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American Journal of Respiratory Cell and Molecular Biology logoLink to American Journal of Respiratory Cell and Molecular Biology
. 2021 Apr;64(4):426–440. doi: 10.1165/rcmb.2020-0302OC

Cigarette Smoke Activates NOTCH3 to Promote Goblet Cell Differentiation in Human Airway Epithelial Cells

Manish Bodas 1, Andrew R Moore 1, Bharathiraja Subramaniyan 1, Constantin Georgescu 2, Jonathan D Wren 2, Willard M Freeman 2, Brent R Brown 1, Jordan P Metcalf 1, Matthew S Walters 1,✉
PMCID: PMC8008804  PMID: 33444514

Abstract

Chronic obstructive pulmonary disease (COPD) is the third leading cause of death in the United States and is primarily caused 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. Primary human bronchial epithelial cells (HBECs) from nonsmokers and smokers with COPD 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 overexpression of the NICD3 (NOTCH3-intracellular domain), or 3) NOTCH3-specific siRNA. Cell differentiation and response to CSE were evaluated by quantitative PCR, Western blotting, immunostaining, and RNA sequencing. We found 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. Overexpression of NICD3 increased the expression of 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 novel potential strategy to control GCMH-related pathologies in smokers and patients with COPD.

Keywords: NOTCH signaling, cigarette smoke, airway epithelial cells, COPD, goblet cell metaplasia or hyperplasia

Clinical Relevance

Increased numbers of mucus-producing secretory (“goblet”) cells, defined as goblet cell metaplasia or hyperplasia (GCMH) in response to cigarette smoke exposure, contributes significantly to chronic obstructive pulmonary disease (COPD) morbidity and mortality. Using an in vitro model of the human airway epithelium, we identify NOTCH3 signaling as one of the mechanisms by which cigarette smoke induces goblet cell differentiation in the airway epithelium. This study furthers our understanding of the underlying mechanisms that regulate goblet cell differentiation in the context of COPD pathophysiology and provides a potential strategy to control GCMH in smokers and patients with COPD.

Chronic obstructive pulmonary disease (COPD) is characterized by a combination of pathological conditions, including chronic bronchitis and emphysema (1, 2), and is the third leading cause of death in the United States (3). Exposure to cigarette smoke (CS) is the primary risk factor for the development and progression of COPD (4). The earliest cigarette smoking–induced changes relevant to the pathogenesis of COPD occur in the airway epithelium (1, 2), a multicellular tissue that covers the luminal surface of the conducting airways from the proximally located trachea to the distal bronchioles (5, 6). This pseudostratified airway epithelium functions as a barrier to protect the lung from harmful environmental factors via the action of specialized cells, including ciliated, secretory (goblet and club), basal, and neuroendocrine cells (5–7). A balance in the percentage of each cell type is critical to maintaining a healthy airway epithelium capable of efficient mucociliary clearance and air flow (6–9). Structural changes in airway epithelial architecture (termed “epithelial remodeling”) play a crucial role in COPD pathogenesis (1, 2). Goblet cell metaplasia or hyperplasia (GCMH) is an epithelial remodeling phenotype characterized by increased numbers of goblet cells (9, 10). These changes culminate in excess mucus production that impairs mucociliary clearance, increases airway resistance, and contributes significantly to COPD morbidity and mortality (1, 9). Therefore, identifying the mechanisms by which CS exposure regulates goblet cell differentiation is critical to developing novel therapeutics to treat COPD-associated GCMH.

The NOTCH signaling pathway plays a vital role in regulating differentiation of the airway epithelium in both humans and mice (11), including goblet cell differentiation (12–23). Activation of canonical NOTCH signaling involves the binding of a plasma membrane-bound ligand (DLL1, DLL3, DLL4, JAG1, or JAG2) to one of the four receptors (NOTCH1–4) located on the plasma membrane of a neighboring cell (24). Upon ligand binding, the receptor is activated by two cleavage events, the final of which is mediated by the γ-secretase enzyme complex that results in the release of the NICD (Notch intracellular domain) into the cytoplasm. The NICD subsequently translocates to the nucleus and induces the transcription of multiple target genes (24). Previous studies have demonstrated the role of NOTCH signaling in the development of GCMH using models involving exposure to allergens, inflammatory cytokines, and viral infections (12, 15, 17–19), but whether NOTCH is involved in CS-induced GCMH is unexplored.

To investigate this, we exposed air–liquid interface (ALI) cultures of primary human bronchial epithelial cells (HBECs) from nonsmokers and patients with COPD to CS extract (CSE). Our data demonstrate that CSE exposure of the airway epithelium generated from nonsmoker and COPD HBECs induces goblet cell differentiation and increases the numbers of MUC5AC+ goblet cells, characteristic of GCMH. CSE exposure induced activation of NOTCH3 signaling and overexpression of the constitutively active NICD3 increased expression of the goblet cell–associated genes SPDEF and MUC5AC, even in the absence of CSE. Finally, siRNA-mediated knockdown of NOTCH3 in nonsmoker and COPD HBECs suppressed CSE-mediated induction of MUC5AC. Therefore, our results identify NOTCH3 signaling as one of the mechanisms by which CS exposure induces goblet cell differentiation in the airway epithelium. This information is critical to understanding the pathophysiology of COPD and provides a potential strategy to control GCMH-related pathologies in smokers and patients with COPD.

Some of the results of these studies have been previously reported in the form of a preprint (bioRxiv, [9 July 2020] https://doi.org/10.1101/2020.07.09.195818).

Methods

Full details of the Methods are provided in the data supplement.

