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Airway multiciliated cells (MCs) maintain respiratory health by clearing mucus and trapped particles through the beating of motile cilia. While it is known that ciliary lengths decrease along the proximal-distal (P-D) axis of the tracheobronchial tree, how this is regulated is unclear. Here, we demonstrate that canonical Notch signaling in MCs plays a critical role in stabilizing ciliary length. Inhibition of Notch signaling in MCs results in ciliary shortening in the trachea, lengthening in the distal airway, and to region-specific alterations in gene expression. We probe how environmental challenges impact MC homeostasis using germ-free and Mycobacterium tuberculosis ( M. tb ) infection models. While germ-free conditions do not perturb ciliary lengths, M. tb infection leads to lengthening of distal airway cilia, correlating with a downregulation of Notch signaling. These findings reveal that ciliary length and the P-D gradient in the airways are actively regulated, with Notch signaling serving as a stabilizing mechanism.

The sebaceous gland (SG) is a vital appendage of the epidermis, and its normal homeostasis and function is crucial for effective maintenance of the skin barrier. Notch signaling is a well-known regulator of epidermal differentiation, and has also been shown to be involved in postnatal maintenance of SGs. However, the precise role of Notch signaling in regulating SG differentiation in the adult homeostatic skin remains unclear. While there is evidence to suggest that Notch1 is the primary Notch receptor involved in regulating the differentiation process, the ligand remains unknown. Using monoclonal therapeutic antibodies designed to specifically inhibit of each of the Notch ligands or receptors, we have identified the Jag2/Notch1 signaling axis as the primary regulator of sebocyte differentiation in mouse homeostatic skin. Mature sebocytes are lost upon specific inhibition of the Jag2 ligand or Notch1 receptor, resulting in the accumulation of proliferative stem/progenitor cells in the SG. Strikingly, this phenotype is reversible, as these stem/progenitor cells re-enter differentiation when the inhibition of Notch activity is lifted. Thus, Notch activity promotes correct sebocyte differentiation, and is required to restrict progenitor proliferation., Competing Interests: SA S.N.F.A. is an employee of Genentech, SC S.C. is an employee of Genentech and holds shares in Roche, KS K.S. is an employee of Genentech and holds shares in Roche, DL D.L. was a Genentech employee, and is currently employed at Roche, and holds shares in Roche, LV L.V. is an employee of Genentech and holds shares in Roche, FP F.P. is an employee of Genentech and holds shares in Roche, CS C.W.S. was a Genentech employee, and holds shares in Roche. C.W.S is currently employed at Gilead Sciences, (© 2024, Abidi et al.)

Lower airway club cells (CCs) serve the dual roles of a secretory cell and a stem cell. Here, we probe how the CC fate is regulated. We find that, in response to acute perturbation of Notch signaling, CCs adopt distinct fates. Although the vast majority transdifferentiate into multiciliated cells, a "variant" subpopulation (v-CCs), juxtaposed to neuroepithelial bodies (NEBs; 5%-10%) and located at bronchioalveolar duct junctions (>80%), does not. Instead, v-CCs transition into lineage-ambiguous states but can revert to a CC fate upon restoration of Notch signaling and repopulate the airways with CCs and multiciliated cells. The v-CC response to Notch inhibition is dependent on localized activation of β-catenin in v-CCs. We propose that the CC fate is stabilized by canonical Notch signaling, that airways are susceptible to perturbations to this pathway, and that NEBs/terminal bronchioles comprise niches that modulate CC plasticity via β-catenin activation to facilitate airway repair post Notch inhibition., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Exacerbations of symptoms represent an unmet need for people with asthma. Bacterial dysbiosis and opportunistic bacterial infections have been observed in, and may contribute to, more severe asthma. However, the molecular mechanisms driving these exacerbations remain unclear. We show here that bacterial lipopolysaccharide (LPS) induces oncostatin M (OSM) and that airway biopsies from patients with severe asthma present with an OSM-driven transcriptional profile. This profile correlates with activation of inflammatory and mucus-producing pathways. Using primary human lung tissue or human epithelial and mesenchymal cells, we demonstrate that OSM is necessary and sufficient to drive pathophysiological features observed in severe asthma after exposure to LPS or Klebsiella pneumoniae . These findings were further supported through blockade of OSM with an OSM-specific antibody. Single-cell RNA sequencing from human lung biopsies identified macrophages as a source of OSM. Additional studies using Osm -deficient murine macrophages demonstrated that macrophage-derived OSM translates LPS signals into asthma-associated pathologies. Together, these data provide rationale for inhibiting OSM to prevent bacterial-associated progression and exacerbation of severe asthma.

