Notch Intracellular Domain Research Articles

Effect of Notch1 signaling on cellular proliferation and apoptosis in human laryngeal carcinoma

BackgroundThe occurrence and development of malignancies include excessive proliferation and apoptosis resistance in tumor cells. This study aimed to identify the effects of Notch1 signaling on proliferation and apoptosis of laryngeal cancer cells in a hypoxic microenvironment.MethodsNotch1 and Ki-67 expression in laryngeal squamous cell carcinoma (LSCC) tissues was detected by immunohistochemistry. The apoptotic index (AI) of LSCC was evaluated by the TUNEL method. Small interfering RNA (siRNA) was used to inhibit Notch1 expression in laryngeal cancer cells. Real-time PCR was used to measure Notch1, Hes1, and Hey1 mRNA expression, and Western blotting was used to measure Notch1 and Notch1 intracellular domain (N1ICD) protein expression. Annexin V-FITC/propidium iodide staining and Cell Counting Kit-8 assays were used to measure cell apoptosis and proliferation, respectively.ResultsNotch1 expression was significantly related to the proliferation index (PI) and AI in LSCC tissues. Hypoxia could induce proliferation and inhibit apoptosis in cancer cells. Notch1 expression and Notch1 signaling activity could be upregulated by hypoxia. Suppressing Notch1 signaling activity in hypoxic cells could decrease proliferation and increase apoptosis.ConclusionsOur study has demonstrated that hypoxia may promote proliferation and inhibit apoptosis of laryngeal cancer cells. Notch1 signaling may play a pivotal role in regulating the proliferation and apoptosis resistance of laryngeal cancer cells under hypoxic conditions.

The Notch signaling pathway in skeletal muscle health and disease.

The Notch signaling pathway is a key regulator of skeletal muscle development and regeneration. Over the past decade, the discoveries of three new muscle disease genes have added a new dimension to the relationship between the Notch signaling pathway and skeletal muscle: MEGF10, POGLUT1, and JAG2. We review the clinical syndromes associated with pathogenic variants in each of these genes, known molecular and cellular functions of their protein products with a particular focus on the Notch signaling pathway, and potential novel therapeutic targets that may emerge from further investigations of these diseases. The phenotypes associated with two of these genes, POGLUT1 and JAG2, clearly fall within the realm of muscular dystrophy, whereas the third, MEGF10, is associated with a congenital myopathy/muscular dystrophy overlap syndrome classically known as early-onset myopathy, areflexia, respiratory distress, and dysphagia. JAG2 is a canonical Notch ligand, POGLUT1 glycosylates the extracellular domain of Notch receptors, and MEGF10 interacts with the intracellular domain of NOTCH1. Additional genes and their encoded proteins relevant to muscle function and disease with links to the Notch signaling pathway include TRIM32, ATP2A1 (SERCA1), JAG1, PAX7, and NOTCH2NLC. There is enormous potential to identify convergent mechanisms of skeletal muscle disease and new therapeutic targets through further investigations of the Notch signaling pathway in the context of skeletal muscle development, maintenance, and disease.

Clinical disease course and survival outcomes following disease recurrence in adenoid cystic carcinoma with and without NOTCH signaling pathway activation

BackgroundAdenoid cystic carcinoma (ACC) is a rare salivary cancer. The highest rates of disease recurrence are in patients with NOTCH pathway activation, reported in up to 20%. Novel drugs targeting NOTCH signaling are under investigation in the recurrent/metastatic (R/M) setting. To understand their clinical utility, there is an urgent need to better characterize the disease course and outcomes following current standard of care treatment. Methods120 patients with R/M ACC underwent clinical review at a single UK Cancer Centre. Patients were retrospectively assessed for tumor NOTCH pathway activation using next generation sequencing (NGS) targeting NOTCH1/2/3 genes and/or NOTCH1 intra-cellular domain (NICD1) immunohistochemistry. Demographic and treatment data were extracted from the clinical notes. Kaplan-Meier survival analysis was performed using log rank test. ResultsNOTCH pathway activation was identified in 13/120 patients (11 %). In 12/101 patients analyzed by NGS, NOTCH1/3 activating somatic mutations were identified, and a further patient was identified with NICD1 diffuse nuclear staining in whom NGS testing was not possible. Patients with NOTCH pathway activation had shorter median RFS (1.1 vs 3.4 years, p = 0.2032) and significantly reduced median OS from diagnosis (4.0 vs 16.3 years, p < 0.0001). There was significantly reduced median OS from time of disease recurrence/metastasis (1.9 vs 9.6 years, p < 0.0001). ConclusionThis study clearly demonstrates a reduction in OS from time of first confirmed disease recurrence/metastasis for patients with NOTCH pathway activated ACC. This provides support for developing new drugs for this sub-group of patients, for whom clinical outcomes are significantly worse and effective treatments are lacking.