Primary HBEC Culture

Primary HBECs were purchased (Lonza) from nonsmokers (catalog number CC-2540) and smokers with COPD (catalog numbers 00195275 and 00195275S). Donor information is summarized in Table E1 in the data supplement. Cells were maintained in BronchiaLife epithelial airway medium (BLEAM) (catalog number LL-0023; Lifeline Cell Technology) supplemented with penicillin (100 IU/ml)–streptomycin (100 μg/ml), as previously described (14).

ALI Culture

Briefly, 1 × 105 HBECs in 100 μl of BLEAM media were seeded in the apical chamber of a Transwell insert (catalog number 3470; Corning) precoated with human type IV collagen (catalog number C7521; Sigma Aldrich) with 1 ml of BLEAM in the basolateral chamber (ALI Day −2). The following day, fresh BLEAM media was replaced in the apical and basolateral chambers (100 μl and 1 ml, respectively). After 2 days of submerged culture, media from the apical chamber was removed to expose the cells to air (ALI Day 0), and 1 ml of HBTEC ALI differentiation medium (catalog number LM-0050; Lifeline Cell Technology) supplemented with penicillin (100 IU/ml)–streptomycin (100 μg/ml) was added to the basolateral chamber. The media was replaced every other day.

CSE and Dibenzazepine Treatments

Cells were treated with CSE (up to 2.5%) via the basolateral chamber for 7 days (ALI Days 0–7 or ALI Days 28–35) with freshly thawed CSE added at each media change. To suppress NOTCH signaling, cells were treated with dibenzazepine (DBZ) (0.1 μM; catalog number 565789; EMD Chemicals Inc.) from ALI Day 28 to 35 in the absence and presence of CSE, with an equal volume of DMSO (catalog number D2650; Sigma Aldrich) used as vehicle control.

RNA Sequencing

The raw sequencing data are publicly available at the Gene Expression Omnibus website (https://www.ncbi.nlm.nih.gov/geo/; accession number GSE152446). Genes that were differentially expressed in response to CSE treatment were determined using a threshold of P < 0.05 on the false-discovery rate (FDR).

Lentivirus-based Overexpression of NICD3

Generation of control or NICD3-expressing replication-deficient lentiviruses was described previously (14).

siRNA-mediated Knockdown of NOTCH3

HBECs were either transfected with 1 pmol of control siRNA (catalog number 4390844) or NOTCH3 siRNA (catalog number 4392420) using Lipofectamine RNAiMax Reagent (catalog number 13778075) and OptiMEM media (catalog number 31985070) (all from Thermo Fisher Scientific) at the time of seeding the cells in ALI culture.

Statistics

Statistical analysis was performed by two-tailed Mann-Whitney U test using the GraphPad Prism version 8.0. A P value of ≤0.05 was considered significant.

Results

In vitro CSE Exposure Promotes Goblet Cell Differentiation

To study how CS exposure induces goblet cell differentiation in the airway epithelium, HBECs from nonsmokers were cultured in vitro at ALI for 28 days and then exposed to different concentrations of CSE for 7 days (Figure 1A). The expression of the oxidative stress–response gene CYP1A1 was significantly upregulated in the presence of 0.5% (10.8-fold) and 2.5% (92.6-fold) CSE in a concentration-dependent manner, confirming that the cells were responding to CSE exposure (Figure 1B). Hematoxylin and eosin staining of paraffin-embedded sections demonstrated that exposure to CSE led to the appearance of a disordered, thickened epithelium compared with untreated cells (Figure 1C). Analysis of cell type–specific marker expression showed no significant change in expression of basal (KRT5) and ciliated (DNAI1) cell markers in CSE (both 0.5% and 2.5%)-treated cells compared with untreated control cells (Figure 1D). However, CSE treatment significantly downregulated the expression of the club cell marker SCGB1A1 (0.59-fold, 0.5% CSE and 0.47-fold, 2.5% CSE) and significantly increased expression of the goblet cell marker MUC5AC (10.4-fold, 0.5% CSE and 19.1-fold, 2.5% CSE) (Figure 1D). To validate that the gene expression changes observed for the cell type–specific markers reflected alterations in cell populations, we quantified the number of basal, ciliated, club, and goblet cells histologically (Figure 1E). Similar to the gene expression data, there were no significant changes in the numbers of basal (KRT5+) and ciliated (acetylated tubulin+) cells in CSE-treated cells compared with untreated control cells. Furthermore, a significant decrease in club cell numbers (SCGB1A1+) was observed for both 0.5% (0.51-fold) and 2.5% (0.45-fold) CSE treatment. However, only treatment with 2.5% CSE resulted in a significant increase in the numbers of MUC5AC+ goblet cells (4.3-fold). These data demonstrate that treatment of in vitro differentiated airway epithelium with 2.5% CSE promotes goblet cell differentiation and induces epithelial remodeling characteristic of GCMH.

Figure 1.

Figure 1.

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In vitro cigarette smoke extract (CSE) exposure of nonsmoker airway epithelium. (A) Schematic of air–liquid interface (ALI) model. Primary human bronchial epithelial cells (HBECs) from nonsmokers were cultured on ALI for 28 days to differentiate into a pseudostratified epithelium containing basal, ciliated, and secretory (club and goblet) cells. At ALI Day 28, the cultures are then untreated or treated for 7 days with CSE. (B) Quantitative PCR (qPCR) of CYP1A1 expression. Data are represented as fold-change in expression compared with untreated cells from n = 4 independent donors. (C) Hematoxylin and eosin staining. Scale bars, 20 μm. (D) qPCR of cell type–specific marker expression. Data are presented as fold-change in expression compared with untreated cells from n = 4 independent donors. (E) Immunofluorescent staining of basal cells (keratin 5 [KRT5], red), ciliated cells (acetylated tubulin, green), club cells (SCGB1A1, red), and goblet cells (MUC5AC, green). Data are presented as fold-change in cell numbers compared with untreated cells from n = 4 independent donors (see Figure E1 for presentation of these data as the percentage of positive cells for each condition). Scale bars, 20 μm. *P < 0.05.

