The Canonical Notch Signaling Pathway: Unfolding the Activation Mechanism

Notch signaling regulates many aspects of metazoan development and tissue renewal. Accordingly, the misregulation or loss of Notch signaling underlies a wide range of human disorders, from developmental syndromes to adult-onset diseases and cancer. Notch signaling is remarkably robust in most tissues even though each Notch molecule is irreversibly activated by proteolysis and signals only once without amplification by secondary messenger cascades. In this Review, we highlight recent studies in Notch signaling that reveal new molecular details about the regulation of ligand-mediated receptor activation, receptor proteolysis, and target selection

Skin-Derived TSLP Triggers Progression from Epidermal-Barrier Defects to Asthma

A skin-derived cytokine with high systemic availability provides a mechanistic explanation for atopic march and highlights a potential therapeutic target for preventing the development of asthma among people with atopic dermatitis

Gamma-secretase composed of PS1/Pen2/Aph1a can cleave notch and amyloid precursor protein in the absence of nicastrin

γ-secretase is a multiprotein intramembrane-cleaving protease with a growing list of protein substrates including the Notch receptors and the amyloid precursor protein. The four components of γ-secretase complex - presenilin (PS), nicastrin (NCT), Pen2, and Aph1 - are all thought to be essential for activity. The catalytic domain resides within PS proteins; NCT has been suggested to be critical for substrate recognition; the contributions of Pen2 and Aph1 remain unclear. The role of NCT has been challenged recently by the observation that a critical residue (E332) in NCT, thought to be essential for γ-secretase activity, is instead involved in complex maturation. Here we report that NCT is dispensable for γ-secretase activity. NCT-independent γ-secretase activity can be detected in two independent NCT-deficient MEF lines, and blocked by the γ-secretase inhibitors DAPT and L-685,458. This catalytic activity requires prior ectodomain shedding of the substrate, and can cleave ligand-activated endogenous Notch receptors, indicating presence at the plasma membrane. siRNA knockdown experiments demonstrated that NCT-independent γ-secretase activity requires the presence of PS1, Pen2 and Aph1a but can tolerate knockdown of PS2 or Aph1b. We conclude that a PS1/Pen2/Aph1a trimeric complex is an active enzyme, displaying similar biochemical properties to those of γ-secretase and roughly 50% of its activity when normalized to PS1 NTF levels. This PS1/Pen2/Aph1a complex, however, is highly unstable. Thus, NCT acts to stabilize γ-secretase, but is not required for substrate recognition

Rapid identification of homologous recombinants and determination of gene copy number with reference/query pyrosequencing (RQPS)

Manipulating the mouse genome is a widespread technology with important applications in many biological fields ranging from cancer research to developmental biology. Likewise, correlations between copy number variations (CNVs) and human diseases are emerging. We have developed the reference-query pyrosequencing (RQPS) method, which is based on quantitative pyrosequencing and uniquely designed probes containing single nucleotide variations (SNVs), to offer a simple and affordable genotyping solution capable of identifying homologous recombinants independent of the homology arm size, determining the micro-amplification status of endogenous human loci, and quantifying virus/transgene copy number in experimental or commercial species. In addition, we also present a simple pyrosequencing-based protocol that could be used for the enrichment of homologous recombinant embryonic stem (ES) cells

γ-Secretase Functions through Notch Signaling to Maintain Skin Appendages but Is Not Required for Their Patterning or Initial Morphogenesis

AbstractThe role of Notch signaling during skin development was analyzed using Msx2-Cre to create mosaic loss-of-function alleles with precise temporal and spatial resolution. We find that γ-secretase is not involved in skin patterning or cell fate acquisition within the hair follicle. In its absence, however, inner root sheath cells fail to maintain their fates and by the end of the first growth phase, the epidermal differentiation program is activated in outer root sheath cells. This results in complete conversion of hair follicles to epidermal cysts that bears a striking resemblance to Nevus Comedonicus. Sebaceous glands also fail to form in γ-secretase-deficient mice. Importantly, mice with compound loss of Notch genes in their skin phenocopy loss of γ-secretase in all three lineages, demonstrating that Notch proteolysis accounts for the major signaling function of this enzyme in this organ and that both autonomous and nonautonomous Notch-dependent signals are involved

A novel non-canonical Notch signaling regulates expression of synaptic vesicle proteins in excitatory neurons

Notch signaling plays crucial roles for cellular differentiation during development through γ-secretase-dependent intramembrane proteolysis followed by transcription of target genes. Although recent studies implicate that Notch regulates synaptic plasticity or cognitive performance, the molecular mechanism how Notch works in mature neurons remains uncertain. Here we demonstrate that a novel Notch signaling is involved in expression of synaptic proteins in postmitotic neurons. Levels of several synaptic vesicle proteins including synaptophysin 1 and VGLUT1 were increased when neurons were cocultured with Notch ligands-expressing NIH3T3 cells. Neuron-specific deletion of Notch genes decreased these proteins, suggesting that Notch signaling maintains the expression of synaptic vesicle proteins in a cell-autonomous manner. Unexpectedly, cGMP-dependent protein kinase (PKG) inhibitor, but not γ-secretase inhibitor, abolished the elevation of synaptic vesicle proteins, suggesting that generation of Notch intracellular domain is dispensable for this function. These data uncover a ligand-dependent, but γ-secretase-independent, non-canonical Notch signaling involved in presynaptic protein expression in postmitotic neurons

