanism regulating expression of OLFM4, under such an inflammatory environment. Methods: Immunostaining using human intestinal tissues were performed to determine the distribution of OLFM4-expressing cells. The expression level of OLFM4 by IECs in response to various inflammatory cytokines was analyzed by RT-PCR or western blot. Also, tetracycline inducible over-expression of Notch1 intracellular domain (NICD1), or Hes1, was employed to examine the involvement of Notch-Hes1 pathway upon OLFM4 expression. Finally, a series of promoter assay was performed to analyze the transcriptional regulation of the human OLFM4 gene. Results: Immunostaining of normal small intestinal and colonic tissues clearly showed the distribution of OLFM4-positive IECs at the lowest part of the crypt, including the crypt base columnar cells. However, in inflamed intestinal tissues of inflammatory bowel disease patients, increased number of OLFM4-expressing IECs was observed, expanding its distribution to the upper part of the crypt. In LS174T cells, stimulation by TNF-α, but not by IL1-β and IFN-γ, significantly up-regulated the mRNA expression of OLFM4. Also, forced expression of both NICD1 and Hes1 significantly up-regulated mRNA and protein expression of OLFM4. Surprisingly, combination of NICD1 or Hes1 over-expression with TNF-α stimulation had synergistic effect upon up-regulation of OLFM4 mRNA expression, reaching up to 2500 fold increase in LS174T cells. Promoter assays using 5' flanking region of the human OLFM4 gene revealed that such a synergistic effect of TNF-α and Notch-Hes1 pathway is mediated through transcriptional regulation, depending on the proximal NF-κB binding site. Consistently, TNF-αmediated up-regulation of NF-κB-dependent transcriptional activity was significantly enhanced by both NICD1 and Hes1 over-expression in LS174T cells. Conclusion: In the inflamed human intestinal mucosa, TNF-α and Notch-Hes1 pathway synergistically up-regulate expression of OLFM4 in IECs. Such an up-regulation of a stem-cell specific genemight be required to promote the regenerative response of the inflamed intestinal environment.

A Novel Role for IL-27 as a Mediator of Intestinal Epithelial Barrier Protection Mediated via Differential Stat Signaling and Induction of Antibacterial and Anti-Inflammatory Proteins

Background & Aims: Recent studies have shown that OLFM4 is a robust marker for intestinal epithelial stem cells. However, the precise distribution of OLFM4-expressing cells within the inflamed human intestinal mucosa has never been described. Also, the molecular mechanism regulating its expression within intestinal epithelial cells (IECs), with regard to the inflammatory environment, remains largely unknown. Our previous studies have shown that NotchHes1 pathway is constitutively activated in IECs of the inflamed intestinal mucosa, and function as a key pathway for lineage-specific gene expression. Thus, we planned the present study to identify the distribution of OLFM4-expressing IECs, and also to reveal the underlying molecular mechanism regulating expression of OLFM4, under such an inflammatory environment. Methods: Immunostaining using human intestinal tissues were performed to determine the distribution of OLFM4-expressing cells. The expression level of OLFM4 by IECs in response to various inflammatory cytokines was analyzed by RT-PCR or western blot. Also, tetracycline inducible over-expression of Notch1 intracellular domain (NICD1), or Hes1, was employed to examine the involvement of Notch-Hes1 pathway upon OLFM4 expression. Finally, a series of promoter assay was performed to analyze the transcriptional regulation of the human OLFM4 gene. Results: Immunostaining of normal small intestinal and colonic tissues clearly showed the distribution of OLFM4-positive IECs at the lowest part of the crypt, including the crypt base columnar cells. However, in inflamed intestinal tissues of inflammatory bowel disease patients, increased number of OLFM4-expressing IECs was observed, expanding its distribution to the upper part of the crypt. In LS174T cells, stimulation by TNF-α, but not by IL1-β and IFN-γ, significantly up-regulated the mRNA expression of OLFM4. Also, forced expression of both NICD1 and Hes1 significantly up-regulated mRNA and protein expression of OLFM4. Surprisingly, combination of NICD1 or Hes1 over-expression with TNF-α stimulation had synergistic effect upon up-regulation of OLFM4 mRNA expression, reaching up to 2500 fold increase in LS174T cells. Promoter assays using 5' flanking region of the human OLFM4 gene revealed that such a synergistic effect of TNF-α and Notch-Hes1 pathway is mediated through transcriptional regulation, depending on the proximal NF-κB binding site. Consistently, TNF-αmediated up-regulation of NF-κB-dependent transcriptional activity was significantly enhanced by both NICD1 and Hes1 over-expression in LS174T cells. Conclusion: In the inflamed human intestinal mucosa, TNF-α and Notch-Hes1 pathway synergistically up-regulate expression of OLFM4 in IECs. Such an up-regulation of a stem-cell specific genemight be required to promote the regenerative response of the inflamed intestinal environment.

