LST-1 is a bifunctional regulator that feeds back on Notch-dependent transcription to regulate C. elegans germline stem cells.

Notch signaling regulates stem cells across animal phylogeny. C. elegans Notch signaling activates transcription of two genes, lst-1 and sygl-1, that encode potent regulators of germline stem cells. The LST-1 protein regulates stem cells in two distinct ways: It promotes self-renewal posttranscriptionally and also restricts self-renewal by a poorly understood mechanism. Its self-renewal promoting activity resides in its N-terminal region, while its self-renewal restricting activity resides in its C-terminal region and requires the Zn finger. Here, we report that LST-1 limits self-renewal by down-regulating Notch-dependent transcription. We detect LST-1 in the nucleus, in addition to its previously known cytoplasmic localization. LST-1 lowers nascent transcript levels at both lst-1 and sygl-1 loci but not at let-858, a Notch-independent locus. LST-1 also lowers levels of two key components of the Notch activation complex, the LAG-1 DNA binding protein and Notch intracellular domain (NICD). Genetically, an LST-1 Zn finger mutant increases Notch signaling strength in both gain- and loss-of-function GLP-1/Notch receptor mutants. Biochemically, LST-1 co-immunoprecipitates with LAG-1 from nematode extracts, suggesting a direct effect. LST-1 is thus a bifunctional regulator that coordinates posttranscriptional and transcriptional mechanisms in a single protein. This LST-1 bifunctionality relies on its bipartite protein architecture and is bolstered by generation of two LST-1 isoforms, one specialized for Notch downregulation. A conserved theme from worms to human is the coupling of PUF-mediated RNA repression together with Notch feedback in the same protein.

Comprehensive analysis of the proximity-dependent nuclear interactome for the oncoprotein NOTCH1 in live cells.

Notch signaling plays a critical role in cell fate decisions in all cell types. Furthermore, gain-of-function mutations in NOTCH1 have been uncovered in many human cancers. Disruption of Notch signaling has recently emerged as an attractive disease treatment strategy. However, the nuclear interaction landscape of the oncoprotein NOTCH1 remains largely unexplored. We therefore employed here a proximity-dependent biotin identification approach to identify in vivo protein associations with the nuclear Notch1 intracellular domain in live cells. We identified a large set of previously reported and unreported proteins that associate with NOTCH1, including general transcription and elongation factors, DNA repair and replication factors, coactivators, corepressors, and components of the NuRD and SWI/SNF chromatin remodeling complexes. We also found that Notch1 intracellular domain associates with protein modifiers and components of other signaling pathways that may influence Notch signal transduction and protein stability such as USP7. We further validated the interaction of NOTCH1 with histone deacetylase 1 or GATAD2B using protein network analysis, proximity-based ligation, in vivo cross-linking and coimmunoprecipitation assays in several Notch-addicted cancer cell lines. Through data mining, we also revealed potential drug targets for the inhibition of Notch signaling. Collectively, these results provide a valuable resource to uncover the mechanisms that fine-tune Notch signaling in tumorigenesis and inform therapeutic targets for Notch-addicted tumors.

FBP1 inhibits NSCLC stemness by promoting ubiquitination of Notch1 intracellular domain and accelerating degradation.

The existence of cancer stem cells is widely acknowledged as the underlying cause for the challenging curability and high relapse rates observed in various tumor types, including non-small cell lung cancer (NSCLC). Despite extensive research on numerous therapeutic targets for NSCLC treatment, the strategies to effectively combat NSCLC stemness and achieve a definitive cure are still not well defined. The primary objective of this study was to examine the underlying mechanism through which Fructose-1,6-bisphosphatase 1 (FBP1), a gluconeogenic enzyme, functions as a tumor suppressor to regulate the stemness of NSCLC. Herein, we showed that overexpression of FBP1 led to a decrease in the proportion of CD133-positive cells, weakened tumorigenicity, and decreased expression of stemness factors. FBP1 inhibited the activation of Notch signaling, while it had no impact on the transcription level of Notch 1 intracellular domain (NICD1). Instead, FBP1 interacted with NICD1 and the E3 ubiquitin ligase FBXW7 to facilitate the degradation of NICD1 through the ubiquitin-proteasome pathway, which is independent of the metabolic enzymatic activity of FBP1. The aforementioned studies suggest that targeting the FBP1-FBXW7-NICD1 axis holds promise as a therapeutic approach for addressing the challenges of NSCLC recurrence and drug resistance.