Genome-Wide Transcriptome Changes in Response to CSE Treatment

To identify candidate genes and/or pathways that regulate goblet cell differentiation in response to CSE, RNA sequencing (RNA-Seq) was performed on untreated and 2.5% CSE–treated airway epithelium 7 days after CSE treatment (ALI Day 35). Comparison of CSE-treated cells with untreated cells identified 273 genes (124 upregulated and 149 downregulated) with significant (FDR-adjusted P < 0.05) expression changes (File E1). Ingenuity Pathway Analysis demonstrated significant enrichment of molecular pathways previously associated with cigarette smoking and smoking-induced lung disease, including HIF1α signaling (10 genes) and aryl hydrocarbon receptor signaling (nine genes) (Figure 2A and File E2) (25, 26). Furthermore, CSE exposure led to expression changes in many genes associated with goblet cell biology (9, 27–30), including inflammatory mediators (CXCL3, CXCL5, IL1β, IL1RN, and IL19), growth factors and signal transducers (AREG, S100A8, S100A9, and S100A12), endoplasmic reticulum stress genes (CREB3L1 and ERN2), mucus hypersecretion genes (AQP5, ATP12A, CLCA2, SLC12A2, SLC26A4, and SLC31A1), and NOTCH signaling genes (JAG2, NOTCH1, and SPDEF) (Figure 2B and File E1).

Figure 2.

Figure 2.

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RNA sequencing (RNA-Seq) analysis to identify CSE-dependent transcriptome changes. Primary HBECs from nonsmokers were cultured on ALI for 28 days to differentiate into a pseudostratified epithelium containing basal, ciliated, and secretory (club and goblet) cells. At ALI Day 28, the cultures were untreated or treated with 2.5% CSE for 7 days then harvested for RNA-Seq analysis (n = 4 independent donors). (A) Pathways enriched in the 273 CSE-responsive gene list on the basis of ingenuity pathway analysis (IPA). Shown are the top 10 IPA-enriched pathways based on P value (log-transformed). (B) Expression changes in genes associated with goblet cell biology present in the 273 CSE-responsive gene list. Data are presented as mean log2 fold-change in expression (2.5% CSE–treated vs. untreated) from n = 4 independent donors. ER = endoplasmic reticulum; GP6 = glycoprotein VI; HIF1α = hypoxia-inducible factor 1-alpha; ILK = integrin-linked kinase.

NOTCH Signaling Activation Regulates CSE-Dependent Goblet Cell Differentiation

NOTCH signaling (12–23) and SPDEF (31) play a critical role in regulation of airway epithelial goblet cell differentiation, with recent studies demonstrating that SPDEF expression is positively regulated by NOTCH signaling (12). Moreover, our RNA-Seq data showed that CSE exposure leads to increased expression of SPDEF, suggestive of increased NOTCH signaling activity (Figure 2B). To determine whether global inhibition of NOTCH signaling by DBZ treatment can suppress CSE-mediated induction of goblet cell differentiation, HBECs from nonsmokers were cultured on ALI for 28 days and then exposed to CSE in the absence or presence of DBZ (Figure 3A). As controls, cells were treated with either DMSO (vehicle control) or DBZ in the absence of CSE. Morphological analysis (via hematoxylin and eosin staining) demonstrated that CSE exposure induced epithelial remodeling, with the appearance of a thickened epithelium (Figure 3B). However, treatment with CSE and DBZ resulted in the appearance of an epithelium similar to DMSO- or DBZ-treated cells, suggesting that CSE-induced changes are regulated in a NOTCH signaling–dependent manner. To further investigate the morphological changes in response to CSE ± DBZ treatment, we quantified the number of basal, ciliated, club, and goblet cells histologically (Figures 3C–3F). Consistent with previous studies demonstrating a role for NOTCH signaling in regulating the balance of ciliated versus secretory cell differentiation in adult airway epithelium (14, 15, 18–20, 32, 33), DBZ treatment alone led to a significant increase (2.01-fold) in ciliated (acetylated tubulin+) cell numbers and a significant decrease in the numbers of both SCGB1A1+ club cells (0.16-fold) and MUC5AC+ goblet cells (0.61-fold) compared with DMSO-treated control cells (Figures 3C–3F). However, no significant changes in basal (KRT5+) cell numbers were observed (Figure 3C). Furthermore, compared with DMSO-treated cells, CSE treatment alone led to no significant changes in basal or ciliated cell numbers (Figures 3C and 3D). As expected, CSE treatment significantly decreased (0.46-fold) the numbers of club cells and significantly increased (3.32-fold) the numbers of goblet cells (Figures 3E–3F). In addition, a comparison of CSE-treated versus CSE and DBZ–treated cells demonstrated no significant changes in the numbers of basal or ciliated cells (Figures 3C and 3D); however, a significant decrease in club cells and goblet cells was observed in CSE and DBZ–treated cells (Figures 3E and 3F). The ability of DBZ to suppress the CSE-mediated increase in goblet cell numbers suggests that CSE promotes goblet cell differentiation via a NOTCH-dependent mechanism.

Figure 3.

Figure 3.