Akt1-dependent expression of angiopoietin 1 and 2 in vascular smooth muscle cells leads to vascular stabilization

Retinal angiogenesis was delayed in VSMC-specific Akt1-deficient mice (Akt1∆SMC) but not in Akt2∆SMC mice. The proliferation of ECs, recruitment of pericytes, and coverage of VSMCs to the endothelium were defective in Akt1∆SMC. The silencing of Akt1 in VSMCs led to the downregulation of angiopoietin 1 (Ang1) and the upregulation of Ang2. The activation of Notch3 in VSMCs was significantly reduced in the retinas of Akt1∆SMC mice. Silencing Akt1 suppressed the activation of Notch3. Moreover, the silencing of Notch3 downregulated Ang1, whereas the overexpression of Notch3 intracellular domain (NICD3) enhanced Ang1 expression. The nuclear localization and transcriptional activity of yes-associated protein (YAP) were affected by the expression level of Akt1. Silencing YAP downregulated Ang2 expression, whereas overexpression of YAP showed the opposite results. Ang1 antibody and Ang2 suppressed endothelial sprouting of wild-type aortic tissues, whereas the Ang2 antibody and Ang1 facilitated the endothelial sprouting of aortic tissues from Akt1∆SMC mice. Finally, severe hemorrhage was observed in Akt1∆SMC mice, which was further facilitated under streptozotocin (STZ)-induced diabetic conditions. Therefore, the Akt1-Notch3/YAP-Ang1/2 signaling cascade in VSMCs might play an essential role in the paracrine regulation of endothelial function.

Notch signaling as a novel target for uterine leiomyosarcoma (259)

<b>Objectives:</b> The Notch signaling pathway is evolutionarily conserved and has been implicated as a potential therapeutic target in several cancers. Uterine leiomyosarcoma (uLMS) is a rare and aggressive cancer with poor survival, higher prevalence in Black women than White women, and few effective adjuvant therapies. The objective of this study was to determine the expression of Notch in uLMS as a foundation for investigating the Notch signaling pathway as a potential therapeutic target in uLMS. <b>Methods:</b> We analyzed previously published mRNA sequencing (seq) datasets on LMS, including uLMS and other sarcoma patient tissue samples for expression of Notch pathway genes. We performed immunostaining for <i>NOTCH1</i> and <i>NOTCH3</i> on uterine tissue from benign hysterectomies (BH, <i>n</i>=3) and uLMS samples (<i>n</i>=30) from a multiple organ LMS tissue array (US Biomax, Inc). <i>NOTCH1</i> expression was measured as a percent positive stain per area, and <i>NOTCH3</i> expression was quantified with an H-Score. We analyzed Notch intracellular domain (ICD) protein expression in human uterine smooth muscle cell lines (hUT-SMC) and uLMS cell lines, SK-UT-1B and SK-LMS-1, by Western blot, and expression of Notch effectors, <i>HES1, HEY1,</i> and <i>NRARP,</i> by qPCR and immunofluorescence. Numerical data were reported as mean ± SEM or median ± interquartile range and compared using unpaired t-test or Mann-Whitney U, with p≤0.05 being statistically significant. <b>Results:</b> mRNA seq analysis revealed similar levels of expression for <i>NOTCH1, 2,</i> and <i>3</i> and <i>HES1</i> in LMS compared to other sarcomas. <i>NOTCH4</i> was not included in the original mRNA seq data and thus could not be evaluated for expression levels. Immunostaining revealed that median H-score of <i>NOTCH3</i> in uLMS was higher than in BH (211.4, IQR: 165.6-239.3 vs 100.3, IQR: 100.0-100.5, p≤0.05), whereas mean expression of <i>NOTCH1</i> was similar (3.7 ± 1.6% vs 1.9 ± 0.5%, p>0.05). Western blot analysis demonstrated that ICD for <i>NOTCH1-4</i> was expressed in hUT-SMC, N1ICD and N2ICD were expressed in SK-UT-1B, and N1ICD, N2ICD, and N4ICD were expressed in SK-LMS-1. mRNA expression of <i>HES1, HEY1,</i> and <i>NRARP</i> and nuclear expression of <i>HES1</i> indicate active Notch signaling in SK-LMS-1 and SK-UT-1B cells. <b>Conclusions:</b> Notch is expressed in uLMS patient tissues and uLMS cell lines. Notch signaling and distinct expression of Notch proteins in uLMS cell lines support the investigation of Notch inhibition as a potential target for treatment options in uLMS. Further studies are warranted to determine the efficacy of targeting Notch inhibition using therapies, such as gamma-secretase inhibitors alone or in combination with a cytotoxic agent.