Targeting endothelial PDGFR-ß facilitates angiogenesis-associated bone formation through the PAK1/NICD axis.

The intricate orchestration of osteoporosis (OP) pathogenesis remains elusive. Mounting evidence suggests that angiogenesis-driven osteogenesis serves as a crucial foundation for maintaining bone homeostasis. This study aimed to explore the potential of the endothelial platelet-derived growth factor receptor-ß (PDGFR-ß) in mitigating bone loss through its facilitation of H-type vessel formation. Our findings demonstrate that the expression level of endothelial PDGFR-ß is reduced in samples obtained from individuals suffering from OP, as well as in ovariectomy mice. Depletion of PDGFR-ß in endothelial cells ameliorates angiogenesis-mediated bone formation in mice. The regulatory influence of endothelial PDGFR-ß on H-type vessels is mediated through the PDGFRß-P21-activated kinase 1-Notch1 intracellular domain signaling cascade. In particular, the endothelium-specific enhancement of PDGFR-ß facilitates H-type vessels and their associated bone formation in OP. Hence, the strategic targeting of endothelial PDGFR-ß emerges as a promising therapeutic approach for the management of OP in the near future.

Kindlin-2 controls angiogenesis through modulating Notch1 signaling.

Kindlin-2 is critical for development and homeostasis of key organs, including skeleton, liver, islet, etc., yet its role in modulating angiogenesis is unknown. Here, we report that sufficient KINDLIN-2 is extremely important for NOTCH-mediated physiological angiogenesis. The expression of KINDLIN-2 in HUVECs is significantly modulated by angiogenic factors such as vascular endothelial growth factor A or tumor necrosis factor α. A strong co-localization of CD31 and Kindlin-2 in tissue sections is demonstrated by immunofluorescence staining. Endothelial-cell-specific Kindlin-2 deletion embryos die on E10.5 due to hemorrhage caused by the impaired physiological angiogenesis. Experiments in vitro show that vascular endothelial growth factor A-induced multiple functions of endothelial cells, including migration, matrix proteolysis, morphogenesis and sprouting, are all strengthened by KINDLIN-2 overexpression and severely impaired in the absence of KINDLIN-2. Mechanistically, we demonstrate that KINDLIN-2 inhibits the release of Notch intracellular domain through binding to and maintaining the integrity of NOTCH1. The impaired angiogenesis and avascular retinas caused by KINDLIN-2 deficiency can be rescued by DAPT, an inhibitor of γ-secretase which releases the intracellular domain from NOTCH1. Moreover, we demonstrate that high glucose stimulated hyperactive angiogenesis by increasing KINDLIN-2 expression could be prevented by KINDLIN-2 knockdown, indicating Kindlin-2 as a potential therapeutic target in treatment of diabetic retinopathy. Our study for the first time demonstrates the significance of Kindlin-2 in determining Notch-mediated angiogenesis during development and highlights Kindlin-2 as the potential therapeutic target in angiogenic diseases, such as diabetic retinopathy.

Ultrasound-assisted efficient and convenient method of extracting valuable metals (Ni, Co, and Cd) from waste Ni-Cd batteries using DL-malic acid.

