The NOTCH3 Downstream Target HEYL Is Required for Efficient Human Airway Basal Cell Differentiation [scRNA-Seq]
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PROVIDER: PRJNA706184 | ENA |
REPOSITORIES: ENA
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Project description:Basal cells (BC) are the resident stem/progenitor cells of the adult pseudostratified airway epithelium, whose differentiation program is orchestrated by the NOTCH signaling pathway. NOTCH3 receptor mediated signaling regulates BC to club cell differentiation; however, the downstream responses that regulate this process are largely unknown. In the present study we utilized an in vitro air-liquid interface model of the human pseudostratified airway epithelium to identify the NOTCH3-dependent downstream genes/pathways that regulate human BC to club cell differentiation. Activation of NOTCH3 signaling in BC via lentivirus-mediated over-expression of the active NOTCH3 intracellular domain (NICD3) promoted club cell differentiation. Bulk RNA-seq analysis of control vs NICD3 -transduced cells, identified 692 NICD3 responsive genes enriched for pathways linked to airway epithelial biology and differentiation including Wnt/β-catenin Signaling. Expression of the classical NOTCH target HEYL increased in response to NOTCH3 activation and positively correlated with the club cell marker SCGB1A1. Further, using single-cell RNA-seq, we report that HEYL+ cells primarily clustered with SCGB1A1+ and NOTCH3+ cells. Moreover, HEYL protein co-localized with SCGB1A1 in ALI cultures in vitro and in the human and mouse airway epithelium in vivo. siRNA-mediated knockdown of HEYL in BC led to changes in epithelial structure including altered morphology and significant reductions in transepithelial electrical resistance and expression of tight junction related genes. Finally, HEYL knockdown significantly reduced the number of SCGB1A1+ club cells, along with a corresponding increase in KRT8+ BC-intermediate cells. Overall, our data identifies NOTCH3-HEYL signaling as a key regulator of BC to club cell differentiation.
2021-03-04 | GSE168127 | GEO
Project description:Basal cells (BC) are the resident stem/progenitor cells of the adult pseudostratified airway epithelium, whose differentiation program is orchestrated by the NOTCH signaling pathway. NOTCH3 receptor mediated signaling regulates BC to club cell differentiation; however, the downstream responses that regulate this process are largely unknown. In the present study we utilized an in vitro air-liquid interface model of the human pseudostratified airway epithelium to identify the NOTCH3-dependent downstream genes/pathways that regulate human BC to club cell differentiation. Activation of NOTCH3 signaling in BC via lentivirus-mediated over-expression of the active NOTCH3 intracellular domain (NICD3) promoted club cell differentiation. Bulk RNA-seq analysis of control vs NICD3 -transduced cells, identified 692 NICD3 responsive genes enriched for pathways linked to airway epithelial biology and differentiation including Wnt/β-catenin Signaling. Expression of the classical NOTCH target HEYL increased in response to NOTCH3 activation and positively correlated with the club cell marker SCGB1A1. Further, using single-cell RNA-seq, we report that HEYL+ cells primarily clustered with SCGB1A1+ and NOTCH3+ cells. Moreover, HEYL protein co-localized with SCGB1A1 in ALI cultures in vitro and in the human and mouse airway epithelium in vivo. siRNA-mediated knockdown of HEYL in BC led to changes in epithelial structure including altered morphology and significant reductions in transepithelial electrical resistance and expression of tight junction related genes. Finally, HEYL knockdown significantly reduced the number of SCGB1A1+ club cells, along with a corresponding increase in KRT8+ BC-intermediate cells. Overall, our data identifies NOTCH3-HEYL signaling as a key regulator of BC to club cell differentiation.
2021-03-04 | GSE168126 | GEO
Project description:The NOTCH3 Downstream Target HEYL Is Required for Efficient Human Airway Basal Cell Differentiation
| PRJNA706178 | ENA
Project description:The NOTCH3 Downstream Target HEYL Is Required for Efficient Human Airway Basal Cell Differentiation [RNA-Seq]
| PRJNA706183 | ENA
Project description: Not available
| S-EPMC8620267 | biostudies-literature
Project description:This SuperSeries is composed of the SubSeries listed below.