Background & Aims: Notch pathway signaling maintains gastric epithelial cell homeostasis by regulating stem cell proliferation and differentiation. We previously identified NOTCH1 and NOTCH2 as the key Notch receptors controlling gastric stem cell function. Here, we identify the niche cells and critical Notch ligand responsible for regulating stem cell proliferation in the distal mouse stomach., Methods: Expression of Notch ligands in the gastric antrum was determined by quantitative reverse-transcriptase polymerase chain reaction and cellular localization was determined by in situ hybridization and immunostaining. The contribution of specific Notch ligands to regulate epithelial cell proliferation in adult mice was determined by inducible gene deletion, or by pharmacologic inhibition using antibodies directed against specific Notch ligands. Mouse gastric organoid cultures were used to confirm that Notch ligand signaling was epithelial specific., Results: Delta-like 1 (DLL1) and Jagged 1 (JAG1) were the most abundantly expressed Notch ligands in the adult mouse stomach, with DLL1 restricted to the antral gland base and JAG1 localized to the upper gland region. Inhibition of DLL1 alone or in combination with other Notch ligands significantly reduced epithelial cell proliferation and the growth of gastric antral organoids, while inhibition of the other Notch ligands, DLL4, JAG1, and JAG2, did not affect proliferation or organoid growth. Similarly, DLL1, and not DLL4, regulated proliferation of LGR5 + antral stem cells, which express the NOTCH1 receptor., Conclusions: DLL1 is the key Notch ligand regulating epithelial cell proliferation in the gastric antrum. We propose that DLL1-expressing cells at the gland base are Notch niche cells that signal to adjacent LGR5 + antral stem cells to regulate stem cell proliferation and epithelial homeostasis., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Systemic Lupus Erythematosus (SLE) is a chronic autoimmune disease representing a serious unmet medical need. The disease is associated with the loss of self-tolerance and exaggerated B cell activation, resulting in autoantibody production and the formation of immune complexes that accumulate in the kidney, causing glomerulonephritis. TLR7, an important mediator of the innate immune response, drives the expression of type-1 interferon (IFN), which leads to expression of type-1 IFN induced genes and aggravates lupus pathology. Because the lysosomal peptide symporter slc15a4 is critically required for type-1 interferon production by pDC, and for certain B cell functions in response to TLR7 and TLR9 signals, we considered it as a potential target for pharmacological intervention in SLE. We deleted the slc15a4 gene in C57BL/6, NZB, and NZW mice and found that pristane-challenged slc15a4-/- mice in the C57BL/6 background and lupus prone slc15a4-/- NZB/W F1 mice were both completely protected from lupus like disease. In the NZB/W F1 model, protection persisted even when disease development was accelerated with an adenovirus encoding IFNα, emphasizing a broad role of slc15a4 in disease initiation. Our results establish a non-redundant function of slc15a4 in regulating both innate and adaptive components of the immune response in SLE pathobiology and suggest that it may be an attractive drug target., Competing Interests: At the time of conducting this research all authors were employees of Genentech Inc. Nico Ghilardi is currently employed at DiCE Molecules. This commercial affiliation with either Genentech or DiCE Molecules does not alter our adherence to PLOS ONE policies on sharing data and materials.