Recycling waste Ni-Cd batteries has received much attention recently because of the serious environmental pollution they cause and to avoid the dissipation of valuable metals. Despite significant research, it is still difficult to efficiently recycle valuable and hazardous metals from waste Ni-Cd batteries in an economical and environmentally friendly manner. This study employed a novel process utilizing ultrasound-assisted leaching to recover Ni, Cd, and Co from waste nickel-cadmium (Ni-Cd) batteries. Organic DL-malic acid served as the leaching agent and H2O2 was employed as an oxidizing agent. The effects of various factors on the recovery efficiency of Ni, Cd, and Co, such as leaching temperature, time, DL-malic acid concentration, pulp density, H2O2 concentration, and ultrasound frequency, were also examined. To predict the chemical compounds present before and after the recycling experiments, the solid residues from the metal extraction were analyzed using XRD, XPS, FE-SEM, and EDS element mapping. Concurrently, ICP-OES was utilized to determine the metal content in the leachate. Under optimized conditions of 90 °C, 90 min, 2M DL-malic acid, 160 mL/g pulp density, and 20% ultrasound frequency, over 83% of Ni, 94% of Cd, and 98% of Co were effectively leached from the waste Ni-Cd battery powder. The leaching kinetics of Ni, Cd, and Co followed the surface chemical reaction control model. The activation energies (Ea) for Ni, Cd, and Co leaching were 21.34, 20.47, and 18.38 kJ/mol, respectively. The findings suggest that ultrasound-assisted leaching is an efficient, cost-effective, environmentally friendly, and sustainable alternative for extracting precious and hazardous metals from waste Ni-Cd batteries. Additionally, it reduces industrial chemical usage and enhances waste management sustainability.

Macrophage PTEN controls STING-induced inflammation and necroptosis through NICD/NRF2 signaling in APAP-induced liver injury.

BACKGROUND: Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) signaling has been known to play a critical role in maintaining cellular and tissue homeostasis, which also has an essential role in the inflammatory response. However, it remains unidentified whether and how the macrophage PTEN may govern the innate immune signaling stimulator of interferon genes (STING) mediated inflammation and hepatocyte necroptosis in APAP-induced liver injury (AILI). METHODS: Myeloid-specific PTEN knockout (PTENM-KO) and floxed PTEN (PTENFL/FL) mice were treated with APAP (400 mg/kg) or PBS. In a parallel in vitro study, bone marrow-derived macrophages (BMMs) were isolated from these conditional knockout mice and transfected with CRISPR/Cas9-mediated Notch1 knockout (KO) or CRISPR/Cas9-mediated STING activation vector followed by LPS (100 ng/ml) stimulation. RESULTS: Here, we report that myeloid-specific PTEN knockout (PTENM-KO) mice were resistant to oxidative stress-induced hepatocellular injury with reduced macrophage/neutrophil accumulation and proinflammatory mediators in AILI. PTENM-KO increased the interaction of nuclear Notch intracellular domain (NICD) and nuclear factor (erythroid-derived 2)-like 2 (NRF2) in the macrophage nucleus, reducing reactive oxygen species (ROS) generation. Mechanistically, it is worth noting that macrophage NICD and NRF2 co-localize within the nucleus under inflammatory conditions. Additionally, Notch1 promotes the interaction of immunoglobulin kappa J region (RBPjκ) with NRF2. Disruption of the Notch1 signal in PTEN deletion macrophages, reduced RBPjκ and NRF2 binding, and activated STING signaling. Moreover, PTENM-KO macrophages with STING activated led to ROS generation and TNF-α release, resulting in hepatocyte necroptosis upon co-culture with primary hepatocytes. CONCLUSIONS: Our findings demonstrate that the macrophage PTEN-NICD/NRF2-STING axis is critical to regulating oxidative stress-induced liver inflammation and necroptosis in AILI and implies the therapeutic potential for managing sterile liver inflammation. Video Abstract.

Modulation of Notch Signaling at Early Stages of Differentiation of Human Induced Pluripotent Stem Cells to Dopaminergic Neurons.

Elaboration of protocols for differentiation of human pluripotent stem cells to dopamine neurons is an important issue for development of cell replacement therapy for Parkinson's disease. A number of protocols have been already developed; however, their efficiency and specificity still can be improved. Investigating the role of signaling cascades, important for neurogenesis, can help to solve this problem and to provide a deeper understanding of their role in neuronal development. Notch signaling plays an essential role in development and maintenance of the central nervous system after birth. In our study, we analyzed the effect of Notch activation and inhibition at the early stages of differentiation of human induced pluripotent stem cells to dopaminergic neurons. We found that, during the first seven days of differentiation, the cells were not sensitive to the Notch inhibition. On the contrary, activation of Notch signaling during the same time period led to significant changes and was associated with an increase in expression of genes, specific for caudal parts of the brain, a decrease of expression of genes, specific for forebrain, as well as a decrease of expression of genes, important for the formation of axons and dendrites and microtubule stabilizing proteins.

