American Journal of Respiratory Cell and Molecular Biology

Excessive mucus production has been linked to many of the pathologic features of respiratory diseases, including obstruction of the airways, decline in lung function, increased rates of mortality, and increased infections. The mucins, MUC5AC and MUC5B, contribute to the viscoelastic properties of mucus, and are found at elevated levels in the airways of individuals with chronic respiratory diseases. The T helper type 2 cell cytokine, IL-13, is known to regulate MUC5AC expression in goblet cells of the airways, although much less is known about the regulation of MUC5B expression. In a study to further understand the mediators of MUC5AC and MUC5B expression, neuregulin (NRG) 1β1 was identified as novel regulator of goblet cell formation in primary cultures of human bronchial epithelial cells (HBECs). NRG1β1 increased expression of MUCAC and MUC5B proteins in a time- and dose-dependent fashion in HBEC cultures. NRG1β1-induced expression of MU5AC and MUC5B was shown to involve v-erb-b2 erythroblastic leukemia viral oncogene homolog (ErbB) and ErbB3 receptors, but not ErbB4 receptors. Treatment of HBECs with inhibitors of p38 mitogen-activated protein kinase, extracellular signal–regulated kinase1/2, and phosphatidylinositol 3-kinase indicated that these kinases were involved in NRG1β1-induced MUC5AC and MUC5B expression. Additionally, NRG1β1 was shown to induce the phosphorylation of the ErbB2 receptor, AKT, and extracellular signal–regulated kinase 1/2. NRG1β1 protein was found increased in the airways of antigen-challenged mice, together with increases in MUC5AC and MUC5B message. Together, these data indicate that NRG1β1 is a novel mediator of MUC5AC and MUC5B expression in HBECs, and may represent a novel therapeutic target for mucus hypersecretion in respiratory diseases.

These studies identify neuregulin 1β1 as a novel mediator of MUC5AC and MUC5B mucin expression. Identification of mechanisms underlying mucin expression has potential therapeutic applications in asthma and chronic obstructive pulmonary disease.

Airway mucus hypersecretion has been linked to several of the hallmark features of respiratory diseases, such as asthma (1) and chronic obstructive pulmonary disease (COPD) (2). Indeed, mucus hypersecretion has been linked to an increase in frequency and duration of infections, decline in lung function, and increases in morbidity and mortality in COPD (24). Furthermore, the progression of COPD has been reported to be strongly associated with accumulation of mucus in the lumen of the small airways (5), and, more recently, mucus occlusion of the small airways has been linked with premature death (6). Whereas in the large airways mucus is produced by goblet cells and submucosal glands, in the small airways the only source of mucus is the goblet cell (7). Perhaps not surprisingly, increased numbers of goblet cells in the bronchiolar epithelium of patients with COPD and chronic bronchitis have been described (8). In the case of asthma, morphometric analysis of lungs from patients who died from a severe acute asthma attack showed increases in goblet cell numbers and mucus in the airway lumen (1). In addition, mucus plugging of the airway lumen has been reported as a major contributing cause of fatal asthma in most patients (9, 10).

The mucus layer plays a beneficial role in healthy individuals, aiding the removal of inhaled exogenous substances, including toxins and bacteria, via the process of mucociliary clearance (11). Mucins MUC5AC and MUC5B are the major gel-forming mucins found in human airway secretions. However, in respiratory diseases such as asthma and COPD, increases in airway mucus containing MUC5AC and MUC5B contribute to airway obstruction (12, 13). One mediator shown to influence MUC5AC gene and protein expression in vitro and in vivo is the T helper type 2 cell cytokine, IL-13 (1417). In primary human bronchial epithelial cell (HBEC) cultures, IL-13 has been shown to induce the expression of the goblet cell mucin MUC5AC (17). Similarly, in transgenic mice expressing IL-13 in airway epithelial cells or in mice given IL-13 intranasally, increased numbers of goblet cells and associated increases in mucin expression are observed (15, 16, 18, 19). Moreover, studies with signal transducer and activator of transcription 6–deficient mice have implicated this transcription factor as playing a central role in mucus production in response to IL-13 (16).

Although much work has focused on the effects of IL-13 on the regulation of MUC5AC expression, the regulation of MUC5B expression in respiratory diseases is much less well characterized. MUC5B has been reported as the major mucin in COPD (20, 21), and, as well as being expressed by submucosal glands, has been shown to be expressed by surface epithelial cells in tissue sections from subjects with COPD and asthma (22). Studies using in vitro epithelial cell cultures have shown that retinoic acid can induce the expression of MUC5B (2325). In another report, cytokines IL-6 and IL-17 were linked with an increase in MUC5B expression, together with increases in MUC5AC in HBEC cultures (26), and, more recently, reactive oxygen species were shown to increase MUC5B expression in HBECs (27).