Notch-dependent and -independent functions of transcription factor RBPJ.

Signal transduction pathways often involve transcription factors that promote activation of defined target gene sets. The transcription factor RBPJ is the central player in Notch signaling and either forms an activator complex with the Notch intracellular domain (NICD) or a repressor complex with corepressors like KYOT2/FHL1. The balance between these two antagonizing RBPJ-complexes depends on the activation state of the Notch receptor regulated by cell-to-cell interaction, ligand binding and proteolytic cleavage events. Here, we depleted RBPJ in mature T-cells lacking active Notch signaling and performed RNA-Seq, ChIP-Seq and ATAC-seq analyses. RBPJ depletion leads to upregulation of many Notch target genes. Ectopic expression of NICD1 activates several Notch target genes and enhances RBPJ occupancy. Based on gene expression changes and RBPJ occupancy we define four different clusters, either RBPJ- and/or Notch-regulated genes. Importantly, we identify early (Hes1 and Hey1) and late Notch-responsive genes (IL2ra). Similarly, to RBPJ depletion, interfering with transcriptional repression by squelching with cofactor KYOT2/FHL1, leads to upregulation of Notch target genes. Taken together, RBPJ is not only an essential part of the Notch co-activator complex but also functions as a repressor in a Notch-independent manner.

Effects of the interaction of Notch and TLR4 pathways on inflammation and heart function in septic heart.

We investigated the role of the interaction between the Notch and Toll-like receptor 4 (TLR4) pathways in septic myocardial injury. The sepsis model was induced in rats with lipopolysaccharide (LPS). Rats were divided into control, LPS, LPS + TAK242 ((6R)-6-[N-(2-chloro-4-fluorophenyl)sulfamoyl]cyclohex-1-ene-1-carboxylate) and LPS + DAPT (N-[N-(3,5-difluorophenacetyl)-l-alanyl]-s-phenylglycinetbutylester) groups. Heart function was evaluated with a Cardiac Doppler ultrasound. Myocardial morphological changes were detected by hematoxylin-eosin staining (H&E). Apoptosis was assessed by a TUNEL assay. The mRNA and protein levels were detected with real-time PCR, Western blot, and immunohistochemistry analysis. We found that heart function in the LPS + TAK242 group was significantly improved, but not in the LPS + DAPT group. LPS + TAK242 had a lower level of degeneration and necrosis of cardiomyocytes and inflammatory cell infiltration, as well as lower apoptosis and caspase-3 expression than the LPS group. Compared with the LPS group, the inflammatory cell infiltration was reduced in the LPS + DAPT group, while the degeneration and necrosis of cardiomyocytes were not obviously improved. Additionally, the expression levels of tumor necrosis factor-α and Interleukin-6, the protein contents of Notch intracellular domain and Hes1, and the P65 nuclear factor kappa-B (NF-κB) to P-P65 NF-κB ratio in LPS + TAK242 group and LPS + DAPT group were significantly lower than those in LPS group. Conclusively, the interaction between TLR4 and Notch signaling pathways enhances the inflammatory response in the septic heart by activating NF-κB. Blocking the TLR4 pathway with TAK242 can improve heart dysfunction and myocardial damage in sepsis, while blocking the Notch pathway with DAPT cannot effectively prevent heart dysfunction and myocardial damage in sepsis.

Melatonin Attenuates Vascular Smooth Muscle Contraction Through the γ-Secretase/Notch Intracellular Domain/Myocardin Pathway.