Delta/Notch-like Epidermal Growth Factor-Related Receptor (DNER), a Potential Prognostic Marker of Gastric Cancer Regulates Cell Survival and Cell Cycle Progression.

Upregulation of the expression of Delta/notch-like epidermal growth factor-related receptor (DNER) and its oncogenic role have been reported in several cancers, including gastric, breast, and prostate cancers. This study aimed to investigate the oncogenic role of DNER and the mechanisms behind its oncogenic role in gastric cancer. Analysis of the RNASeq data of gastric cancer tissues obtained from the TCGA database revealed that the expression of DNER was associated with the pathology of advanced gastric cancer and the prognosis of patients. DNER expression was increased upon stem cell-enriching cancer spheroid culture. Knockdown of DNER expression inhibited cell proliferation and invasion, induced apoptosis, enhanced chemosensitivity, and decreased spheroid formation of SNU-638 gastric cancer cells. DNER silencing elevated the expression of p53, p21cip/waf, and p27, and increased G1 phase cells at the expense of S phase cells. Knockdown of p21cip/waf expression in the DNER-silenced cells partially restored cell viability and S phase progression. DNER silencing also induced the apoptosis of SNU-638 cells. While both cleaved caspases-8 and 9 were detected in adherent cells, only cleaved caspase-8 was found to have increased in spheroid-cultured cells, suggesting a distinct activation pattern of caspase activation depending on the growth condition. Knockdown of p53 expression rescued the DNER-silenced cells from apoptosis and partially restored cell viability. In contrast, overexpression of the Notch intracellular domain (NICD) decreased the expression of p53, p21cip/waf, and cleaved caspase-3 in DNER-silenced cells. Moreover, NICD expression fully reverted the cell viability reduction, arrest in the G1 phase, and elevated apoptosis caused by DNER silencing, thereby suggesting activation of Notch signaling by DNER. Expression of a membrane-unbound mutant of mDNER also decreased cell viability and induced apoptosis. On the other hand, TGF-ß signals were found to be involved in DNER expression in both adherent and spheroid-cultured cells. DNER could therefore be a link connecting TGF-ß signaling to Notch signaling. Taken together, DNER regulates cell proliferation, survival, and invasive capacity of the gastric cancer cells through the activation of Notch signaling, which may facilitate tumor progression into an advanced stage. This study provides evidences suggesting that DNER could be a potential prognostic marker, a therapeutic target, and a drug candidate in the form of a cell-free mutant.

Alcohol drinking inhibits NOTCH-PAX9 signaling in esophageal squamous epithelial cells.

Alcohol drinking has been established as a major risk factor for esophageal diseases. Our previous study showed that ethanol exposure inhibited PAX9 expression in human esophageal squamous epithelial cells in vitro and in vivo. In this study, we aimed to investigate the molecular pathways through which alcohol drinking suppresses PAX9 in esophageal squamous epithelial cells. We first demonstrated the inhibition of NOTCH by ethanol exposure in vitro. NOTCH regulated PAX9 expression in KYSE510 and KYSE410 cells in vitro and in vivo. RBPJ and NOTCH intracellular domain (NIC) D1 ChIP-PCR confirmed Pax9 as a direct downstream target of NOTCH signaling in mouse esophagus. NOTCH inhibition by alcohol drinking was further validated in mouse esophagus and human tissue samples. In conclusion, ethanol exposure inhibited NOTCH signaling and thus suppressed PAX9 expression in esophageal squamous epithelial cells in vitro and in vivo. Our data support a novel mechanism of alcohol-induced esophageal injury through the inhibition of NOTCH-PAX9 signaling. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Selective Recovery of Cadmium, Cobalt, and Nickel from Spent Ni-Cd Batteries Using Adogen® 464 and Mesoporous Silica Derivatives.