In a study to further establish mediators involved in MUC5AC and MUC5B production, we identified neuregulin (NRG) 1β1 as a regulator of goblet cell formation in vitro. In this study, we have investigated the signaling involved in NRG1β1-induced goblet cell formation. Our data suggest that NRG1β1 represents a potential therapeutic target for modulating MUC5AC and MUC5B expression in respiratory diseases.

Materials

NRG1α and NRG1β1 were purchased from R&D Systems (Abingdon, UK), and IL-13 was purchased from Peprotech (London, UK). The phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002, the p38 mitogen-activated protein kinase (MAPK) inhibitors, SB203580 and SB202190, and extracellular signal–regulated kinase (ERK) 1/2 SL327 inhibitor were purchased from Calbiochem (Nottingham, UK). The inhibitor, AEE788 (28), was synthesized by Novartis Institutes of Biomedical Research (Horsham, UK).

Cell Culture

HBECs (donors 2F1578, 2F1688, and 2F1341; Lonza group Ltd., Basel, Switzerland) were cultured in bronchial epithelial cell growth medium (Lonza) supplemented with the provided singlequots. For differentiation, cells were grown on 0.4-μm pore size, 12 mm Transwell inserts (Corning, Corning, NY) at a cell density of 8.25 × 104 cells per insert in differentiation medium. Differentiation medium contained 50% bronchial epithelial cell growth medium and 50% Dulbecco's modified Eagle's medium (Invitrogen, Paisley, UK), and was supplemented with 52 μg/ml bovine pituitary extract, 5 μg/ml insulin, 0.5 μg/ml hydrocortisone, 10 μg/ml transferrin, 0.5 μg/ml epinephrine, 0.5 ng/ml human epidermal growth factor (EGF), 50 μg/ml gentamicin, and 50 nM retinoic acid. Cells were maintained submerged for 7 days, then grown at air–liquid interface (ALI) for the remaining 7 days. Cells were treated with cytokines or growth factors during the ALI culture period, added to the basolateral chamber of the Transwell inserts. Cytokines analyzed were IL-1α, -2, -3, -4, -7, -9, -10, -11, -15, -17C, -18, -20, -22, -25, -26, and -27, TNF-α, TNF-β, granulocyte–colony-stimulating factor (G-CSF), granulocyte macrophage–CSF (GM-CSF), cardiotrophin, and leukemia inhibitory factor (LIF) (R&D Systems), and IL-13 (Peprotech). Growth factors analyzed were bone morphogenetic protein (BMP)-2, -4, and -7, fibroblast growth factor (FGF)-1, -9, -19, insulin-like growth factor (IGF)-I and -II, macrophage-colony stimulating factor (M-CSF), NRG1α and -1β1, platelet-derived growth factor (PDGF)-AA, PDGF-AB, PDGF-BB, transforming growth factor (TGF)-α, -β1, -β2, and -β3, and were also from R&D Systems. Cytokine and growth factors were tested on HBECs at two concentrations based on previous literature reports of cell-based functional effects, and were in the range of 1–100 ng/ml. Where included, inhibitors or antibody were added to the basolateral chamber of the Transwell inserts for 2 hours before the addition of NRG1β1, and were included in the culture medium for the duration of the experiment. HBECs were treated with compound or antibody in combination with NRG1β1 for 7 days at ALI, replacing the NRG1β1 together with fresh antibody or compound every time the medium was replenished (every 2–3 d). Concentrations of inhibitor tested were based on previous literature reports, where possible, indicating no adverse toxicity after prolonged treatment (17, 29, 30).

For antibody treatment of cells in combination with NRG1β1, v-erb-b2 erythroblastic leukemia viral oncogene homolog (ErbB) receptor (AF1129), and ErbB3 receptor antibodies (MAB3841) were from R&D Systems, and ErbB4 receptor antibody (MS-304-P1ABX) was from Lab Vision (Runcorn, UK).