Inositol 1, 4, 5-trisphosphate (IP3) signaling-mediated calcium release drives the contraction of vascular smooth muscles and hence regulates blood vessel volume and blood pressure. Melatonin supplementation has been suggested to be beneficial for hypertension. To determine whether the blood pressure-lowering effect of melatonin was accounted for by IP3 signaling, we evaluated the vasoconstriction response and IP3 signaling in isolated mouse thoracic aortic rings during melatonin incubation. C57BL/6 mice were given intraperitoneal injections daily with melatonin, and the systolic blood pressure and contractility of aortic rings from melatonin-treated mice were decreased, and the contraction suppression effect of melatonin was attributed to the impaired expression of contractile proteins in vascular smooth muscle cells rather than IP3 signaling. Our results further showed that melatonin increased the expression of γ-secretase, which could cleave and release the notch intracellular domain, and the notch intracellular domain prevented the transcription of contractile genes by interfering with the interaction between serum response factor and myocardin, the master regulator of contractile protein. In this article, we report a novel mechanism by which melatonin regulates smooth muscle contractility that does not depend on IP3 signaling.

Hydroxylation of the NOTCH1 intracellular domain regulates Notch signaling dynamics

Notch signaling plays a pivotal role in the development and, when dysregulated, it contributes to tumorigenesis. The amplitude and duration of the Notch response depend on the posttranslational modifications (PTMs) of the activated NOTCH receptor – the NOTCH intracellular domain (NICD). In normoxic conditions, the hydroxylase FIH (factor inhibiting HIF) catalyzes the hydroxylation of two asparagine residues of the NICD. Here, we investigate how Notch-dependent gene transcription is regulated by hypoxia in progenitor T cells. We show that the majority of Notch target genes are downregulated upon hypoxia. Using a hydroxyl-specific NOTCH1 antibody we demonstrate that FIH-mediated NICD1 hydroxylation is reduced upon hypoxia or treatment with the hydroxylase inhibitor dimethyloxalylglycine (DMOG). We find that a hydroxylation-resistant NICD1 mutant is functionally impaired and more ubiquitinated. Interestingly, we also observe that the NICD1-deubiquitinating enzyme USP10 is downregulated upon hypoxia. Moreover, the interaction between the hydroxylation-defective NICD1 mutant and USP10 is significantly reduced compared to the NICD1 wild-type counterpart. Together, our data suggest that FIH hydroxylates NICD1 in normoxic conditions, leading to the recruitment of USP10 and subsequent NICD1 deubiquitination and stabilization. In hypoxia, this regulatory loop is disrupted, causing a dampened Notch response.

Activation of NOTCH signaling via DLL1 is mediated by APE1-redox-dependent NF-κB activation in oesophageal adenocarcinoma

ObjectiveOesophageal adenocarcinoma (EAC) arises in the setting of Barrett’s oesophagus, an intestinal metaplastic precursor lesion that can develop in patients with chronic GERD. Here, we investigated the role of acidic...

Abstract 1107: Notch activation drives resistance to Kras(G12C) inhibition in lung adenocarcinoma

Abstract Approximately one-third of lung adenocarcinoma (LUAD) tumors contain driver mutations in the Kras GTPase, most commonly occurring as Kras(G12C) activating mutations. Despite the promising development of covalent G12C small molecule inhibitors, early-phase clinical trial data indicate most patients will likely demonstrate disease progression through either intrinsic or acquired resistance. Thus, there is an urgent need to identify such resistance mechanisms to targeting Kras. Our lab previously discovered a unique requirement for Notch3 in tumor-propagating cells from the KrasLSL-G12D p53fl/fl LUAD mouse model as well as in patient-derived xenografts. Here we report activated Notch3 as a novel driver of resistance to Kras(G12C) inhibition in LUAD cell lines. Aiming to study the Notch3 transcriptional signature in LUAD, we developed an inducible model of Notch3 intracellular domain (NICD) expression in Kras-mutant LUAD cell lines. Our data demonstrate that activated NICD modulates downstream RTK signaling pathways to suppress both cell cycle arrest and apoptosis in response to G12C inhibitor treatment. We subsequently performed RNA-sequencing to identify Notch-dependent genes driving resistance and in parallel, used Cut&amp;Run technology to interrogate the global chromatin binding profiles of Notch3 and associated histone marks in NICD-expressing cell lines. We integrate our transcriptomic and epigenomic data to reveal that NICD acts as a key regulator of Rho family genes, which in turn support cell survival upon Kras inhibition. We additionally find enrichment of cell division and morphological signatures in inhibitor treated NICD cell lines and we elucidate the mechanism of direct Notch target genes in mediating the apoptotic response. Finally, we conduct a focused in vitro CRISPRi screen in the H358 LUAD cell line to identify the subset of Notch target genes required for NICD-induced resistance. Our results define a Notch3 transcriptional landscape in LUAD and demonstrate a role for Notch signaling in promoting resistance to small molecule Kras inhibitors. Citation Format: Elizabeth E. Hwang, Alex Lee, Stanley Leung, Marcus Breese, Alejandro Sweet-Cordero. Notch activation drives resistance to Kras(G12C) inhibition in lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1107.