Spent Ni-Cd batteries are now considered an important source for many valuable metals. The recovery of cadmium, cobalt, and nickel from spent Ni-Cd Batteries has been performed in this study. The optimum leaching process was achieved using 20% H2SO4, solid/liquid (S/L) 1/5 at 80 °C for 6 h. The leaching efficiency of Fe, Cd, and Co was nearly 100%, whereas the leaching efficiency of Ni was 95%. The recovery of the concerned elements was attained using successive different separation techniques. Cd(II) ions were extracted by a solvent, namely, Adogen® 464, and precipitated as CdS with 0.5% Na2S solution at pH of 1.25 and room temperature. The extraction process corresponded to pseudo-2nd-order. The prepared PTU-MS silica was applied for adsorption of Co(II) ions from aqueous solution, while the desorption process was performed using 0.3 M H2SO4. Cobalt was precipitated at pH 9.0 as Co(OH)2 using NH4OH. The kinetic and thermodynamic parameters were also investigated. Nickel was directly precipitated at pH 8.25 using a 10% NaOH solution at ambient temperature. FTIR, SEM, and EDX confirm the structure of the products.

Dynamic binding of RBPJ is determined by Notch signaling status.

Notch signaling plays crucial roles in mediating cell fate choices in all metazoans largely by specifying the transcriptional output of one cell in response to a neighboring cell. The DNA-binding protein RBPJ is the principle effector of this pathway in mammals and, together with the transcription factor moiety of Notch (NICD), regulates the expression of target genes. The prevalent view presumes that RBPJ statically occupies consensus binding sites while exchanging repressors for activators in response to NICD. We present the first specific RBPJ chromatin immunoprecipitation and high-throughput sequencing study in mammalian cells. To dissect the mode of transcriptional regulation by RBPJ and identify its direct targets, whole-genome binding profiles were generated for RBPJ; its coactivator, p300; NICD; and the histone H3 modifications H3 Lys 4 trimethylation (H3K4me3), H3 Lys 4 monomethylation (H3K4me1), and histone H3 Lys 27 acetylation (H3K27ac) in myogenic cells under active or inhibitory Notch signaling conditions. Our results demonstrate dynamic binding of RBPJ in response to Notch activation at essentially all sites co-occupied by NICD. Additionally, we identify a distinct set of sites where RBPJ recruits neither NICD nor p300 and binds DNA statically, irrespective of Notch activity. These findings significantly modify our views on how RBPJ and Notch signaling mediate their activities and consequently impact on cell fate decisions.

RHOQ is induced by DLL4 and regulates angiogenesis by determining the intracellular route of the Notch intracellular domain.

Angiogenesis, the formation of new blood vessels by endothelial cells, is a finely tuned process relying on the balance between promoting and repressing signalling pathways. Among these, Notch signalling is critical in ensuring appropriate response of endothelial cells to pro-angiogenic stimuli. However, the downstream targets and pathways effected by Delta-like 4 (DLL4)/Notch signalling and their subsequent contribution to angiogenesis are not fully understood. We found that the Rho GTPase, RHOQ, is induced by DLL4 signalling and that silencing RHOQ results in abnormal sprouting and blood vessel formation both in vitro and in vivo. Loss of RHOQ greatly decreased the level of Notch signalling, conversely overexpression of RHOQ promoted Notch signalling. We describe a new feed-forward mechanism regulating DLL4/Notch signalling, whereby RHOQ is induced by DLL4/Notch and is essential for the NICD nuclear translocation. In the absence of RHOQ, Notch1 becomes targeted for degradation in the autophagy pathway and NICD is sequestered from the nucleus and targeted for degradation in lysosomes.

Localization of DLL1- and NICD-positive osteoblasts in cortical bone during postnatal growth in rats.

The long bone midshaft expands by forming primary osteons at the periosteal surface of cortical bone in humans and rodents. Osteoblastic bone formation in the vascular cavity in the center of primary osteons is delayed during cortical bone development. The mechanisms of the formation of primary osteons is not fully understood, however. Focusing on NOTCH1 signaling, an inhibitory signaling on osteoblastic bone formation, our immunohistochemical analysis revealed Delta like1 (DLL1), a ligand of NOTCH1, and the NOTCH1 intracellular domain (NICD, an activated form of NOTCH1) immunoreactivity, in the cuboidal osteoblasts lining the bone surface in the vascular cavity of primary osteons during postnatal growth in rats. Interestingly, five days after treatment of primary osteoblasts with ascorbic acid and ß glycerophosphate, protein levels of both DLL1 and NICD increased transiently, indicating that DLL1 activates NOTCH1 in primary cultured osteoblasts. Thus, the results imply that DLL1-NOTCH1 signaling in osteoblasts is associated with primary osteonal bone formation.