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Treatment with the γ-secretase inhibitor dibenzazepine (DBZ) suppresses CSE-induced goblet cell differentiation. (A) Schematic of ALI model. Primary HBECs from nonsmokers were cultured on ALI for 28 days to differentiate into a pseudostratified epithelium containing basal, ciliated, and secretory (club and goblet) cells. At ALI Day 28, the cultures were treated with DMSO (vehicle), DBZ (0.1 μM), DMSO + CSE (2.5%), and CSE + DBZ for 7 days. (B) Hematoxylin and eosin staining. (C–F) Immunofluorescent staining of basal cells (KRT5, red) (C), ciliated cells (acetylated tubulin, green) (D), club cells (SCGB1A1, red) (E), and goblet cells (MUC5AC, green) (F). Data are presented as fold-change in cell numbers compared with DMSO-treated cells from n = 4 independent donors (see Figure E2 for presentation of these data as the percentage of positive cells for each condition). Scale bars, 20 μm. *P < 0.05.

CSE Exposure Promotes NOTCH3 Signaling Activation to Regulate Goblet Cell Differentiation

Signaling via the NOTCH1-3 receptors and the NOTCH ligands DLL1, JAG1, and JAG2 plays an important role in regulating differentiation of the adult airway epithelium in response to injury and environmental insult (12, 14, 15, 17–20, 22, 23, 32–36). Therefore, we assessed the effect of CSE exposure on expression (mRNA levels) of these genes. No significant changes in expression were observed for DLL1, JAG1, NOTCH2, and NOTCH3 in response to CSE (Figure 4A). However, consistent with our RNA-Seq data (Figure 2B), a significant decrease in the expression of JAG2 (0.64-fold) and NOTCH1 (0.56-fold) was observed in response to CSE (Figure 4A). Activation of canonical ligand–dependent NOTCH signaling results in proteolytic cleavage of the NOTCH receptor at the intracellular transmembrane region and subsequent release of the NICD into the cytoplasm that can enter the nucleus to induce transcription (24). Therefore, to assess NOTCH signaling activation in response to CSE, we quantified the protein levels of the NICD from NOTCH1, 2, and 3 in whole cell lysates by Western blot analysis. Consistent with the gene expression patterns of NOTCH1 and NOTCH2 (Figure 4A), CSE treatment resulted in a significant decrease in NICD1 protein levels and no consistent change in NICD2 protein levels compared with untreated cells (Figures 4B and 4C). In contrast to NICD1 and NICD2, a significant increase in NICD3 protein levels was observed in CSE-treated cells (Figures 4B and 4C), despite there being no change in NOTCH3 mRNA levels (Figure 4A). Furthermore, confocal immunofluorescent staining demonstrated increased NOTCH3 protein levels in both the cytoplasm and nucleus of CSE-treated versus untreated cells (Figure 4D). The increased nuclear staining of NOTCH3 NICD is indicative of increased NOTCH signaling, suggesting that CSE exposure might promote goblet cell differentiation via activation of NOTCH3-dependent signaling. Analysis of cells 2 days after CSE treatment (ALI Days 28–30) revealed changes in mRNA expression of cell type–specific markers and NOTCH receptors consistent with those observed after 7 days of exposure (Figure E3). However, Western blot analysis of the same time point showed a significant increase in NICD3 levels in response to CSE but no change in the levels of NICD1 (Figure E3). These data suggest that CSE-dependent activation of NOTCH3 signaling is an early event in development of GCMH and precedes changes to NOTCH1 signaling. To investigate the expression of NOTCH3 in goblet cells, we performed colocalization of NOTCH3 with MUC5AC (Figure 4E). In both untreated and CSE-treated cells, NOTCH3 was expressed in a subset of cells that surrounded areas of MUC5AC+ goblet cells. However, CSE treatment led to colocalization of NOTCH3 with some MUC5AC+ cells. Consistent with our in vitro findings, we also observed increased staining of both NOTCH3 and MUC5AC in airway sections from smokers compared with nonsmokers (Figure E4). Single-cell RNA-Seq studies of in vitro ALI cultures have shown that NOTCH3 expression is predominantly enriched in club cells and a subpopulation of basal cells (termed “supra or secretory-primed cells”) that are characterized by the expression of the marker KRT13 (23, 37). However, we detected no significant difference in the number of KRT13+ cells with CSE treatment (Figure E5), suggesting that this cell population may not be important for the development of CSE-dependent goblet cell differentiation. To determine the ability of NOTCH3 signaling to promote goblet cell differentiation in the absence of CSE, HBECs from nonsmokers were infected with control lentivirus or lentivirus overexpressing NICD3 and harvested 7 days after infection (Figure 4E). Compared with control lentivirus–infected cells, overexpression of NICD3 led to a significant increase in expression of SPDEF (2.47-fold) and MUC5AC (7.04-fold), demonstrating that constitutive NOTCH3 activation can promote goblet cell differentiation (Figure 4F). In addition, these data demonstrate that SPDEF is a downstream target of NOTCH3 signaling.

Figure 4.

Figure 4.

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CSE treatment induces NOTCH3 activation. (A–E) Primary HBECs from nonsmokers were cultured on ALI for 28 days to differentiate into a pseudostratified epithelium containing basal, ciliated, and secretory (club and goblet) cells. At ALI Day 28, the cultures were untreated or treated for 7 days with 2.5% CSE then harvested for analysis. (A) qPCR of NOTCH ligand (DLL1, JAG1, and JAG2) and receptor (NOTCH1, NOTCH2, and NOTCH3) expression in untreated and CSE-treated cells. Data are presented as fold-change in expression compared with untreated cells from n = 4 independent donors. (B) Western analysis of the NICD (NOTCH1–3 receptor intracellular domain) in untreated and CSE-treated cells. β-tubulin was used as a loading control. (C) Quantification of NOTCH1–3 NICD protein levels. Data are presented as the ratio of NICD/β-tubulin protein levels between untreated and CSE-treated cells from n = 4 independent donors. (D) Immunofluorescent staining and confocal microscopy of NOTCH3 (red) and nuclei (blue, DAPI) in untreated and CSE-treated cells. Scale bars, 10 μm. White arrows indicate nuclear staining of NOTCH3. (E) Immunofluorescent staining of NOTCH3 (red), MUC5AC (green), and nuclei (blue, DAPI) in untreated and CSE-treated cells. Scale bars, 20 μm. (F) Primary HBECs from nonsmokers were infected on ALI with either control lentivirus (Lenti-Control) or lentivirus expressing the activated NOTCH3 NICD (Lenti-NICD3). After 7 days of culture (ALI Day 7), the cells were harvested. qPCR of SPDEF and MUC5AC expression in Lenti-Control and Lenti-NICD3–infected cells. Data are presented as fold-change in expression compared with Lenti-Control cells in n = 6 independent donors (single ALI well analyzed for gene expression per experiment). *P < 0.05 and **P < 0.005.