ERBB3 is a pseudokinase domain-containing member of the ERBB family of receptor tyrosine kinases (RTKs). Following ligand binding, ERBB receptors homo- or hetero-dimerize, leading to a head-to-tail arrangement of the intracellular kinase domains, where the "receiver" kinase domain of one ERBB is activated by the "activator" domain of the other ERBB in the dimer. In ERBB3, a conserved valine at codon 943 (V943) in the kinase C-terminal domain has been shown to be important for its function as an "activator" kinase in vitro . Here we report a knock-in mouse model where we have modified the endogenous Erbb3 allele to allow for tissue-specific conditional expression of Erbb3 V943R ( Erbb3 CKI-V943R ). Additionally, we generated an Erbb3 D850N ( Erbb3 CKI-D850N ) conditional knock-in mouse model where the conserved aspartate in the DFG motif of the pseudokinase domain was mutated to abolish any potential residual kinase activity. While Erbb3 D850N/D850N animals developed normally, homozygous Erbb3 V943R/V943R expression during development resulted in embryonic lethality. Further, tissue specific expression of Erbb3 V943R/V943R in the mammary gland epithelium following its activation using MMTV-Cre resulted in delayed elongation of the ductal network during puberty. Single-cell RNA-seq analysis of Erbb3 V943R/V943R mammary glands showed a reduction in a specific subset of fibrinogen-producing luminal epithelial cells., Competing Interests: The authors from Genentech hold company shares/options., (© 2020 Genentech, Inc. Advanced Genetics published by Wiley Periodicals LLC.)

Rationale: Declining lung function in patients with interstitial lung disease is accompanied by epithelial remodeling and progressive scarring of the gas-exchange region. There is a need to better understand the contribution of basal cell hyperplasia and associated mucosecretory dysfunction to the development of idiopathic pulmonary fibrosis (IPF). Objectives: We sought to decipher the transcriptome of freshly isolated epithelial cells from normal and IPF lungs to discern disease-dependent changes within basal stem cells. Methods: Single-cell RNA sequencing was used to map epithelial cell types of the normal and IPF human airways. Organoid and air-liquid interface cultures were used to investigate functional properties of basal cell subtypes. Measurements and Main Results: We found that basal cells included multipotent and secretory primed subsets in control adult lung tissue. Secretory primed basal cells include an overlapping molecular signature with basal cells obtained from the distal lung tissue of IPF lungs. We confirmed that NOTCH2 maintains undifferentiated basal cells and restricts basal-to-ciliated differentiation, and we present evidence that NOTCH3 functions to restrain secretory differentiation. Conclusions: Basal cells are dynamically regulated in disease and are specifically biased toward the expansion of the secretory primed basal cell subset in IPF. Modulation of basal cell plasticity may represent a relevant target for therapeutic intervention in IPF.

Prevailing dogma holds that cell-cell communication through Notch ligands and receptors determines binary cell fate decisions during progenitor cell divisions, with differentiated lineages remaining fixed. Mucociliary clearance in mammalian respiratory airways depends on secretory cells (club and goblet) and ciliated cells to produce and transport mucus. During development or repair, the closely related Jagged ligands (JAG1 and JAG2) induce Notch signalling to determine the fate of these lineages as they descend from a common proliferating progenitor. In contrast to such situations in which cell fate decisions are made in rapidly dividing populations, cells of the homeostatic adult airway epithelium are long-lived, and little is known about the role of active Notch signalling under such conditions. To disrupt Jagged signalling acutely in adult mammals, here we generate antibody antagonists that selectively target each Jagged paralogue, and determine a crystal structure that explains selectivity. We show that acute Jagged blockade induces a rapid and near-complete loss of club cells, with a concomitant gain in ciliated cells, under homeostatic conditions without increased cell death or division. Fate analyses demonstrate a direct conversion of club cells to ciliated cells without proliferation, meeting a conservative definition of direct transdifferentiation. Jagged inhibition also reversed goblet cell metaplasia in a preclinical asthma model, providing a therapeutic foundation. Our discovery that Jagged antagonism relieves a blockade of cell-to-cell conversion unveils unexpected plasticity, and establishes a model for Notch regulation of transdifferentiation.

The hierarchical relationships between stem cells and progenitors that guide mammary gland morphogenesis are still poorly defined. While multipotent basal stem cells have been found within the myoepithelial compartment, the in vivo lineage potential of luminal progenitors is unclear. Here we used the expression of the Notch1 receptor, previously implicated in mammary gland development and tumorigenesis, to elucidate the hierarchical organization of mammary stem/progenitor cells by lineage tracing. We found that Notch1 expression identifies multipotent stem cells in the embryonic mammary bud, which progressively restrict their lineage potential during mammary ductal morphogenesis to exclusively generate an ERαneg luminal lineage postnatally. Importantly, our results show that Notch1-labelled cells represent the alveolar progenitors that expand during pregnancy and survive multiple successive involutions. This study reveals that postnatal luminal epithelial cells derive from distinct self-sustained lineages that may represent the cells of origin of different breast cancer subtypes.