Abstract 6308: Antitumor effects of RBPJ Inhibitor-1 on osteosarcoma cells

Abstract Osteosarcoma (OS) is the most common primary malignancy of the bone in adolescents and children with limited treatment options. Drug resistance to current therapy together with severe side effects urgently calls for alternative treatment strategies targeting critical cancer signaling pathways. Recently, our and other studies suggest that hyperactivation of Notch signaling contributes to the pathogenesis of human and murine OS, and its inhibition may be a therapeutic approach for the treatment of OS. Notch pathophysiological activity involves activated Notch (i.e. NICD, the notch intracellular domain) forming a nuclear complex with DNA-binding factors and coactivators such as recombination signal binding protein for immunoglobulin kappa J region (RBPJ). This evolutionary conserved mechanism is involved in the maintenance and survival of cancer stem cells, which can govern various cell activities like metastasis and chemoresistance. This study aims to examine the in vitro effects of targeting the NICD-RBPJ interaction using a small molecular inhibitor of the RBPJ (RIN1) on osteosarcoma cell lines. We found that treatment of 10 µM RIN1 on SJSA-1 (ATCC, CRL-2098), a human osteosarcoma cell line with a high Notch activity, can significantly decrease cell proliferation at 48 and 72 hours (h). We also found that at 10 µM of RIN1 affects SJSA-1 migration using a Boyden chamber-based cell system at 24 h, a time point where we find minimal contribution from an effect on cell proliferation. Treatment with RIN1 can block SJSA-1 clonogenicity, but we showed that it has limited ability to block SJSA-1 cell invasion. Treatment with RIN1 does not initiate or enhance the osteoblast-like differentiation of SJSA-1. Notch target genes HEY1 and CyclinD1 was significantly modulated in SJSA-1 cells upon treatment in a Notch-independent manner. Interestingly, treatment with RIN1 on SJSA-1 decreases SOX2 protein expression, a well-known cancer stem cell marker, and upregulates tumor suppressor RB1 protein expression. Ongoing studies include additional cellular function assays and RNA-seq transcriptomic analysis of RIN1’s effect on two Notch-dependent cancer cell lines including MB 157 (ATCC, CRL-7721) and T29 in its activating context. In summary, RIN1 has been shown to have a significant effect on the different cell behaviors of human osteosarcoma, which may eventually facilitate with the treatment of patients that have metastatic disease. Citation Format: Dakota Callahan, Leetoria Hinojosa, Haydee M. Torres, Jianning Tao. Antitumor effects of RBPJ Inhibitor-1 on osteosarcoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6308.

RNF144B stimulates the proliferation and inhibits the apoptosis of human spermatogonial stem cells via the FCER2/NOTCH2/HES1 pathway and its abnormality is associated with azoospermia.

Studies on gene regulation and signaling transduction pathways of human spermatogonial stem cells (SSCs) are of the utmost significance for unveiling molecular mechanisms underlying human spermatogenesis and gene therapy of male infertility. We have demonstrated, for the first time, that RNF144B stimulated cell proliferation and inhibited the apoptosis of human SSCs. The target of RNF144B was identified as FCER2 by RNA sequencing. We revealed that RNF144B interacted with FCER2 by immunoprecipitation. Consistently, overexpression of FCER2 reversed the phenotype of proliferation and apoptosis of human SSCs caused by RNF144B knockdown. Interestingly, FCER2 pulled down N2ICD (NOTCH2 intracellular domain), while N2ICD could bind to FCER2 in human SSCs. The levels of NOTCH2, FCER2, HES1, and HEY1 were reduced by RNF144B siRNA in human SSCs. Significantly, RNF144B was expressed at a lower level in nonobstructive azoospermia (NOA) patients than in the obstructive azoospermia (OA) patients with normal spermatogenesis, and 52 patients with heterozygous mutations of RNF144B were detected in 1,000 NOA patients. These results implicate that RNF144B promotes the proliferation of human SSCs and suppresses their apoptosis via the FCER2/NOTCH2/HES1 pathway and that the abnormality of RNF144B is associated with spermatogenesis failure. This study thus provides novel molecular mechanisms regulating the fate determinations of human SSCs, and it offers new biomarkers for the diagnosis and treatment of male infertility.

