A regulatory T cell Notch4-GDF15 axis licenses tissue inflammation in asthma.

Elucidating the mechanisms that sustain asthmatic inflammation is critical for precision therapies. We found that interleukin-6- and STAT3 transcription factor-dependent upregulation of Notch4 receptor on lung tissue regulatory T (Treg) cells is necessary for allergens and particulate matter pollutants to promote airway inflammation. Notch4 subverted Treg cells into the type 2 and type 17 helper (TH2 and TH17) effector T cells by Wnt and Hippo pathway-dependent mechanisms. Wnt activation induced growth and differentiation factor 15 expression in Treg cells, which activated group 2 innate lymphoid cells to provide a feed-forward mechanism for aggravated inflammation. Notch4, Wnt and Hippo were upregulated in circulating Treg cells of individuals with asthma as a function of disease severity, in association with reduced Treg cell-mediated suppression. Our studies thus identify Notch4-mediated immune tolerance subversion as a fundamental mechanism that licenses tissue inflammation in asthma.

Notch4 signaling limits regulatory T-cell-mediated tissue repair and promotes severe lung inflammation in viral infections.

A cardinal feature of COVID-19 is lung inflammation and respiratory failure. In a prospective multi-country cohort of COVID-19 patients, we found that increased Notch4 expression on circulating regulatory T (Treg) cells was associated with disease severity, predicted mortality, and declined upon recovery. Deletion of Notch4 in Treg cells or therapy with anti-Notch4 antibodies in conventional and humanized mice normalized the dysregulated innate immunity and rescued disease morbidity and mortality induced by a synthetic analog of viral RNA or by influenza H1N1 virus. Mechanistically, Notch4 suppressed the induction by interleukin-18 of amphiregulin, a cytokine necessary for tissue repair. Protection by Notch4 inhibition was recapitulated by therapy with Amphiregulin and, reciprocally, abrogated by its antagonism. Amphiregulin declined in COVID-19 subjects as a function of disease severity and Notch4 expression. Thus, Notch4 expression on Treg cells dynamically restrains amphiregulin-dependent tissue repair to promote severe lung inflammation, with therapeutic implications for COVID-19 and related infections.

NOTCH4 potentiates the IL-13 induced genetic program in M2 alternative macrophages through the AP1 and IRF4-JMJD3 axis.

IL-13 signaling polarizes macrophages to an M2 alternatively activated phenotype, which regulates tissue repair and anti-inflammatory responses. However, an excessive activation of this pathway leads to severe pathologies, such as allergic airway inflammation and asthma. In this work, we identified NOTCH4 receptor as an important modulator of M2 macrophage activation. We show that the expression of NOTCH4 is induced by IL-13, mediated by Janus kinases and AP1 activity, probably mediated by the IL-13Rα1 and IL-13Rα2 signaling pathway. Furthermore, we demonstrate an important role for NOTCH4 signaling in the IL-13 induced gene expression program in macrophages, including various genes that contribute to pathogenesis of the airways in asthma, such as ARG1, YM1, CCL24, IL-10, or CD-163. We also demonstrate that NOTCH4 signaling modulates IL-13-induced gene expression by increasing IRF4 activity, mediated, at least in part, by the expression of the histone H3K27me3 demethylase JMJD3, and by increasing AP1-dependent transcription. In summary, our results provide evidence for an important role of NOTCH4 signaling in alternative activation of macrophages by IL-13 and suggest that NOTCH4 may contribute to the increased severity of lesions in M2 inflammatory responses, such as allergic asthma, which points to NOTCH4 as a potential new target for the treatment of these pathologies.

Notch1 and Notch4 core binding domain peptibodies exhibit distinct ligand-binding and anti-angiogenic properties.