Getting a Notch closer to renal dysfunction: activated Notch suppresses expression of the adaptor protein Disabled-2 in tubular epithelial cells.

Reactivation of Notch signaling in kidneys of animal models and patients with chronic kidney disease (CKD) has been shown to contribute to epithelial injury and fibrosis development. Here, we investigated the mechanisms of Notch-induced injury in renal epithelial cells. We performed genome-wide transcriptome analysis to identify Notch target genes using an in vitro system of cultured tubular epithelial cells expressing the intracellular domain of Notch1. One of the top downregulated genes was Disabled-2 ( Dab2). With the use of Drosophila nephrocytes as a model system, we found that Dab (the Drosophila homolog of Dab2) knockdown resulted in a significant filtration defect, indicating that loss of Dab2 plays a functional role in kidney disease development. We showed that Dab2 expression in cultured tubular epithelial cells is involved in endocytic regulation and that it also protects cells from TGF-ß-induced epithelial-to-mesenchymal transition. In vivo correlation studies indicated its additional role in renal ischemia-induced injury. Together, these data suggest that Dab2 plays a versatile role in the kidney and may impact on acute and CKDs.-Schütte-Nütgen, K., Edeling, M., Mendl, G., Krahn, M. P., Edemir, B., Weide, T., Kremerskothen, J., Michgehl, U., Pavenstädt, H. Getting a Notch closer to renal dysfunction: activated Notch suppresses expression of the adaptor protein Disabled-2 in tubular epithelial cells.

Molecular interaction of NFκB and NICD in monocyte-macrophage differentiation is a target for intervention in atherosclerosis.

The activation of two transcription factors, NFκB and NICD (notch intracellular domain), plays a crucial role in different stages of atherosclerotic disease progression, from early endothelial activation by modified lipids like oxidized low-density lipoprotein (oxyLDL) to the imminent rupture of the atherosclerotic plaque. Inflammatory mediators and the notch pathway proteins were upregulated in atherogenic diet-induced rats and the same was confirmed by the differentiation of monocyte to macrophage on exposure to oxyLDL. The inflammatory transcription factor NFκB and the notch signaling transcription factor NICD were analysed for their molecular interaction in monocyte to macrophage differentiation. Inhibition of NFκB by dexamethasone in monocyte to macrophage differentiation resulted in a concomitant downregulation of NICD, whereas inhibition of NICD by N-(N-[3, 5-difluorophenacetyl])-l-alanyl)-S-phenylglycine t-butyl ester (DAPT), a γ-secretase inhibitor, did not significantly influence the expression of NFκB, but downregulated macrophage differentiation. These findings revealed that NFκB inhibition using dexamethasone regulated NICD, which turned down macrophage differentiation. Thus, inhibition of both NFκB-NICD is a potential target for intervention in atherosclerosis.

Cell cycle regulation of oscillations yields coupling of growth and form in a computational model of the presomitic mesoderm.

A striking example of coupling between growth and form occurs during the segmentation of the vertebrate embryo. During segmentation, pairs of segments, one on either side of the anterior-posterior axis, bud off from the presomitic mesoderm (PSM) at regular intervals in time. In the clock and wavefront model, a multicellular oscillator regulates the time at which the next pair of segments form whilst a wavefront regulates their spatial location. In most mathematical models of segmentation, it is assumed that cells in the PSM are oscillators that have a constant natural frequency. Based on recent experimental findings, here we propose a model in which the natural oscillation frequency of each PSM cell is a function of its position in the cell cycle. Given adequate oscillator coupling and that cells in the PSM are randomly distributed in the cell cycle, we find that the emergent oscillator frequency is a weighted average of the constituent oscillator frequencies with the weightings dependent on the fraction of cells in a given cell cycle state. Here, we show that such a model can allow for coupling between pattern formation and growth rate in PSM tissue.