Tissue clearing and subsequent imaging of transparent organs is a powerful method to analyze fluorescently labeled cells and molecules in 3D, in intact organs. Unlike traditional histological methods, where the tissue of interest is sectioned for fluorescent imaging, 3D imaging of cleared tissue allows examination of labeled cells and molecules in the entire specimen. To this end, optically opaque tissues should be rendered transparent by matching the refractory indices throughout the tissue. Subsequently, the tissue can be imaged at once using laser-scanning microscopes to obtain a complete high-resolution 3D image of the specimen. A growing list of tissue clearing protocols including 3DISCO, CLARITY, Sca/e, ClearT2, and SeeDB provide new ways for researchers to image their tissue of interest as a whole. Among them, 3DISCO is a highly reproducible and straightforward method, which can clear different types of tissues and can be utilized with various microscopy techniques. This protocol describes this straightforward procedure and presents its various applications. It also discusses the limitations and possible difficulties and how to overcome them.

The identity of mammary stem and progenitor cells remains poorly understood, mainly as a result of the lack of robust markers. The Notch signaling pathway has been implicated in mammary gland development as well as in tumorigenesis in this tissue. Elevated expression of the Notch3 receptor has been correlated to the highly aggressive "triple negative" human breast cancer. However, the specific cells expressing this Notch paralogue in the mammary gland remain unknown. Using a conditionally inducible Notch3-CreERT2(SAT) transgenic mouse, we genetically marked Notch3-expressing cells throughout mammary gland development and followed their lineage in vivo. We demonstrate that Notch3 is expressed in a highly clonogenic and transiently quiescent luminal progenitor population that gives rise to a ductal lineage. These cells are capable of surviving multiple successive pregnancies, suggesting a capacity to self-renew. Our results also uncover a role for the Notch3 receptor in restricting the proliferation and consequent clonal expansion of these cells.

Notch signalling is implicated in stem and progenitor cell fate control in numerous organs. Using conditional in vivo genetic labelling we traced the fate of cells expressing the Notch2 receptor paralogue and uncovered the existence of two previously unrecognized mammary epithelial cell lineages that we term S (Small) and L (Large). S cells appear in a bead-on-a-string formation and are embedded between the luminal and basal/myoepithelial layers in a unique reiterative pattern, whereas single or paired L cells appear among ductal and alveolar cells. Long-term lineage tracing and functional studies indicate that S and L cells regulate ipsi- and contralateral spatial placement of tertiary branches and formation of alveolar clusters. Our findings revise present models of mammary epithelial cell hierarchy, reveal a hitherto undescribed mechanism regulating branching morphogenesis and may have important implications for identification of the cell-of-origin of distinct breast cancer subtypes.

Introduction: In this study we evaluated the immunohistochemical expression of the receptors Notch 1 and Notch 2, the ligand Delta 1, and the transcription factors HES 1 and HES 5 in the epithelium of well-defined periapical cysts., Methods: Immunohistochemistry was carried out on 55 formalin-fixed and paraffin-embedded, well-defined periapical cysts with minimum inflammation, obtained from the archival tissue database of the Department of Oral Pathology and Surgery. Western blotting was performed to evaluate the specificity of the anti-Notch antibody and the expression of Notch signaling in 5 fresh-frozen periapical cysts. The levels of staining intensity were estimated by the performance of a semiautomated image analysis system. Descriptive statistic of mean values obtained by computerized image analysis method was performed., Results: Immunostaining reaction of all Notch signaling components was observed in the cytoplasm and/or the cytoplasmic membrane in the majority of epithelial cells of periapical cysts. Nuclear staining was observed occasionally in all cases. Notch 2 showed strong staining in 52.83% of the cases, followed by Notch 1 (35.85%), HES 1 and HES 5 moderate staining in 72.73% and 57.69% of the cases, respectively, and Delta 1 weak staining in 58.33% of the cases. No statistical correlation was found between the antibodies and the sex or the age of the study group., Conclusions: Notch is an evolutionarily conserved signaling mechanism that regulates cell fate decisions during development and postnatal life in organisms as diverse as worms, flies, and humans. The present observations indicate that Notch pathway is active downstream in the lining epithelium of periapical cysts, suggesting an involvement of this pathway in periapical cyst growth and expansion., (Copyright © 2011 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