The Notch signaling pathway is an important therapeutic target for the treatment of inflammatory diseases and cancer. We previously created ligand-specific inhibitors of Notch signaling comprised of Fc fusions to specific EGF-like repeats of the Notch1 extracellular domain, called Notch decoys, which bound ligands, blocked Notch signaling, and showed anti-tumor activity with low toxicity. However, the study of their function depended on virally mediated expression, which precluded dosage control and limited clinical applicability. We have refined the decoy design to create peptibody-based Notch inhibitors comprising the core binding domains, EGF-like repeats 10-14, of either Notch1 or Notch4. These Notch peptibodies showed high secretion properties and production yields that were improved by nearly 100-fold compared to previous Notch decoys. Using surface plasmon resonance spectroscopy coupled with co-immunoprecipitation assays, we observed that Notch1 and Notch4 peptibodies demonstrate strong but distinct binding properties to Notch ligands DLL4 and JAG1. Both Notch1 and Notch4 peptibodies interfere with Notch signaling in endothelial cells and reduce expression of canonical Notch targets after treatment. While prior DLL4 inhibitors cause hyper-sprouting, the Notch1 peptibody reduced angiogenesis in a 3-dimensional in vitro sprouting assay. Administration of Notch1 peptibodies to neonate mice resulted in reduced radial outgrowth of retinal vasculature, confirming anti-angiogenic properties. We conclude that purified Notch peptibodies comprising EGF-like repeats 10-14 bind to both DLL4 and JAG1 ligands and exhibit anti-angiogenic properties. Based on their secretion profile, unique Notch inhibitory activities, and anti-angiogenic properties, Notch peptibodies present new opportunities for therapeutic Notch inhibition.

The Clinical Application of Immunohistochemical Expression of Notch4 Protein in Patients with Colon Adenocarcinoma.

The Notch signalling pathway is one of the most conserved and well-characterised pathways involved in cell fate decisions and the development of many diseases, including cancer. Among them, it is worth noting the Notch4 receptor and its clinical application, which may have prognostic value in patients with colon adenocarcinoma. The study was performed on 129 colon adenocarcinomas. Immunohistochemical and fluorescence expression of Notch4 was performed using the Notch4 antibody. The associations between the IHC expression of Notch4 and clinical parameters were analysed using the Chi2 test or Chi2Yatesa test. The Kaplan-Meier analysis and the log-rank test were used to verify the relationship between the intensity of Notch4 expression and the 5-year survival rate of patients. Intracellular localisation of Notch4 was detected by the use of the immunogold labelling method and TEM. 101 (78.29%) samples had strong Notch4 protein expression, and 28 (21.71%) samples were characterised by low expression. The high expression of Notch4 was clearly correlated with the histological grade of the tumour (p < 0.001), PCNA immunohistochemical expression (p < 0.001), depth of invasion (p < 0.001) and angioinvasion (p < 0.001). We can conclude that high expression of Notch4 is correlated with poor prognosis of colon adenocarcinoma patients (log-rank, p < 0.001).

Androgen receptor suppresses vasculogenic mimicry in hepatocellular carcinoma via circRNA7/miRNA7-5p/VE-cadherin/Notch4 signalling.

Androgen receptor (AR) can suppress hepatocellular carcinoma (HCC) invasion and metastasis at an advanced stage. Vasculogenic mimicry (VM), a new vascularization pattern by which tumour tissues nourish themselves, is correlated with tumour progression and metastasis. Here, we investigated the effect of AR on the formation of VM and its mechanism in HCC. The results suggested that AR could down-regulate circular RNA (circRNA) 7, up-regulate micro RNA (miRNA) 7-5p, and suppress the formation of VM in HCC Small hairpin circR7 (ShcircR7) could reverse the impact on VM and expression of VE-cadherin and Notch4 increased by small interfering AR (shAR) in HCC, while inhibition of miR-7-5p blocked the formation of VM and expression of VE-cadherin and Notch4 decreased by AR overexpression (oeAR) in HCC. Mechanism dissection demonstrated that AR could directly target the circR7 host gene promoter to suppress circR7, and miR-7-5p might directly target the VE-cadherin and Notch4 3'UTR to suppress their expression in HCC. In addition, knockdown of Notch4 and/or VE-cadherin revealed that shVE-cadherin or shNotch4 alone could partially reverse the formation of HCC VM, while shVE-cadherin and shNotch4 together could completely suppress the formation of HCC VM. Those results indicate that AR could suppress the formation of HCC VM by down-regulating circRNA7/miRNA7-5p/VE-Cadherin/Notch4 signals in HCC, which will help in the design of novel therapies against HCC.

Human NOTCH4 is a key target of RUNX1 in megakaryocytic differentiation.