To investigate whether CSE removal after exposure alters NOTCH3 activation and goblet cell (MUC5AC+) numbers, ALI cultures of nonsmoker HBECs were treated with CSE for 7 days (ALI Days 28–35), followed by the removal of CSE for an additional 7 days (ALI Days 35–42). As expected, CSE exposure significantly increased the numbers of MUC5AC+ cells (2.11-fold) and the intensity of NOTCH3 staining in ALI Day 35 cultures (Figure E6). However, compared with ALI Day 35 CSE-treated cultures, removal of CSE for an additional 7 days significantly reduced the numbers of MUC5AC+ cells (0.43-fold) and led to a reduction in NOTCH3 staining intensity (Figure E6). Combined, these data suggest that continuous CSE exposure is required to induce NOTCH3 signaling activation and maintain goblet cell numbers.

siRNA-mediated Knockdown of NOTCH3 Suppresses CSE-mediated Induction of Goblet Cell Differentiation

Because CSE induces NOTCH3/NICD3 activation (Figures 4B–4D) and overexpression of NICD3 induces goblet cell differentiation (Figure 4F), we next tested the role of NOTCH3 signaling in regulating CSE-dependent induction of goblet cell differentiation. Because of difficulties in achieving high knockdown efficiency with siRNA in fully differentiated airway epithelium (e.g., ALI Day 28), we adopted an alternative approach. Nonsmoker HBECs were transfected with either control siRNA or NOTCH3-specific siRNA at the time of seeding the cells on ALI culture and then treated with CSE for 7 days from ALI Day 0 to ALI Day 7. Quantitative PCR analysis was then performed to measure NOTCH3 knockdown efficiency, response to CSE (CYP1A1 expression), and goblet cell differentiation (SPDEF and MUC5AC expression). Because of the variability in differentiation kinetics and response to CSE between the different HBEC donors, the data are presented as individual experiments (Figure 5). Compared with siRNA control–transfected cells, we consistently observed >70% knockdown of NOTCH3 mRNA expression in siRNA NOTCH3–transfected cells in the absence and presence of CSE. These data were also confirmed at the protein level (Figure E7). Across three independent experiments, CSE treatment of siRNA control–transfected cells resulted in increased expression of the oxidative-response gene CYP1A1, suggesting that the cells were responding to CSE (Figure 5). Furthermore, CSE exposure induced goblet cell differentiation as assessed by increased expression of SPDEF and MUC5AC. Similar to siRNA control cells, CSE treatment of siRNA NOTCH3–transfected cells increased CYP1A1 expression to comparable amounts. However, knockdown of NOTCH3 reduced CSE-induced goblet cell differentiation, as evidenced by the decreased expression of SPDEF and MUC5AC. Combined, these data suggest that CSE exposure induces goblet cell differentiation in the human airway epithelium via activation of NOTCH3 signaling.

Figure 5.

Figure 5.

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siRNA-mediated knockdown of NOTCH3 suppresses CSE-dependent induction of SPDEF and MUC5AC. Primary HBECs from nonsmokers were either transfected with siCon or siN3 during seeding on ALI culture. At ALI Day 0, the cultures were either untreated or treated for 7 days with 2.5% CSE, and then harvested for qPCR analysis of NOTCH3, CYP1A1, SPDEF, and MUC5AC. For each donor (experiments 1–3) the data are presented as mean relative expression compared with Actin-Beta (ACTB) from n = 3 ALI wells. Error bars indicate the SEM. ND = not detected; siCon = control siRNA; siN3 = NOTCH3-specific siRNA.

CSE Exposure Activates NOTCH3 Signaling in COPD Cells to Regulate Goblet Cell Differentiation