The conserved role of Notch signaling in controlling intestinal cell fate specification and homeostasis has been extensively studied. Nevertheless, the precise identity of the cells in which Notch signaling is active and the role of different Notch receptor paralogues in the intestine remain ambiguous, due to the lack of reliable tools to investigate Notch expression and function in vivo. We generated a new series of transgenic mice that allowed us, by lineage analysis, to formally prove that Notch1 and Notch2 are specifically expressed in crypt stem cells. In addition, a novel Notch reporter mouse, Hes1-EmGFP(SAT), demonstrated exclusive Notch activity in crypt stem cells and absorptive progenitors. This roster of knock-in and reporter mice represents a valuable resource to functionally explore the Notch pathway in vivo in virtually all tissues.

The synthesis of M(I)(CO)(3)(NNO) (M = Re, (99m)Tc) complexes conjugated to the antitumor agent 2-(4'-aminophenyl)benzothiazole and to its 6-methyl derivative, as well as their in vitro and in vivo biological evaluation as breast cancer radiopharmaceuticals, is reported. The Re complexes displayed under the fluorescence microscope clear uptake by the sensitive to the 2-(4'-aminophenyl)benzothiazole pharmacophore breast cancer cell lines MCF-7 and T47D, while uptake by less sensitive lines and by normal fibroblasts was much weaker. In accordance, uptake of the corresponding radioactive (99m)Tc complexes was clearly higher in the breast cancer cell lines MCF-7 and MDA-231 compared to normal fibroblasts. Biodistribution of the (99m)Tc complexes in SCID mice bearing MCF-7 xenografts showed appreciable tumor uptake. A tumor/muscle ratio of 2.2 was measured for the complex conjugated to 2-(4'-aminophenyl)benzothiazole that led to successful tumor imaging. The results render the 2-(4'-aminophenyl)benzothiazole complexes potential candidates for imaging ((99m)Tc) and targeted radiotherapy ((188)Re) of breast cancer.

GHRH, besides its neuroendocrine action in controlling the release of GH from the pituitary, stimulates the growth of various cancers in vivo and in vitro by direct mechanism(s). However, the molecular mechanism that mediates these proliferative effects of GHRH in extrapituitary tissues remains poorly characterized. In the present study, we investigated whether the tumor suppressor p21/waf1 is involved in the mediation of the proliferative effects of GHRH in A549 human lung cancer epithelial cells. Exposure of A549 cells to the GHRH antagonist JMR-132 caused a significant inhibition in the rate of cell proliferation. In A549 cells, GHRH suppressed while JMR-132 increased the levels of p21 expression in a dose-dependent manner. This suggests that GHRH could regulate p21 levels. We then evaluated whether p21 is required in A549 cells for the regulation of cell proliferation by GHRH. To this end, we knocked-down p21 expression in A549 cells by siRNA and assessed the effects of antagonist JMR-132 on cell proliferation. We found that the loss of p21 expression abolished the anti-proliferative effects of JMR-132. Suppression of p21 expression by siRNA in human HT29 colon cancer cells and non-transformed mouse osteoblasts KS483 also blocked the anti-proliferative effects of JMR-132 suggesting that the regulation of cell proliferation by GHRH is p21 dependent. These results shed light on the molecular mechanism of action of GHRH antagonists in tumor tissues and suggest that the antineoplastic activity of GHRH antagonists could be considered for the treatment of cancers expressing p21.

The efficacy of chemotherapy is usually viewed as the outcome of cancer-cell-autonomous processes while the contribution of stroma is being overseen. Here we show that p53 mutations in stromal fibroblasts, a genetic lesion that is detectable in primary breast, prostate and probably other cancers, while they accelerate tumorigenesis they also sensitize tumours against conventional chemotherapy by doxorubicin and cis-platinum. The mechanism by which p53 of stromal fibroblasts affects the response of a tumour against chemotherapy is likely to involve the induction of senescence in the fibroblasts which in turns results in the production of growth factors acting onto the cancer cells by paracrine mechanisms. Our findings identify stromal fibroblasts as important modulators of the efficacy of anticancer therapy., ((c) 2008 Wiley-Liss, Inc.)