Megakaryocytes (MKs) in adult marrow produce platelets that play important roles in blood coagulation and hemostasis. Monoallelic mutations of the master transcription factor gene RUNX1 lead to familial platelet disorder (FPD) characterized by defective MK and platelet development. However, the molecular mechanisms of FPD remain unclear. Previously, we generated human induced pluripotent stem cells (iPSCs) from patients with FPD containing a RUNX1 nonsense mutation. Production of MKs from the FPD-iPSCs was reduced, and targeted correction of the RUNX1 mutation restored MK production. In this study, we used isogenic pairs of FPD-iPSCs and the MK differentiation system to identify RUNX1 target genes. Using integrative genomic analysis of hematopoietic progenitor cells generated from FPD-iPSCs, and mutation-corrected isogenic controls, we identified 2 gene sets the transcription of which is either up- or downregulated by RUNX1 in mutation-corrected iPSCs. Notably, NOTCH4 expression was negatively controlled by RUNX1 via a novel regulatory DNA element within the locus, and we examined its involvement in MK generation. Specific inactivation of NOTCH4 by an improved CRISPR-Cas9 system in human iPSCs enhanced megakaryopoiesis. Moreover, small molecules known to inhibit Notch signaling promoted MK generation from both normal human iPSCs and postnatal CD34+ hematopoietic stem and progenitor cells. Our study newly identified NOTCH4 as a RUNX1 target gene and revealed a previously unappreciated role of NOTCH4 signaling in promoting human megakaryopoiesis. Our work suggests that human iPSCs with monogenic mutations have the potential to serve as an invaluable resource for discovery of novel druggable targets.

FKBPL and its peptide derivatives inhibit endocrine therapy resistant cancer stem cells and breast cancer metastasis by downregulating DLL4 and Notch4.

BACKGROUND: Optimising breast cancer treatment remains a challenge. Resistance to therapy is a major problem in both ER- and ER+ breast cancer. Tumour recurrence after chemotherapy and/or targeted therapy leads to more aggressive tumours with enhanced metastatic ability. Self-renewing cancer stem cells (CSCs) have been implicated in treatment resistance, recurrence and the development of metastatic disease. METHODS: In this study, we utilised in vitro, in vivo and ex vivo breast cancer models using ER+ MCF-7 and ER- MDA-MB-231 cells, as well as solid and metastatic breast cancer patient samples, to interrogate the effects of FKBPL and its peptide therapeutics on metastasis, endocrine therapy resistant CSCs and DLL4 and Notch4 expression. The effects of FKBPL overexpression or peptide treatment were assessed using a t-test or one-way ANOVA with Dunnett's multiple comparison test. RESULTS: We demonstrated that FKBPL overexpression or treatment with FKBPL-based therapeutics (AD-01, pre-clinical peptide /ALM201, clinical peptide) inhibit i) CSCs in both ER+ and ER- breast cancer, ii) cancer metastasis in a triple negative breast cancer metastasis model and iii) endocrine therapy resistant CSCs in ER+ breast cancer, via modulation of the DLL4 and Notch4 protein and/or mRNA expression. AD-01 was effective at reducing triple negative MDA-MB-231 breast cancer cell migration (n ≥ 3, p < 0.05) and invasion (n ≥ 3, p < 0.001) and this was translated in vivo where AD-01 inhibited breast cancer metastasis in MDA-MB-231-lucD3H1 in vivo model (p < 0.05). In ER+ MCF-7 cells and primary breast tumour samples, we demonstrated that ALM201 inhibits endocrine therapy resistant mammospheres, representative of CSC content (n ≥ 3, p < 0.05). Whilst an in vivo limiting dilution assay, using SCID mice, demonstrated that ALM201 alone or in combination with tamoxifen was very effective at delaying tumour recurrence by 12 (p < 0.05) or 21 days (p < 0.001), respectively, by reducing the number of CSCs. The potential mechanism of action, in addition to CD44, involves downregulation of DLL4 and Notch4. CONCLUSION: This study demonstrates, for the first time, the pre-clinical activity of novel systemic anti-cancer therapeutic peptides, ALM201 and AD-01, in the metastatic setting, and highlights their impact on endocrine therapy resistant CSCs; both areas of unmet clinical need.

Notch4 Negatively Regulates the Inflammatory Response to Mycobacterium tuberculosis Infection by Inhibiting TAK1 Activation.