Immunohistochemical Detection of MUC5AC and MUC5B Protein

After 7 days (unless otherwise stated) of differentiation at ALI, the apical surface of the HBECs was washed gently with PBS and fixed with 10% neutral buffered formalin, and wax embedded. Inserts were sectioned at 3-μm thickness and stained with either a MUC5AC (clone 45M1, 1:1,000 dilution; Labvision) or a MUC5B (1:2,000 dilution) monoclonal antibody using a 3,3′-diaminobenzidine (DAB) map protocol on a Ventana XT immunostainer (Ventana, Tuscon, AZ). Incubations with primary antibodies were performed at 37°C for 20 minutes (MUC5AC) or 60 minutes (MUC5B). Secondary antibodies were a Universal secondary antibody for MUC5AC (Ventana) or goat anti-mouse IgG2b for MUC5B (Southern Biotech, Birmingham, AL). All antibodies were diluted in Tris-buffered saline (TBS) (pH 7.6). Sections were counterstained with 1% (wt/vol) alcian blue in 3% (wt/vol) acetic acid (pH 2.5), and nuclei were lightly stained with hematoxylin. The areas of MUC5AC and MUC5B staining were assessed using a Zeiss Axioplan 2 microscope (10× magnification; Carl Zeiss AG, München, Germany) with a KS400 image analyzer (Image Associates, Bicester, UK). A total of 14 fields were scored for each sample. Data are presented as goblet cell density, which was defined as the ratio of stained area (μm2) to length (μm) of epithelium scored, as described previously (17). A custom-prepared monoclonal antibody against MUC5B was raised against a synthetic peptide, SWYNGHRPEPGLG, and was obtained from the Hybridoma Core Laboratory, Interdisciplinary Center for Biotechnology Research, University of Florida (Gainesville, FL). The specificity of the MUC5B antibodies were confirmed by ELISA using purified MUC5AC and MUC5B proteins provided by D. Thornton (University of Manchester, Manchester, UK).

Quantitative RT-PCR

Total RNA was extracted from HBECs using the RNeasy kit (Qiagen, Crawley, UK). cDNA was prepared for 1 μg total RNA using the first-strand cDNA synthesis kit (Roche Diagnostics, Burgess Hill, UK) using random hexamer primers. Quantitative RT-PCR (QRT-PCR) was used to analyze MUC5AC and MUC5B expression with the following primers and probes (from Applied Biosystems, Foster City, CA): MUC5AC (sense 5′-TGGGAGTCCAGGTCATGTTCT-3′, antisense 5′-CGTGCGGCACTCATCCTT-3′, and probe 5′-ACTTGCACCAACGACAG-3′); MUC5B (sense 5′-GGGATCTTCCTGGTCATCGA-3′, antisense 5′-GCTACGCGTGGCAAAGTCAT-3′, and probe 5′-AGCGTGTTCATCCGACTG-3′). Reactions were performed in a 96-well plate using an ABI7900 Sequence Detection System (Applied Biosystems) with the following conditions: 1 cycle of 50°C for 2 minutes, 1 cycle of denaturation (95°C for 10 min) followed by 40 cycles of denaturation (95°C for 15 s), and annealing/extension (60°C for 1 min). Expression values were interpolated from an HBEC cDNA standard curve and normalized to the housekeeping gene, Hypoxanthine phosphoribosyl transferase 1 (HPRT) (Applied Biosystems).

For quantitative analysis of MUC5AC and MUC5B expression in mouse lung tissue, RNA was isolated from lung tissue from a mouse ovalbumin (OVA) model of allergen-induced goblet cell formation (31). The studies reported here conform to the United Kingdom Animals (Scientific Procedures) Act 1986. BALB/c mice were sensitized with OVA over 2 weeks and given a daily challenge of OVA (50 mg/ml) for 2 consecutive days, as described previously (31). RNA was prepared from mouse lung tissue using the reagents and protocols supplied in the Qiagen RNeasy miniprep kit. Frozen lung tissue (20 mg) was homogenized in 600 μl of RLT buffer (Qiagen) using a polytron homogenizer (Kinematica AG, Littau-Lucerne, Switzerland). PCR reaction mixtures contained 1× SYBR Green PCR Master Mix (Sigma-Aldrich, Poole, UK), 400 nM of each forward and reverse primer, and 200 ng of cDNA template. Samples were analyzed in duplicate using an ABI7900 Sequence Detection System. For analysis of mucin gene expression, the primers used were MUC5AC 5′-CAGCCGAGAGGAGGGTTTGATCT-3′ and 5′-AGTCTCTCTCCGCTCCTCTCAAT-3′, MUC5B 5′-AGGAAGACCAGTGTGTTTGTC-3′ and 5′-GTCCTCATTGAAGAAGGGCTG-3′, and β-actin 5′-TGTGATGGTGGGAATGGGTCAG-3′ and 5′-TTTGATGTCACGCACGATTTCC-3′. The quantitative PCR program was as follows: 50°C for 2 minutes, 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds, 60°C for 15 seconds, and 72°C for 30 seconds. Expression values are shown normalized to the housekeeping gene, β-actin.