Alterations in NOTCH pathway components have been identified in the airways of smokers with and without COPD relative to healthy nonsmokers (13, 33, 38, 39). Furthermore, HBECs from patients with COPD have been shown to have an altered differentiation capacity compared with those from healthy control subjects (40–42). Therefore, from a therapeutic perspective, it is critical to determine whether CSE exposure impacts goblet cell differentiation in COPD airway epithelium in a similar manner to that of healthy nonsmokers. No significant difference in NOTCH3 mRNA and NICD3 protein levels was observed between cells from nonsmokers and cells from smokers with COPD (Figure E8), suggesting that direct exposure of cells to CSE is required to induce NOTCH3 activation. To this end, HBECs from smokers with COPD were cultured in vitro on the ALI for 28 days and then exposed to 2.5% CSE for 7 days (Figure 6). Although all the HBEC donors with COPD differentiated into a pseudostratified epithelium, consistent with other studies (42), the COPD airway epithelium had significantly lower numbers of ciliated cells compared with the nonsmoker HBEC cultures at ALI Day 35 (Figure E9). However, CSE exposure of COPD airway epithelium led to the appearance of a remodeled epithelium (Figure 6A) and a significant increase in the expression of the oxidative stress–response gene CYP1A1 (102.5-fold) (Figure 6B) similar to that observed in nonsmoker airway epithelium (Figures 1B and 1C). Expression of the basal (KRT5) and ciliated (DNAI1) cell markers did not change in the presence of CSE, whereas expression of the club cell marker SCGB1A1 was significantly decreased (0.29-fold) with a corresponding significant increase in expression of the goblet cell markers MUC5AC (17.8-fold) and SPDEF (4.1-fold) (Figure 6B). The CSE-dependent changes in cell-type–specific marker gene expression were then validated by quantifying the number of basal, ciliated, club, and goblet cells histologically (Figure 6C). Corroborating the gene expression data, there were no significant changes in the numbers of basal (KRT5+) and ciliated (acetylated tubulin+) cells in CSE-treated ALI cultures compared with untreated cultures. Furthermore, a significant decrease in the numbers of SCGB1A1+ club cells (0.38-fold) and a corresponding significant increase in MUC5AC+ goblet cells (2.97-fold) were observed with CSE treatment (Figure 6C). To characterize the CSE-dependent transcriptome changes in COPD airway epithelium, RNA-Seq was performed on untreated or 2.5% CSE–treated cells 7 days after treatment (ALI Day 35). Our analysis identified 230 genes (140 upregulated and 90 downregulated) with significant (FDR-adjusted P < 0.05) expression changes (File E3). Molecular pathways associated with the 230 CSE-responsive genes included those previously associated with cigarette smoking and COPD, including LXR/RXR activation (14 genes), aryl hydrocarbon receptor signaling (10 genes), and xenobiotic metabolism signaling (11 genes) (File E4) (25, 26). Comparison of the 230 CSE-responsive genes identified in COPD airway epithelium with the 273 CSE-responsive genes identified in nonsmoker airway epithelium revealed 125 genes common to both groups (File E5). Within this 125 gene set were many of the genes associated with goblet cell biology altered in nonsmoker cells (Figure 2B), including inflammatory mediators (CXCL3, CXCL5, IL1β, IL1RN and IL19), endoplasmic reticulum stress genes (CREB3L1 and ERN2), mucus hypersecretion genes (SLC12A2 and SLC26A4) and NOTCH signaling genes (NOTCH1 and SPDEF) (Figure 6D). Importantly, the direction of CSE-induced gene expression changes for these genes was conserved in nonsmoker and COPD airway epithelium (Figures 2B and 6D), suggesting a shared mechanism of CSE-dependent goblet cell differentiation.

Figure 6.

Figure 6.

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In vitro CSE exposure of chronic obstructive pulmonary disease (COPD) airway epithelium induces goblet cell differentiation. (A–C) Primary HBECs from smokers with COPD were cultured on ALI for 28 days to differentiate into a pseudostratified epithelium containing basal, ciliated, and secretory (club and goblet) cells. At ALI Day 28, the cultures are then untreated or treated for 7 days with 2.5% CSE before harvest. (A) Hematoxylin and eosin staining. Scale bars, 20 μm. (B) qPCR of CYP1A1, KRT5, DNAI1, SCGB1A1, MUC5AC, and SPDEF expression in untreated and CSE-treated cells. Data are presented as fold-change in expression compared with untreated cells from n = 4 independent donors. (C) Immunofluorescent staining of basal cells (KRT5, red), ciliated cells (acetylated tubulin, green), club cells (SCGB1A1, red), and goblet cells (MUC5AC, green). Data are represented as fold-change in cell numbers compared with untreated cells from n = 4 independent donors (see Figure E10 for presentation of these data as the percentage of positive cells for each condition). Scale bars, 20 μm. SEM. *P < 0.05. (D) RNA-Seq analysis of 2.5% CSE-treated versus untreated COPD cells. Expression changes in genes associated with goblet cell biology present in the 230 CSE-responsive genes and altered by CSE exposure of nonsmoker airway epithelium. Data are presented as mean log2 fold-change in expression (2.5% CSE–treated vs. untreated) from n = 4 independent donors.

On the basis of our demonstration that CSE induces goblet cell differentiation in nonsmoker cells in a NOTCH-dependent manner, we quantified gene expression of the NOTCH ligands (DLL1, JAG1, and JAG2) and receptors (NOTCH1, NOTCH2, and NOTCH3) in CSE-treated versus untreated COPD airway epithelium. Similar to nonsmoker airway epithelium (Figure 4A), no significant changes in expression were observed for DLL1, JAG1, NOTCH2, and NOTCH3 (Figure 7A), whereas CSE significantly decreased the expression of JAG2 (0.63-fold) and NOTCH1 (0.66-fold) (Figure 7A). We next investigated NICD1–3 protein levels in response to CSE by Western blot analysis (Figures 7B and 7C). Consistent with the results from nonsmoker airway epithelium (Figures 4B and 4C), CSE treatment led to increased protein levels of NICD3, decreased levels of NICD1, and no consistent change in NICD2 (Figures 7B and 7C). To determine the role of NOTCH3 signaling in regulating CSE-dependent induction of goblet cell differentiation in COPD airway epithelial cells, we performed siRNA-mediated knockdown of NOTCH3 on ALI culture in the absence and presence of CSE using the same strategy described for nonsmoker HBECs. Compared with siRNA control–transfected cells, we consistently observed >80% knockdown efficiency of NOTCH3 mRNA expression in siRNA NOTCH3–transfected cells in the absence and presence of CSE. These data were also confirmed at the protein level (Figure E7). From three independent experiments (presented individually), CSE treatment of cells transfected with either siRNA control or siRNA NOTCH3 resulted in increased expression of the oxidative-response gene CYP1A1 (Figure 7D). We were unable to reliably detect MUC5AC in untreated cells at ALI Day 7; however, CSE exposure induced goblet cell differentiation with increased expression of MUC5AC (all three experiments) and a partial induction of SPDEF (two of three experiments) (Figure 7D). Moreover, knockdown of NOTCH3 suppressed CSE-dependent induction of SPDEF and MUC5AC (Figure 7D). Combined, these data suggest that CSE exposure of COPD airway epithelium induces goblet cell differentiation in a NOTCH3-dependent manner.