Correlation of Immunological and Molecular Profiles with Response to Crizotinib in Alveolar Soft Part Sarcoma: An Exploratory Study Related to the EORTC 90101 "CREATE" Trial.

Alveolar soft part sarcoma (ASPS) is a rare subtype of soft tissue sarcoma characterized by an unbalanced translocation, resulting in ASPSCR1-TFE3 fusion that transcriptionally upregulates MET expression. The European Organization for Research and Treatment of Cancer (EORTC) 90101 “CREATE” phase II trial evaluated the MET inhibitor crizotinib in ASPS patients, achieving only limited antitumor activity. We performed a comprehensive molecular analysis of ASPS tissue samples collected in this trial to identify potential biomarkers correlating with treatment outcome. A tissue microarray containing 47 ASPS cases was used for the characterization of the tumor microenvironment using multiplex immunofluorescence. DNA isolated from 34 available tumor samples was analyzed to detect recurrent gene copy number alterations (CNAs) and mutations by low-coverage whole-genome sequencing and whole-exome sequencing. Pathway enrichment analysis was used to identify diseased-associated pathways in ASPS sarcomagenesis. Kaplan–Meier estimates, Cox regression, and the Fisher’s exact test were used to correlate histopathological and molecular findings with clinical data related to crizotinib treatment, aiming to identify potential factors associated with patient outcome. Tumor microenvironment characterization showed the presence of PD-L1 and CTLA-4 in 10 and 2 tumors, respectively, and the absence of PD-1 in all specimens. Apart from CD68, other immunological markers were rarely expressed, suggesting a low level of tumor-infiltrating lymphocytes in ASPS. By CNA analysis, we detected a number of broad and focal alterations. The most common alteration was the loss of chromosomal region 1p36.32 in 44% of cases. The loss of chromosomal regions 1p36.32, 1p33, 1p22.2, and 8p was associated with shorter progression-free survival. Using whole-exome sequencing, 13 cancer-associated genes were found to be mutated in at least three cases. Pathway enrichment analysis identified genetic alterations in NOTCH signaling, chromatin organization, and SUMOylation pathways. NOTCH4 intracellular domain dysregulation was associated with poor outcome, while inactivation of the beta-catenin/TCF complex correlated with improved outcome in patients receiving crizotinib. ASPS is characterized by molecular heterogeneity. We identify genetic aberrations potentially predictive of treatment outcome during crizotinib therapy and provide additional insights into the biology of ASPS, paving the way to improve treatment approaches for this extremely rare malignancy.

IL-33 enhances Jagged1 mediated NOTCH1 intracellular domain (NICD) deubiquitination and pathological angiogenesis in proliferative retinopathy

Pathological retinal neovascularization (NV) is a clinical manifestation of various proliferative retinopathies, and treatment of NV using anti-VEGF therapies is not selective, as it also impairs normal retinal vascular growth and function. Here, we show that genetic deletion or siRNA-mediated downregulation of IL-33 reduces pathological NV in a murine model of oxygen-induced retinopathy (OIR) with no effect on the normal retinal repair. Furthermore, our fluorescent activated cell sorting (FACS) data reveals that the increase in IL-33 expression is in endothelial cells (ECs) of the hypoxic retina and conditional genetic deletion of IL-33 in retinal ECs reduces pathological NV. In vitro studies using human retinal microvascular endothelial cells (HRMVECs) show that IL-33 induces sprouting angiogenesis and requires NFkappaB-mediated Jagged1 expression and Notch1 activation. Our data also suggest that IL-33 enhances de-ubiquitination and stabilization of Notch1 intracellular domain via its interaction with BRCA1-associated protein 1 (BAP1) and Numb in HRMVECs and a murine model of OIR.