Tuberculosis, caused by Mycobacterium tuberculosis infection, remains a global threat to human health, but knowledge of the molecular mechanisms underlying the pathogenesis of tuberculosis is still limited. Although Notch4, a member of the Notch receptor family, is involved in the initiation of mammary tumors, its function in M. tuberculosis infection remains unclear. In this study, we found that Notch4-deficient mice were more resistant to M. tuberculosis infection, with a much lower bacterial burden and fewer pathological changes in the lungs. Notch4 inhibited M. tuberculosis-induced production of proinflammatory cytokines by interaction with TAK1 and inhibition of its activation. Furthermore, we found that Notch intracellular domain 4 prevented TRAF6 autoubiquitination and suppressed TRAF6-mediated TAK1 polyubiquitination. Finally, Notch inhibitors made mice more resistant to M. tuberculosis infection. These results suggest that Notch4 is a negative regulator of M. tuberculosis-induced inflammatory response, and treatment with a Notch inhibitor could serve as a new therapeutic strategy for tuberculosis.

NOTCH4 regulates colorectal cancer proliferation, invasiveness, and determines clinical outcome of patients.

Notch signal has complex roles in human malignancies, which might be attributed to the diversity of Notch receptors. Here, we set out to identify the association of NOTCH4 with colorectal cancer (CRC). In the hospital-based study cohort, we investigated NOTCH4 mRNA levels in primary CRC, as well as its association with clinicopathologic characteristics. Besides, NOTCH4 cDNA and siRNA was transfected into colorectal cancer cell line to elucidate its impact on tumor cell proliferation and migration. Results revealed a statistically significant lower expression of NOTCH4 mRNA in tumor specimens compared with that in control. NOTCH4 level in CRC was found to be related to tumor differentiation, invasion, and node metastasis. Moreover, it was demonstrated that NOTCH4 mRNA level could be an independent prognostic factor for both disease-free and overall survival of CRC patients. Overexpression of NOTCH4 in CRC cell lines suppressed tumor cell proliferation, migration, and invasion, while induced apoptosis. In the opposite, the malignant behavior of CRC cells was enhanced by NOTCH4 knockdown. These results demonstrated for the first time that NOTCH4 expression was decreased in CRC, which could determine tumor proliferation, relapse, and prognosis.

Notch-4 silencing inhibits prostate cancer growth and EMT via the NF-κB pathway.

Epithelial-mesenchymal transition (EMT) is implicated in the metastasis of human prostate cancer (PCa). Notch signaling has been established as a regulator of EMT. Notch-4 has emerged as a mammary proto-oncogene and a target in several cancers. However, the role and the mechanism of action of Notch-4 in PCa are still unclear. In the present study, we first observed a marked increase in Notch-4 expression in the PCa cell lines DU145, PC3 and LnCAP compared with the non-malignant prostate epithelial cell line RWPE1. Knocking down the expression of Notch-4 suppressed the viability and proliferation in the PCa cell lines DU145 and PC3. Also, further study showed that a decline in Notch-4 significantly promoted apoptosis in PC3 cells. Notch-4 silencing also resulted in decreased cell migration and invasion and affected the expression of EMT markers. We hypothesized that Notch-4 ablation suppresses the activity of NF-κB, so we used PMA to stimulate NF-κB p50 and p65 activation in PC3 cells. The results indicate that PMA treatment impaired the action of Notch-4 ablation in the biology of PC3 cells including cell growth, apoptosis, migration, invasion and EMT. The results of the present study show that RNAi targeting against Notch-4 expression suppresses PCa progression.

Analysis of the microvascular morphology and hemodynamics of breast cancer in mice using SPring-8 synchrotron radiation microangiography.