Notch signaling promotes angiogenesis and improves cardiac function after myocardial infarction.

Currently, the role of Notch signaling during myocardial infarction (MI) remains controversy. In this study we used in vitro and in vivo approaches to investigate the role of Notch signaling in MI. Using cultured human umbilical vein endothelial cells exposed to hypoxia/reoxygenation (H/R), we demonstrated that H/R inhibited the proliferation, VEGF secretion, and tube formation of HUVECs, and these effects were correlated with the inhibition of Notch signaling. Furthermore, these effects were antagonized by overexpression of NICD but aggravated by knockdown of NICD. In addition, in MI model rats we found that heart dysfunction and angiogenesis in model rats was partly improved by NICD overexpression but was aggravated by knockdown of NICD. In conclusion, these data demonstrate that Notch signaling is downregulated in H/R injury in the hearts. Artificial activation of Notch signaling could promote myocardial survival and angiogenesis and improve cardiac function following H/R injury.

Modulation of basal cell fate during productive and transforming HPV-16 infection is mediated by progressive E6-driven depletion of Notch.

In stratified epithelia such as the epidermis, homeostasis is maintained by the proliferation of cells in the lower epithelial layers and the concomitant loss of differentiated cells from the epithelial surface. These differentiating keratinocytes progressively stratify and form a self-regenerating multi-layered barrier that protects the underlying dermis. In such tissue, the continual loss and replacement of differentiated cells also limits the accumulation of oncogenic mutations within the tissue. Inactivating mutations in key driver genes, such as TP53 and NOTCH1, reduce the proportion of differentiating cells allowing for the long-term persistence of expanding mutant clones in the tissue. Here we show that through the expression of E6, HPV-16 prevents the early fate commitment of human keratinocytes towards differentiation and confers a strong growth advantage to human keratinocytes. When E6 is expressed either alone or with E7, it promotes keratinocyte proliferation at high cell densities, through the combined inactivation of p53 and Notch1. In organotypic raft culture, the activity of E6 is restricted to the basal layer of the epithelium and is enhanced during the progression from productive to abortive or transforming HPV-16 infection. Consistent with this, the expression of p53 and cleaved Notch1 becomes progressively more disrupted, and is associated with increased basal cell density and reduced commitment to differentiation. The expression of cleaved Notch1 is similarly disrupted also in HPV-16-positive cervical lesions, depending on neoplastic grade. When taken together, these data depict an important role of high-risk E6 in promoting the persistence of infected keratinocytes in the basal and parabasal layers through the inactivation of gene products that are commonly mutated in non-HPV-associated neoplastic squamous epithelia. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Atrophin protein RERE positively regulates Notch targets in the developing vertebrate spinal cord.

The Notch signaling pathway controls cell fate decision, proliferation, and other biological functions in both vertebrates and invertebrates. Precise regulation of the canonical Notch pathway ensures robustness of the signal throughout development and adult tissue homeostasis. Aberrant Notch signaling results in profound developmental defects and is linked to many human diseases. In this study, we identified the Atrophin family protein RERE (also called Atro2) as a positive regulator of Notch target Hes genes in the developing vertebrate spinal cord. Prior studies have shown that during early embryogenesis in mouse and zebrafish, deficit of RERE causes various patterning defects in multiple organs including the neural tube. Here, we detected the expression of RERE in the developing chick spinal cord, and found that normal RERE activity is needed for proper neural progenitor proliferation and neuronal differentiation possibly by affecting Notch-mediated Hes expression. In mammalian cells, RERE co-immunoprecipitates with CBF1 and Notch intracellular domain (NICD), and is recruited to nuclear foci formed by over-expressed NICD1. RERE is also necessary for NICD to activate the expression of Notch target genes. Our findings suggest that RERE stimulates Notch target gene expression by preventing degradation of NICD protein, thereby facilitating the assembly of a transcriptional activating complex containing NICD, CBF1/RBPjκ in vertebrate, Su(H) in Drosophila melanogaster, Lag1 in C. elegans, and other coactivators.