RT-PCR Analysis of ErbB Receptor Expression

For analysis of ErbB receptor expression in cDNA prepared from differentiated HBECs, PCR reactions were prepared as follows: PCR reaction mixtures contained 12.5 μl HotStar Taq Master mix (Qiagen), 50 pmol of each forward and reverse primer (Table 1), 50 ng cDNA, and water to a final volume of 25 μl in 0.2-ml, thin-walled PCR tubes. Control PCR reactions were performed with primers specific to the housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase, using primers shown in Table 1. PCR cycling conditions were as follows: denaturation at 95°C for 15 minutes, 35 cycles of denaturation 94°C for 15 seconds, annealing at 55°C for 15 seconds, and extension at 72°C for 45 seconds, followed by a final extension of 5 minutes at 72°C. PCR products were analyzed on 2% (wt/vol) agarose gels.

TABLE 1. RT-PCR PRIMERS FOR ANALYSIS OF ErbB RECEPTOR EXPRESSION


Gene

Primer Sequences

Size of PCR Product (bp)
ErbB1Forward, 5′-GTCCTCATTGCCCTCAACACAG-3′326
Reverse, 5′-CCATTGGGACAGCTTGGATCAC-3′
ErbB2Forward, 5′-CAGTTACCAGTGCCAATATCC-3′250
Reverse, 5′-TTGTGCAGAATTCGTCCCC-3′
ErbB3Forward, 5′-ACTCTGAATGGCCTGAGTG-3′253
Reverse, 5′-CAAACTTCCCATCGTAGACC-3′
ErbB4Forward, 5′-ACCAGCATTGAGCACAACC-3′368
Reverse, 5′-CGTCCACATCCTGAACTACC-3′
GAPDHForward, 5′-CCACCCATGGCAAATTCCATGGCA-3′598

Reverse, 5′-TCTAGACGGCAGGTCAGGTCCACC-3′

Definition of abbreviations: Erb, v-erb-b2 erythroblastic leukemia viral oncogene homolog; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

NRG1β1 ELISA

NRG1β1 protein was quantified in bronchoalveolar lavage (BAL) supernatant from the mouse OVA model (31) using an NRG1β1 ELISA kit (R&D Systems), according to the manufacturer's protocol.

Western Blotting Analysis

For analysis of ErbB receptor expression, HBECs were lysed in ice-cold lysis buffer that contained 50 mM Tris (pH 7.5), 150 mM NaCl, 0.65% (vol/vol) NP-40 supplemented with Complete Protease Inhibitor Cocktail (Roche Diagnostics) and lysates cleared by centrifugation at 16,000 × g for 5 minutes at 4°C. Protein concentration was determined using a Micro BCA Protein Assay kit (Perbio, Cramlington, UK) according to the manufacturer's instructions. Cleared lysates were denatured at 70°C for 10 minutes in 1× NuPAGE sample buffer (Invitrogen) and equal amounts of protein from samples resolved using Bis-Tris NuPAGE polyacrylamide gels with MOPS running buffer (Invitrogen). Proteins were transferred to Immobilon-P Polyvinylidene fluoride (PVDF) membranes (Millipore, Watford, UK) in NuPAGE transfer buffer (Invitrogen). For HBECs treated with NRG1β1 in combination with inhibitors protein lysates were prepared in CeLyA lysis buffer CLB1 (Zeptosens, Witterswil, Switzerland), and were diluted in 1× NuPAGE sample buffer without denaturation for SDS-PAGE analysis. For immunoprobing, primary antibodies were used at 1:1,000 dilution, except where indicated. For analysis of ErbB receptor expression, all primary antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA) and were rabbit polyclonal antibodies, except where indicated as follows: epidermal growth factor receptor (EGFR) (1,005); ErbB2 (C-18); ErbB3 (C-17); ErbB4 (C-18); and glyceraldehyde 3-phosphate dehydrogenase, a mouse monoclonal primary antibody (6C5). For analysis of protein phosphorylation, all antibodies were from Cell Signaling and were rabbit polyclonal antibodies, except where indicated as follows: phospho-AKT (Ser473; 1/500); AKT; phospho-ERK1/2 (Thr202/Tyr204); ERK1/2 and phospho-ErbB2 (Tyr1221/1222), a rabbit monoclonal antibody (6B12). The secondary antibodies were AlexaFluor 680–conjugated anti-rabbit secondary antibody (Invitrogen) or IRDye 800–conjugated anti-mouse secondary antibody (Tebu-bio, Peterborough, UK); and were used at 1:2,000 or 1:5,000 dilution. Membranes were blocked in PBS containing 0.1% (vol/vol) Tween-20 and 5% (wt/vol) Blotto (Santa Cruz Biotechnology) for 4 hours at room temperature, followed by overnight incubation at 4°C with primary antibodies diluted in blocking buffer. For phospho-ERK1/2, ERK1/2, and phospho-ErbB2, immunoprobing was performed as described previously here, except that TBS was used in place of PBS, and incubations with primary antibody were performed overnight in TBS, 5% (wt/vol) BSA. Membranes were washed in PBS or TBS containing 0.1% (vol/vol) Tween-20 before incubation with respective infrared dye–conjugated secondary antibodies diluted in Odyssey blocking buffer (50% [vol/vol] PBS, 50% [vol/vol] Odyssey buffer [LI-COR Biosciences, Cambridge, UK]) supplemented with 0.1% (vol/vol) Tween-20 and 0.01% (wt/vol) SDS at room temperature for 1 hour in the dark. After incubation with the secondary antibodies, membranes were again washed in the dark before being imaged using a LI-COR Odyssey infrared imaging system (LI-COR Biosciences).