The Extracellular Domain of Notch2 Increases Its Cell-Surface Abundance and Ligand Responsiveness during Kidney Development

SummaryNotch2, but not Notch1, plays indispensable roles in kidney organogenesis, and Notch2 haploinsufficiency is associated with Alagille syndrome. We proposed that proximal nephron fates are regulated by a threshold that requires nearly all available free Notch intracellular domains (NICDs) but could not identify the mechanism that explains why Notch2 (N2) is more important than Notch1 (N1). By generating mice that swap their ICDs, we establish that the overall protein concentration, expression domain, or ICD amino acid composition does not account for the differential requirement of these receptors. Instead, we find that the N2 extracellular domain (NECD) increases Notch protein localization to the cell surface during kidney development and is cleaved more efficiently upon ligand binding. This context-specific asymmetry in NICD release efficiency is further enhanced by Fringe. Our results indicate that an elevated N1 surface level could compensate for the loss of N2 signal in specific cell contexts

Randomized trial of calcipotriol combined with 5-fluorouracil for skin cancer precursor immunotherapy

BACKGROUND. Actinic keratosis is a precursor to cutaneous squamous cell carcinoma. Long treatment durations and severe side effects have limited the efficacy of current actinic keratosis treatments. Thymic stromal lymphopoietin (TSLP) is an epithelium-derived cytokine that induces a robust antitumor immunity in barrier-defective skin. Here, we investigated the efficacy of calcipotriol, a topical TSLP inducer, in combination with 5-fluorouracil (5-FU) as an immunotherapy for actinic keratosis. METHODS. The mechanism of calcipotriol action against skin carcinogenesis was examined in genetically engineered mouse models. The efficacy and safety of 0.005% calcipotriol ointment combined with 5% 5-FU cream were compared with Vaseline plus 5-FU for the field treatment of actinic keratosis in a randomized, double-blind clinical trial involving 131 participants. The assigned treatment was self-applied to the entirety of the qualified anatomical sites (face, scalp, and upper extremities) twice daily for 4 consecutive days. The percentage of reduction in the number of actinic keratoses (primary outcome), local skin reactions, and immune activation parameters were assessed. RESULTS. Calcipotriol suppressed skin cancer development in mice in a TSLP-dependent manner. Four-day application of calcipotriol plus 5-FU versus Vaseline plus 5-FU led to an 87.8% versus 26.3% mean reduction in the number of actinic keratoses in participants (P < 0.0001). Importantly, calcipotriol plus 5-FU treatment induced TSLP, HLA class II, and natural killer cell group 2D (NKG2D) ligand expression in the lesional keratinocytes associated with a marked CD4(+) T cell infiltration, which peaked on days 10–11 after treatment, without pain, crusting, or ulceration. CONCLUSION. Our findings demonstrate the synergistic effects of calcipotriol and 5-FU treatment in optimally activating a CD4(+) T cell–mediated immunity against actinic keratoses and, potentially, cancers of the skin and other organs. TRIAL REGISTRATION. ClinicalTrials.gov NCT02019355. FUNDING. Not applicable (investigator-initiated clinical trial)

The anatomical distribution of genetic associations

Deeper understanding of the anatomical intermediaries for disease and other complex genetic traits is essential to understanding mechanisms and developing new interventions. Existing ontology tools provide functional, curated annotations for many genes and can be used to develop mechanistic hypotheses; yet information about the spatial expression of genes may be equally useful in interpreting results and forming novel hypotheses for a trait. Therefore, we developed an approach for statistically testing the relationship between gene expression across the body and sets of candidate genes from across the genome. We validated this tool and tested its utility on three applications. First, we show that the expression of genes in associated loci from GWA studies implicates specific tissues for 57 out of 98 traits. Second, we tested the ability of the tool to identify novel relationships between gene expression and phenotypes. Specifically, we experimentally confirmed an underappreciated prediction highlighted by our tool: that white blood cell count – a quantitative trait of the immune system – is genetically modulated by genes expressed in the skin. Finally, using gene lists derived from exome sequencing data, we show that human genes under selective constraint are disproportionately expressed in nervous system tissues

Genetic Mosaic Analysis Indicates That the Bulb Region of Coat Hair Follicles Contains a Resident Population of Several Active Multipotent Epithelial Lineage Progenitors

AbstractThe hair follicle represents an excellent model system for exploring the properties of lineage-forming units in a dynamic epithelium containing multiple cell types. During its growth (anagen) phase, the proximal–distal axis of the mouse coat hair (pelage) follicle provides a historical record of all epithelial lineages generated from its resident stem cell population. An unresolved question in the field is whether the bulb region of anagen pelage follicles contains multipotential progenitors and whether their individual contribution to cellular census fluctuates over time. To address this issue, chimeric follicles were harvested in midanagen from three types of genetic mosaic mouse models. Analysis of the distribution of genotypic markers, including digital three-dimensional reconstruction of serially sectioned chimeric follicles, revealed that on average the bulb contains four or fewer active progenitors, each capable of giving rise to all six follicular epithelial fates. Moreover, analysis of mosaic pelage, as well as cultured whisker follicles provided evidence that bulb-associated progenitors can give rise to expanding descendant clones during midanagen, leading to the conclusion that the bulb contains dormant or symmetrically dividing stem cells. This latter feature resembles the behavior of hematopoietic stem cells after bone marrow transplantation, and raises the question of whether this property may be shared by stem cells in other self-renewing epithelia