Tumor vasculature is characterized by morphological and functional abnormalities. However, analysis of the dynamics in blood flow is still challenging because of limited spatial and temporal resolution. Synchrotron radiation (SR) microangiography above the K-edge of the iodine contrast agent can provide high-contrast imaging of microvessels in time orders of milliseconds. In this study, mice bearing the human breast cancer cell lines MDAMB231 and NOTCH4 overexpression in MDAMB231 (MDAMB231NOTCH4+) and normal mice were assessed using SR microangiography. NOTCH is transmembrane protein that has crucial roles for vasculogenesis, angiogenesis and tumorigenesis, and NOTCH4 is considered to be a cause of high-flow arteriovenous shunting. A subgroup of mice received intravenous eribulin treatment, which is known to improve intratumor core circulation (MDAMB231_eribulin). Microvessel branches from approximately 200 µm to less than 20 µm in diameter were observed within the same visual field. The mean transition time (MTT) was measured as a dynamic parameter and quantitative analysis was performed. MTT in MDAMB231 was longer than that in normal tissue, and MDAMB231NOTCH4+ showed shorter MTT [5.0 ± 1.4 s, 3.6 ± 1.0 s and 3.6 ± 1.1 s (mean ± standard deviation), respectively]. After treatment, average MTT was correlated to tumor volume (r = 0.999) in MDAMB231_eribulin, while in contrast there was no correlation in MDAMB231 (r = -0.026). These changes in MTT profile are considered to be driven by the modulation of intratumoral circulation dynamics. These results demonstrate that a SR microangiography approach enables quantitative analysis of morphological and dynamic characteristics of tumor vasculature in vivo. Further studies will reveal new findings concerning vessel function in tumors.

Regulation of Notch-mediated transcription by a bovine herpesvirus 1 encoded protein (ORF2) that is expressed in latently infected sensory neurons.

Bovine herpesvirus 1 (BoHV-1) is an Alphaherpesvirinae subfamily member that establishes life-long latency in sensory neurons. The latency-related RNA (LR-RNA) is abundantly expressed during latency. An LR mutant virus containing stop codons at the amino-terminus of open reading frame (ORF)2 does not reactivate from latency and replicates less efficiently in tonsils and trigeminal ganglia. ORF2 inhibits apoptosis, interacts with Notch family members, and interferes with Notch-dependent transcription suggesting ORF2 expression enhances survival of infected neurons. The Notch signaling pathway is crucial for neuronal differentiation and survival suggesting that interactions between ORF2 and Notch family members regulate certain aspects of latency. Consequently, for this study, we compared whether ORF2 interfered with the four mammalian Notch family members. ORF2 consistently interfered with Notch1-3-mediated transactivation of three cellular promoters. Conversely, Notch4-mediated transcription was not consistently inhibited by ORF2. Electrophoretic shift mobility assays using four copies of a consensus-DNA binding site for Notch/CSL (core binding factor (CBF)-1, Suppressor of Hairless, Lag-2) as a probe revealed ORF2 interfered with Notch1 and 3 interactions with a CSL family member bound to DNA. Additional studies demonstrated ORF2 enhances neurite sprouting in mouse neuroblastoma cells that express Notch1-3, but not Notch4. Collectively, these studies indicate that ORF2 inhibits Notch-mediated transcription and signaling by interfering with Notch interacting with CSL bound to DNA.

Mechanoresponsive ETS1 causes endothelial dysfunction and arterialization in varicose veins via NOTCH4/DLL4 signaling.

Varicose veins are the most common venous disorder in humans and are characterized by hemodynamic instability due to valvular insufficiency and orthostatic lifestyle factors. It is unclear how changes in biomechanical signals cause aberrant remodeling of the vein wall. Our previous studies suggest that Notch signaling is implicated in varicose vein arterialization. In the arterial system, mechanoresponsive ETS1 is a transcriptional activator of the endothelial Notch, but its involvement in sensing disrupted venous flow and varicose vein formation has not been investigated. Here, we use human varicose veins and cultured human venous endothelial cells to show that disturbed venous shear stress activates ETS1-NOTCH4/DLL4 signaling. Notch components were highly expressed in the neointima, whereas ETS1 was upregulated in all histological layers of varicose veins. In vitro microfluidic flow-based studies demonstrate that even minute changes in venous flow patterns enhance ETS1-NOTCH4/DLL4 signaling. Uniform venous shear stress, albeit an inherently low-flow system, does not induce ETS1 and Notch proteins. ETS1 activation under altered flow was mediated primarily by MEK1/2 and, to a lesser extent, by MEK5 but was independent of p38 MAP kinase. Endothelial cell-specific ETS1 knockdown prevented disturbed flow-induced NOTCH4/DLL4 expression. TK216, an inhibitor of ETS-family, prevented the acquisition of arterial molecular identity and loss of endothelial integrity in cells exposed to the ensuing altered shear stress. We conclude that ETS1 senses blood flow disturbances and may promote venous remodeling by inducing endothelial dysfunction. Targeting ETS1 rather than downstream Notch proteins could be an effective and safe strategy to develop varicose vein therapies.