Optogenetic control of NOTCH1 signaling

The Notch signaling pathway is a crucial regulator of cell differentiation as well as tissue organization, whose deregulation is linked to the pathogenesis of different diseases. NOTCH1 plays a key role in breast cancer progression by increasing proliferation, maintenance of cancer stem cells, and impairment of cell death. NOTCH1 is a mechanosensitive receptor, where mechanical force is required to activate the proteolytic cleavage and release of the Notch intracellular domain (NICD). We circumvent this limitation by regulating Notch activity by light. To achieve this, we have engineered an optogenetic NOTCH1 receptor (optoNotch) to control the activation of NOTCH1 intracellular domain (N1ICD) and its downstream transcriptional activities. Using optoNotch we confirm that NOTCH1 activation increases cell proliferation in MCF7 and MDA-MB-468 breast cancer cells in 2D and spheroid 3D cultures, although causing distinct cell-type specific migratory phenotypes. Additionally, optoNotch activation induced chemoresistance on the same cell lines. OptoNotch allows the fine-tuning, ligand-independent, regulation of N1ICD activity and thus a better understanding of the spatiotemporal complexity of Notch signaling.BvUWa4_Br16BQ-qQYt4TAvVideo

Notch ankyrin domain: evolutionary rise of a thermodynamic sensor

Notch signalling pathway plays a key role in metazoan biology by contributing to resolution of binary decisions in the life cycle of cells during development. Outcomes such as proliferation/differentiation dichotomy are resolved by transcriptional remodelling that follows a switch from Notchon to Notchoff state, characterised by dissociation of Notch intracellular domain (NICD) from DNA-bound RBPJ. Here we provide evidence that transitioning to the Notchoff state is regulated by heat flux, a phenomenon that aligns resolution of fate dichotomies to mitochondrial activity. A combination of phylogenetic analysis and computational biochemistry was utilised to disclose structural adaptations of Notch1 ankyrin domain that enabled function as a sensor of heat flux. We then employed DNA-based micro-thermography to measure heat flux during brain development, followed by analysis in vitro of the temperature-dependent behaviour of Notch1 in mouse neural progenitor cells. The structural capacity of NICD to operate as a thermodynamic sensor in metazoans stems from characteristic enrichment of charged acidic amino acids in β-hairpins of the ankyrin domain that amplify destabilising inter-residue electrostatic interactions and render the domain thermolabile. The instability emerges upon mitochondrial activity which raises the perinuclear and nuclear temperatures to 50 °C and 39 °C, respectively, leading to destabilization of Notch1 transcriptional complex and transitioning to the Notchoff state. Notch1 functions a metazoan thermodynamic sensor that is switched on by intercellular contacts, inputs heat flux as a proxy for mitochondrial activity in the Notchon state via the ankyrin domain and is eventually switched off in a temperature-dependent manner.4Fh46PNx-YRBNHf862t6mSVideo abstract

Notch1/Hes1‑PTEN/AKT/IL‑17A feedback loop regulates Th17 cell differentiation in mouse psoriasis‑like skin inflammation.

IL-17A, the effector cytokine of T helper (Th) 17 cells, plays a crucial role in the pathogenesis of psoriasis. The Notch1 and PI3K/AKT signaling pathways are implicated in Th17 cell differentiation and IL-17A production. The present study aimed to evaluate the regulatory effect of the Notch1/hairy and enhancer of split 1 (Hes1)-PTEN/AKT/IL-17A feedback loop on Th17 cell differentiation via the PI3K/AKT inhibitor LY294002 in a mouse model of psoriasis. Mice were randomly divided into 3 groups: a control group, a model group [5% imiquimod (IMQ)-induced group] and an intervention group (5% IMQ-induced plus LY294002-treated group). Skin structural characteristics were recorded and evaluated by hematoxylin and eosin staining. The weights of the spleens and inguinal lymph nodes were measured. Th17 cell percentage, as well as the mRNA and protein expression levels of Notch1, Notch1 intracellular domain (NICD1), Hes1, PTEN, AKT, phosphorylated (p)-AKT, mTOR complex 1 (mTORC1), p-mTORC1, S6 kinase (S6K)1, S6K2 and IL-17A were detected in skin samples of the three experimental groups. Additionally, splenic mononuclear cells from model mice were treated by 10 and 50 µM LY294002 to further evaluate its regulatory effect on Notch1/Hes1-PTEN/AKT/IL-17A feedback loop. Increased Th17 cell percentage, increased expression of Notch1, NICD1, Hes1, AKT, p-AKT, mTORC1, p-mTORC1, S6K1, S6K2 and IL-17A, and decreased PTEN levels were observed in model mice alongside marked psoriasis-like skin inflammation, splenomegaly and lymphadenopathy. LY294002 treatment significantly alleviated the severity of psoriasis-like skin inflammation in the intervention mice, attenuated the degree of epidermal hyperplasia and dermal inflammatory cell infiltration, and mitigated splenomegaly and lymphadenopathy. In addition, LY294002 treatment reversed the increased Th17 cell percentage, as well as the increased expression of Notch1, NICD1, Hes1, AKT, p-AKT, mTORC1, p-mTORC1, S6K1, S6K2 and IL-17A, and the decreased expression of PTEN. In vitro study from 5% IMQ-induced mouse splenic mononuclear cells presented that high dose of LY294002 exerted more obviously regulatory effect on Notch1/Hes1-PTEN/AKT/IL-17A feedback loop. The current findings suggested that the Notch1/Hes1-PTEN/AKT/IL-17A feedback loop regulates Th17 cell differentiation within the disease environment of psoriasis. Blocking the Notch1/Hes1-PTEN/AKT/IL-17A feedback loop may thus be a potential therapeutic method for management of psoriatic inflammation.