Figure 7.

Figure 7.

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CSE induces SPDEF and MUC5AC expression in COPD airway epithelium in a NOTCH3-dependent manner. (A–C) Primary HBECs from smokers with COPD were cultured on ALI for 28 days to differentiate into a pseudostratified epithelium containing basal, ciliated, and secretory (club and goblet) cells. At ALI Day 28, the cultures were untreated or treated for 7 days with 2.5% CSE and then harvested for analysis. (A) qPCR of NOTCH ligand (DLL1, JAG1, and JAG2) and receptor (NOTCH1, NOTCH2, and NOTCH3) expression in untreated and CSE-treated cells. Data are presented as fold-change in expression compared with untreated cells from n = 4 independent donors. *P < 0.05. (B) Western analysis of the NOTCH1–3 NICD in untreated and CSE-treated cells. β-tubulin was used as a loading control. (C) Quantification of NOTCH1–3 NICD protein levels. Data are presented as ratio of NICD/β-tubulin protein levels between untreated and CSE-treated cells from n = 4 independent donors. *P < 0.05. (D) Primary HBECs from smokers with COPD were transfected with either siCon or siN3 during seeding on ALI culture. At ALI Day 0, the cultures were either untreated or treated for 7 days with 2.5% CSE and then harvested for qPCR analysis of NOTCH3, CYP1A1, SPDEF, and MUC5AC. For each donor (experiments 1–3), the data are presented as mean relative expression compared with ACTB from n = 3 ALI wells. Error bars indicate SEM.

Discussion

The NOTCH signaling pathway plays a crucial role in regulating cell fate decisions of multiple cell types in the human and murine airway epithelium, including basal, ciliated, club, and goblet cells (11). Here, we demonstrate that CSE exposure of airway epithelium generated from nonsmoker-derived and smoker with COPD–derived HBECs on ALI culture promotes GCMH in a NOTCH-dependent manner. Further analysis of specific NOTCH components revealed that CSE exposure increased NICD3 protein levels and nuclear localization, indicative of increased NOTCH3 activation. Moreover, constitutive overexpression of NICD3 was sufficient to induce expression of the goblet cell–promoting transcription factor SPDEF and MUC5AC even in the absence of CSE, whereas siRNA-mediated knockdown of NOTCH3 in differentiating nonsmoker and COPD smoker HBECs suppressed CSE-mediated induction of SPDEF and MUC5AC. Overall, these data demonstrate that activation of NOTCH3 signaling regulates airway epithelial goblet cell differentiation in response to CSE, leading to enhanced goblet cell differentiation and the development of GCMH.

It is well established that the NOTCH pathway plays a critical role in regulating goblet cell differentiation of the airway epithelium in both humans and mice during homeostasis and in response to injury or environmental insult (12–23). Previous studies have shown that interactions between JAG1–2 ligands and NOTCH1–3 receptors regulate the development of GCMH in the asthmatic airway epithelium and in response to inflammatory cytokines, allergens, and viral infections (12, 15, 17–19), whereas their role in the CSE-induced GCMH model was unknown. Reid and colleagues (12) recently showed that NOTCH3 expression is upregulated in the airway epithelium of subjects with asthma and that it regulates MUC5AC expression, whereas Jing and colleagues (17) demonstrated that rhinovirus infection regulates development of GCMH in COPD airway epithelial cells in a NOTCH3-dependent manner. Our study expands on these findings by demonstrating that CSE exposure activates NOTCH3 signaling to induce goblet cell differentiation and MUC5AC expression in both nonsmoker and COPD airway epithelial cells.

Basal cells are the stem/progenitor cells of the airway epithelium in both humans and mice and are capable of generating club cells, which in turn can undergo differentiation into goblet or ciliated cell lineages (5, 6). The NOTCH-dependent differentiation of basal cells into a pseudostratified airway epithelium is a multistep process, with cell fate decisions involving a coordinated response between different combinations of NOTCH ligand–receptor signaling events (11, 14, 15, 20, 22, 23, 33, 36, 43). Work by Mori and colleagues (33) demonstrated that NOTCH3 activation in mice leads to priming of basal cell differentiation into early/intermediate club cells, which in turn can differentiate into goblet cells or ciliated phenotypes through secondary NOTCH signaling. Similarly, Carraro and colleagues (23) showed that NOTCH3 activity in human cells is required to maintain a subpopulation of basal cells that are capable of differentiating into mature goblet cells. Previous work from our laboratory demonstrated that constitutive activation of NOTCH3 signaling via NICD3 increases both club and goblet cell differentiation of nonsmoker HBECs on ALI culture (14). In addition, work from Reid and colleagues (12) showed that NOTCH3 activation regulates MUC5AC expression. Our data demonstrate that CSE exposure of ALI Day 28 differentiated airway epithelium leads to increased numbers of goblet cells (MUC5AC+) and decreased numbers of club cells (SCGB1A1+), suggesting that CSE-dependent activation of NOTCH3 signaling in club cells may promote further differentiation of this cell population into goblet cells. Furthermore, siRNA-mediated knockdown of NOTCH3 prevented CSE-mediated induction of goblet cell differentiation. In contrast to our study, recent work by Carraro and colleagues (23) demonstrated that inhibition of NOTCH3 signaling promoted goblet cell differentiation of secretory-primed basal cells. This discrepancy may be related to differences in the ALI culture systems and time points analyzed in both studies. In addition, Carraro and colleagues enriched for specific basal cell populations (CD66+ and CD66−) via cell sorting before ALI culture, whereas no cell sorting was used in our study. Therefore, the epithelial cell populations present in each study are potentially different and may respond differentially to either NOTCH3 inhibition or CSE treatment. Thus, further studies are required to clarify how CSE impacts the differentiation of individual cell populations and the timing of NOTCH3 signaling events that regulate this process.