Endothelial Rbpj deletion normalizes Notch4-induced brain arteriovenous malformation in mice.

Upregulation of Notch signaling is associated with brain arteriovenous malformation (bAVM), a disease that lacks pharmacological treatments. Tetracycline (tet)-regulatable endothelial expression of constitutively active Notch4 (Notch4*tetEC) from birth induced bAVMs in 100% of mice by P16. To test whether targeting downstream signaling, while sustaining the causal Notch4*tetEC expression, induces AVM normalization, we deleted Rbpj, a mediator of Notch signaling, in endothelium from P16, by combining tet-repressible Notch4*tetEC with tamoxifen-inducible Rbpj deletion. Established pathologies, including AV connection diameter, AV shunting, vessel tortuosity, intracerebral hemorrhage, tissue hypoxia, life expectancy, and arterial marker expression were improved, compared with Notch4*tetEC mice without Rbpj deletion. Similarly, Rbpj deletion from P21 induced advanced bAVM regression. After complete AVM normalization induced by repression of Notch4*tetEC, virtually no bAVM relapsed, despite Notch4*tetEC re-expression in adults. Thus, inhibition of endothelial Rbpj halted Notch4*tetEC bAVM progression, normalized bAVM abnormalities, and restored microcirculation, providing proof of concept for targeting a downstream mediator to treat AVM pathologies despite a sustained causal molecular lesion.

JAG1-NOTCH4 mechanosensing drives atherosclerosis.

Endothelial cell (EC) sensing of disturbed blood flow triggers atherosclerosis, a disease of arteries that causes heart attack and stroke, through poorly defined mechanisms. The Notch pathway plays a central role in blood vessel growth and homeostasis, but its potential role in sensing of disturbed flow has not been previously studied. Here, we show using porcine and murine arteries and cultured human coronary artery EC that disturbed flow activates the JAG1-NOTCH4 signaling pathway. Light-sheet imaging revealed enrichment of JAG1 and NOTCH4 in EC of atherosclerotic plaques, and EC-specific genetic deletion of Jag1 (Jag1ECKO) demonstrated that Jag1 promotes atherosclerosis at sites of disturbed flow. Mechanistically, single-cell RNA sequencing in Jag1ECKO mice demonstrated that Jag1 suppresses subsets of ECs that proliferate and migrate. We conclude that JAG1-NOTCH4 sensing of disturbed flow enhances atherosclerosis susceptibility by regulating EC heterogeneity and that therapeutic targeting of this pathway may treat atherosclerosis.

The NOTCH4-GATA4-IRG1 axis as a novel target in early-onset colorectal cancer.

The early onset of colorectal cancer (CRC) in individuals younger than 50 years is an emerging phenomenon, and obesity is a strong risk factor. Inflammatory mechanisms are mediated by immune cells, with macrophages and their phenotypical changes playing a significant role in CRC. Obesity-related hormones, such as leptin and adiponectin, affect macrophage polarization and cytokine expression. Macrophage metabolism, and therefore polarization, directly affects tumor progression and survival in patients with CRC. Altered obesity-related hormone levels induce phosphoinositide kinase-3 (PI3K)/serine-threonine-protein kinase (AKT) activation in colon cancer, causing increased cell survival, hyperplasia, and proliferation. Investigating the effects of obesity-related mechanisms on PI3K/Akt signaling can provide new insights for targeting mechanisms in CRC and obesity among the young. Central molecules for the control of cell proliferation, differentiation, and tumorigenesis within the gastrointestinal tract include downstream targets of the PI3K/AKT pathway, such as Neurogenic locus notch homolog 4 (Notch4) and GATA binding proteins (GATA). Leptin and adiponectin both alter gene expression within this pathway, thereby affecting TAM-mediated CRC progression. Our goal is to introduce the NOTCH4-GATA4-IRG1 axis as a link between inflammation and sporadic CRC and to discuss this pathway as a new potential immunotherapeutic target in individuals affected with obesity and early-onset CRC.

NOTCH4 Exhibits Anti-Inflammatory Activity in Activated Macrophages by Interfering With Interferon-γ and TLR4 Signaling.