2021-03-04 | GSE168128 | GEO
Project description:Chronic obstructive pulmonary disease (COPD) is the 3rd leading cause of death in the United States and is caused primarily by cigarette smoking. Increased numbers of mucus-producing secretory (“goblet”) cells defined as goblet cell metaplasia or hyperplasia (GCMH), contributes significantly to COPD pathophysiology. The objective of this study was to determine whether NOTCH signaling regulates goblet cell differentiation in response to cigarette smoke. To this end, primary human bronchial epithelial cells (HBECs) from nonsmokers and COPD smokers were differentiated in vitro on air-liquid interface and exposed to cigarette smoke extract (CSE) for 7 days. NOTCH signaling activity was modulated using (1) the NOTCH/γ-secretase inhibitor Dibenzazepine (DBZ), (2) lentiviral over-expression of the NOTCH3-intracellular domain (NICD3) or (3) NOTCH3-specific siRNA. Cell differentiation and response to CSE were evaluated by qPCR, Western blotting, immunostaining and RNA-Seq. Our data demonstrate that CSE exposure of nonsmoker airway epithelium induced goblet cell differentiation characteristic of GCMH. Treatment with DBZ suppressed CSE-dependent induction of goblet cell differentiation. Furthermore, CSE induced NOTCH3 activation, as revealed by increased NOTCH3 nuclear localization and elevated NICD3 protein levels. Over-expression of NICD3 increased expression of the goblet cell associated genes SPDEF and MUC5AC, whereas NOTCH3 knockdown suppressed CSE-mediated induction of SPDEF and MUC5AC. Finally, CSE exposure of COPD airway epithelium induced goblet cell differentiation in a NOTCH3-dependent manner. These results identify NOTCH3 activation as one of the important mechanisms by which cigarette smoke induces goblet cell differentiation, thus providing a potential strategy to control GCMH-related pathologies in smokers and patients with COPD.
2020-06-16 | GSE152446 | GEO
Project description:Airway basal cells (BC) function as progenitor cells capable of differentiating into ciliated and secretory cells to replenish the airway epithelium during physiological turnover and repair. The objective of this study was to define the role of Notch signaling in regulating human airway BC differentiation into a pseudostratified mucociliated epithelium. Notch inhibition with γ-secretase inhibitors demonstrated Notch activation is essential for BC differentiation into secre-tory cells and ciliated cells, but more so for the secretory lineage. Sustained Notch activation via lentivirus expression of the intracellular domain of each Notch receptor (NICD1-4) demonstrated that the Notch 2 and 4 pathways have little effect on BC differentiation, while activation of the Notch1 or 3 pathways has a major influence, with persistent expression of NICD1 or 3 resulting in a skewing toward secretory cell differentiation with a parallel decrease in ciliated cell differentiation. These observations provide insights into the control of the balance of BC differentiation into the secretory vs ciliated cell lineage, a balance that is critical for maintaining the normal function of the airway epithelium in barrier defense against the inhaled environment. Array-based expression profiling of the Notch signaling pathway genes specifically in human airway basal cells.
2015-04-13 | E-GEOD-53537 | biostudies-arrayexpress
Project description:Loss of arterial smooth muscle cells (SMCs) and abnormal accumulation of the extracellular domain of the NOTCH3 (Notch3ECD) receptor are the two core features of CADASIL, the most common genetic cerebral small vessel disease. Although CADASIL is known to be caused by highly stereotyped dominant mutations in NOTCH3, the relationship between NOTCH3 receptor activity, Notch3ECD accumulation and arterial SMC loss has remained elusive, hampering the development of disease-modifying therapies. Using novel histopathological and multiscale imaging modalities, we quantified previously undetectable CADASIL-driven focal arterial SMC loss in the central nervous system of mice expressing the archetypal Arg169Cys CADASIL mutation. Notably, we found more severe arterial pathology and greater Notch3ECD accumulation in transgenic model mice overexpressing the mutation on a homozygous wild-type Notch3 background (TgNotch3R169C) than in knock-in model Notch3R170C/R170C mice expressing this mutation without a wild-type Notch3 copy. We further showed that wild-type Notch3ECD co-aggregated with mutant Notch3ECD and that elimination of one copy of wild-type Notch3 in TgNotch3R169C mice was sufficient to attenuate Notch3ECD accumulation and arterial pathology. Importantly, RNA sequencing revealed no substantial change in the expression of Notch3-regulated genes in TgNotch3R169C brain arteries. Collectively, our results provide compelling evidence that Notch3ECD accumulation, involving mutant and wild-type NOTCH3, and not aberrant NOTCH3 signaling, is the key driver of arterial SMC loss in CADASIL, thus identifying NOTCH3-lowering approaches as candidate therapeutic strategies.
2024-04-17 | GSE242794 | GEO