Notch‐Hes1 pathway contributes to the cochlear prosensory formation potentially through the transcriptional down‐regulation of p27Kip1

The Notch signaling pathway has a crucial role in the differentiation of hair cells and supporting cells by mediating "lateral inhibition" via the ligands Delta-like1 (Dll1) and Jagged2 (Jag2) and the effectors Hes1 and Hes5 during mammalian inner ear development. Recently, another Notch ligand, Jagged1 (Jag1)-dependent Notch activation, has been revealed to be important for the determination of the prosensory region in the earlier stage before cell differentiation. However, little is known about the effectors of the Notch pathway in this context. P27(Kip1), a cyclin-dependent kinase inhibitor, is also known to demarcate the prosensory region in the cochlear primordium, which consists of the sensory progenitors that have completed their terminal mitoses. Hes1 reportedly promotes precursor cell proliferation through the transcriptional down-regulation of p27(Kip1) in the thymus, liver, and brain. In this study, we observed Hes1 as a mediator between the Notch signaling pathway and the regulation of proliferation of sensory precursor cells by p27(Kip1) in the developing cochlea. We showed that Hes1, but not Hes5, was weakly expressed at the time of onset of p27(Kip1). The expression pattern of Hes1 prior to cell differentiation was similar to that of activated Notch1. P27(Kip1) was up-regulated and BrdU-positive S-phase cells were reduced in the developing cochlear epithelium of Hes1 null mice. These results suggest that the Notch-Hes1 pathway may contribute to the adequate proliferation of sensory precursor cells via the potential transcriptional down-regulation of p27(Kip1) expression and play a pivotal role in the correct prosensory determination.

The bHLH gene Hes1 regulates differentiation of multiple cell types.

For embryos that have small pancreas and lack brain, eyes and thymus, the defects are caused by mutation of a single gene, Hes1. Hes1 encodes a basic helix-loop-helix (bHLH) transcriptional repressor and functionally antagonizes positive bHLH genes such as the neuronal determination gene, Mash1. Misexpression of Hes1 inhibits cell differentiation and keeps cells at the precursor stage or proliferative stage. Conversely, in the absence of Hes1, the expression of positive bHLH genes is upregulated and cells differentiate prematurely without sufficient cell growth. As a result, the development of many tissues such as the brain, eye and pancreas is severely affected. Thus, Hes1 regulates tissue morphogenesis by maintaining undifferentiated cells. In the case of T cell development, Hes1 mutation leads to defects of expansion of early T cell precursors and thereby suppresses T cell fate specification. Thus, Hes1 promotes differentiation of some cell types in addition to maintenance of the undifferentiated state. Interestingly, Hes1 expression is controlled by the transmembrane protein Notch, which is activated by the ligands expressed on the surface of neighboring cells. Taken together, these results indicate that the Notch-Hes1 pathway, which is controlled by cell-cell interaction, plays an essential role in differentiation of many cell types.