NOTCH4 is a member of the NOTCH family of receptors whose expression is intensively induced in macrophages after their activation by Toll-like receptors (TLR) and/or interferon-γ (IFN-γ). In this work, we show that this receptor acts as a negative regulator of macrophage activation by diminishing the expression of proinflammatory cytokines, such as IL-6 and IL-12, and costimulatory proteins, such as CD80 and CD86. We have observed that NOTCH4 inhibits IFN-γ signaling by interfering with STAT1-dependent transcription. Our results show that NOTCH4 reprograms the macrophage response to IFN-γ by favoring STAT3 versus STAT1 phosphorylation without affecting their expression levels. This lower activation of STAT1 results in diminished transcriptional activity and expression of STAT1-dependent genes, including IRF1, SOCS1 and CXCL10. In macrophages, NOTCH4 inhibits the canonical NOTCH signaling pathway induced by LPS; however, it can reverse the inhibition exerted by IFN-γ on NOTCH signaling, favoring the expression of NOTCH-target genes, such as Hes1. Indeed, HES1 seems to mediate, at least in part, the enhancement of STAT3 activation by NOTCH4. NOTCH4 also affects TLR signaling by interfering with NF-κB transcriptional activity. This effect could be mediated by the diminished activation of STAT1. These results provide new insights into the mechanisms by which NOTCH, TLR and IFN-γ signal pathways are integrated to modulate macrophage-specific effector functions and reveal NOTCH4 acting as a new regulatory element in the control of macrophage activation that could be used as a target for the treatment of pathologies caused by an excess of inflammation.

Reciprocal activation of HEY1 and NOTCH4 under SOX2 control promotes EMT in head and neck squamous cell carcinoma.

Several comprehensive studies have demonstrated that the NOTCH pathway is altered in a bimodal manner in head and neck squamous cell carcinoma (HNSCC). In a previous study, it was found that the NOTCH4/HEY1 pathway was specifically upregulated in HNSCC and promoted epithelial­mesenchymal transition (EMT), and that HEY1 activation supported SOX2 expression. However, the interactions in this pathway have not yet been fully elucidated. The present study investigated the NOTCH4/HEY1/SOX2 axis in HNSCC using in vitro models and the Cancer Genome Atlas (TCGA) database. To explore the association, reporter and ChIP RT­qPCR assays using SOX2­overexpressing (SOX2­OE) cells were performed. The association between NOTCH4 and HEY1 was examined in the same manner using HEY1­overexpressing (HEY1­OE) cells. The results of the in vitro experiments indicated that HEY1 promoted EMT in the HNSCC cells. Furthermore, the overexpression of HEY1 also promoted sphere formation and increased murine xenograft tumorigenicity. Reporter assays and ChIP RT­qPCR experiments indicated that SOX2 regulated HEY1 expression via direct binding of the HEY1 promoter. HEY1 expression significantly correlated with SOX2 expression in primary lung SCC and other SCCs using the TCGA database. HEY1 also regulated NOTCH4 expression to create a positive reciprocal feedback loop. On the whole, the present study demonstrates that HEY1 expression in HNSCC is regulated via the promotion of SOX2 and promotes EMT. The NOTCH4/HEY1 pathway is specifically upregulated via a positive reciprocal feedback loop mediated by the HEY1­medaited regulation of NOTCH4 transcription, and SOX2 correlates with HEY1 expression in SCC from other primary sites.

Rare variant analysis in eczema identifies exonic variants in DUSP1, NOTCH4 and SLC9A4.

Previous genome-wide association studies revealed multiple common variants involved in eczema but the role of rare variants remains to be elucidated. Here, we investigate the role of rare variants in eczema susceptibility. We meta-analyze 21 study populations including 20,016 eczema cases and 380,433 controls. Rare variants are imputed with high accuracy using large population-based reference panels. We identify rare exonic variants in DUSP1, NOTCH4, and SLC9A4 to be associated with eczema. In DUSP1 and NOTCH4 missense variants are predicted to impact conserved functional domains. In addition, five novel common variants at SATB1-AS1/KCNH8, TRIB1/LINC00861, ZBTB1, TBX21/OSBPL7, and CSF2RB are discovered. While genes prioritized based on rare variants are significantly up-regulated in the skin, common variants point to immune cell function. Over 20% of the single nucleotide variant-based heritability is attributable to rare and low-frequency variants. The identified rare/low-frequency variants located in functional protein domains point to promising targets for novel therapeutic approaches to eczema.