Cdk5-mediated JIP1 phosphorylation regulates axonal outgrowth through Notch1 inhibition

BackgroundActivated Cdk5 regulates a number of processes during nervous system formation, including neuronal differentiation, growth cone stabilization, and axonal growth. Cdk5 phosphorylates its downstream substrates located in axonal growth cones, where the highly expressed c-Jun N-terminal kinase (JNK)-interacting protein1 (JIP1) has been implicated as another important regulator of axonal growth. In addition, stringent control of the level of intracellular domain of Notch1 (Notch1-IC) plays a regulatory role in axonal outgrowth during neuronal differentiation. However, whether Cdk5-JIP1-Notch1 cooperate to regulate axonal outgrowth, and the mechanism of such joint contribution to this pathway, is presently unknown, and here we explore their potential interaction.ResultsOur interactome screen identified JIP1 as an interactor of p35, a Cdk5 activator, and we sought to explore the relationship between Cdk5 and JIP1 on the regulation of axonal outgrowth. We demonstrate that JIP1 phosphorylated by Cdk5 at Thr205 enhances axonal outgrowth and a phosphomimic JIP1 rescues the axonal outgrowth defects in JIP1−/− and p35−/− neurons. Axonal outgrowth defects caused by the specific increase of Notch1 in JIP1−/− neurons are rescued by Numb-mediated inhibition of Notch1. Finally, we demonstrate that Cdk5 phosphorylation of JIP1 further amplifies the phosphorylation status of yet another Cdk5 substrate E3-ubiquitin ligase Itch, resulting in increased Notch1 ubiquitination.ConclusionsOur findings identify a potentially critical signaling axis involving Cdk5-JIP1-Itch-Notch1, which plays an important role in the regulation of CNS development. Future investigation into the way this pathway integrates with additional pathways regulating axonal growth will further our knowledge of normal central nervous system development and pathological conditions.

Presenilin enhancer 2 is crucial for the transition of apical progenitors into neurons but into not basal progenitors in the developing hippocampus.

Recent evidence has shown that presenilin enhancer 2 (Pen2; Psenen) plays an essential role in corticogenesis by regulating the switch of apical progenitors (APs) to basal progenitors (BPs). The hippocampus is a brain structure required for advanced functions, including spatial navigation, learning and memory. However, it remains unknown whether Pen2 is important for hippocampal morphogenesis. To address this question, we generated Pen2 conditional knockout (cKO) mice, in which Pen2 is inactivated in neural progenitor cells (NPCs) in the hippocampal primordium. We showed that Pen2 cKO mice exhibited hippocampal malformation and decreased population of NPCs in the neuroepithelium of the hippocampus. We found that deletion of Pen2 neither affected the proliferative capability of APs nor the switch of APs to BPs in the hippocampus, and that it caused enhanced transition of APs to neurons. We demonstrated that expression of the Notch1 intracellular domain (N1ICD) significantly increased the population of NPCs in the Pen2 cKO hippocampus. Collectively, this study uncovers a crucial role for Pen2 in the maintenance of NPCs during hippocampal development.