Prior studies identified alterations in NOTCH signaling components at the mRNA, protein, and epigenetic level in the small airway epithelium of smokers with and without COPD relative to that of nonsmokers (13, 33, 38, 39). A study by Tilley and colleagues (38) reported decreased NOTCH3 mRNA in the small airway epithelium in smokers versus nonsmokers, suggesting that NOTCH signaling was suppressed in response to smoking. However, changes in the protein concentrations of active NOTCH3 receptor (NICD3) were not assessed. In support of our study, CS increased the concentrations of NOTCH3 protein in human lung adenocarcinoma both in vitro and in vivo (44). Furthermore, 24 weeks of CS exposure of mice increased NOTCH signaling activity in lung lymphoid tissue (45). Importantly, the mechanism by which CSE activates NOTCH3 signaling to induce goblet cell differentiation remains unknown. Canonical NOTCH signaling is dependent on cell–cell contact and requires binding of a ligand on one cell to the receptor on a neighboring cell (24). Studies in both mice and humans identified the JAG1 and JAG2 ligands as the major regulators of cell differentiation in airway epithelium (18, 19, 22, 33, 35, 43). However, in our study, CSE decreased the expression of JAG2 in the airway epithelium but had no effect on JAG1 expression. Prior work from our laboratory showed that the overexpression of JAG1 in an immortalized airway basal epithelial cell line led to increased secretory cell differentiation (22). Therefore, one possibility is that decreased JAG2 expression in response to CSE exposure leads to increased availability of JAG1 and subsequent activation of JAG1–NOTCH3–dependent signaling to promote goblet cell differentiation.

CS exposure may also regulate NOTCH3 activation via noncanonical ligand-independent mechanisms (24). It is noteworthy that CSE exposure increased NICD3 protein levels (and nuclear localization) with no change in the expression of NOTCH3 mRNA, suggesting that CSE regulates NOTCH3 protein levels post-transcriptionally. Direct interaction of post–secretase-cleaved NOTCH3 fragments with the E3-ubiquitin ligase WWP2 leads to monoubiquitination of NICD3, which promotes its sorting to and degradation in lysosomes, leading to suppression of NOTCH3 signaling (46). Therefore, CSE exposure may induce NOTCH3 activation via increasing NICD3 half-life by preventing its ubiquitination and subsequent degradation in lysosomes. Although we do not observe any changes in WWP2 expression in response to CSE, it is plausible that CSE alters the expression or activity of other E3-ubiquitin ligases that regulate NICD3 ubiquitination and stability. Alternatively, cellular redox status has been shown to regulate NOTCH3 protein concentrations through lysosome-dependent protein degradation (47). Treatment of cancer cells with the antioxidant N-acetyl-cysteine leads to increased NOTCH3 degradation through lysosome-dependent pathways with no change in NOTCH3 mRNA concentrations (48). Moreover, CS exposure induces high amounts of oxidative stress because of the cumulative effect of intrinsic reactive oxygen species present in the smoke, and increased cellular reactive oxygen species production (49, 50). Based on this knowledge, further studies are warranted to evaluate whether CS-induced oxidative stress in the airway epithelium leads to impairment of lysosome-dependent NOTCH3 protein degradation.

In summary, our data demonstrate that CSE-induced NOTCH3 activation is a key regulator of GCMH development in the airway epithelium derived from both nonsmokers and subjects with COPD. Thus, targeting NOTCH3 signaling could be used as a novel therapeutic strategy to control GCMH in smokers with and without COPD.

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Acknowledgments

Acknowledgment

The authors thank Drs. Linda Thompson, Dean Dawson, Lorin Olson, and Xiao-Hong Sun at the Oklahoma Medical Research Foundation for discussions, guidance, and support. They also thank the Imaging Core Facility at the Oklahoma Medical Research Foundation for assistance in sample processing and confocal microscopy.

Footnotes

Supported by National Institute of General Medical Sciences (NIGMS) COBRE Grant (GM103636, Project 4), Oklahoma Center for Adult Stem Cell (OCASCR) Research Grant, Oklahoma Shared Clinical and Translational Resources Pilot Project Grant (U54GM104938), College of Medicine Alumni Association Research Grant, and Presbyterian Health Foundation 3D Bio-Printing Seed Grant and New Investigator Seed Grant (M.S.W.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Author Contributions: Conception of study and experimental design: M.B. and M.S.W. Performed experiments: M.B., A.R.M., B.S., and M.S.W. Data analysis and/or interpretation: M.B., A.R.M., B.S., C.G., J.D.W., W.M.F., B.R.B., J.P.M., and M.S.W. Writing and preparation of original draft manuscript: M.B. and M.S.W. All authors have reviewed, critiqued and approved the final manuscript.

This article has a data supplement, which is accessible from this issue’s table of contents at www.atsjournals.org.

Originally Published in Press as DOI: 10.1165/rcmb.2020-0302OC on January 14, 2021

Author disclosures are available with the text of this article at www.atsjournals.org.

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rcmb.2020-0302OC_disclosures.pdf (239.6KB, pdf)

Articles from American Journal of Respiratory Cell and Molecular Biology are provided here courtesy of American Thoracic Society

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