Statistical Analysis

Data are shown as the means (±SEM) and represent triplicate wells from at least two independent experiments. Data were analyzed using Student's t tests. Differences were considered significant between control and treatment groups for P values less than 0.05.

NRG1β1 Induces Goblet Cell Formation in HBEC Cultures

In an in vitro screen for regulators of goblet cell formation using a human airway epithelial cell system, a panel of 22 cytokines (IL-1α, -2, -3, -4, -7, -9, -10, -11, -15, -17C, -18, -20, -22, -24, -26, and -27, TNF-α and -β, G-CSF, GM-CSF, cardiotrophin-1, and LIF) and 18 growth factors (BMP-2, -4, and -7, FGF-1, -9, and -19, IGF-I and -II, M-CSF, NRG1α and -1β1, PDFG-AA, PDGF-AB and -BB, and TGFα, -β1, -β2, and -β3) were analyzed for their effects on MUC5AC protein expression. HBECs were treated for 7 days at ALI in the presence of the mediator at two concentrations, and effects on MUC5AC protein analyzed by immunohistochemistry using a monoclonal antibody against MUC5AC. Cells were also counterstained with alcian blue to look for effects of the mediator on additional mucins. IL-13 (Figure 1A) and IL-4 (data not shown) were found to stimulate goblet cell formation in HBECs, confirming previous observations (16). In addition to IL-13 and -4, only NRG1α and -1β1 had any effects on either alcian blue or MUC5AC staining. The growth factor, NRG1β1, caused a dose-dependent and significant increase in MUC5AC-positive goblet cells (Figure 1A), giving a threefold increase in MUC5AC protein when tested at 50 nM compared with the vehicle control. NRG1β1 also caused a dose-dependent increase in MUC5AC message (Figure 1C). In contrast, the closely related isoform, NRG1α, had no significant effect on MUC5AC protein and message (Figures 1B and 1D). The MUC5AC antibody–stained sections of NRG1β1-treated HBECs were counterstained with alcian blue and a subset of goblet-like cells, which did not stain with the MUC5AC antibody, were noted in the cultures, suggesting that these cells expressed other mucins (Figure 1F). NRG1β1 also caused an increased thickening of the epithelial cultures compared with untreated cells (Figure 1F). MUC5AC protein induced by NRG1β1 treatment of HBEC cultures was analyzed by immunohistochemistry over a time course at ALI. After treatment of HBECs for 7 days at ALI, expression of MUC5AC increased significantly over the untreated controls, and continued to increase up to 14 days at ALI (Figure 1E).

MUC5B Expression Stimulated by NRG1β1

To further characterize the effects of NRG1β1 on mucin gene expression, effects on MUC5B were examined. In contrast to IL-13, which had no effect on MUC5B gene expression, NRG1β1 caused a dose-dependent increase in MUC5B gene and protein expression (Figures 2A, 2C, and 2E). Although NRG1α had no effect on MUC5AC protein in HBEC cultures, it caused a significant increase in MUC5B gene and protein expression (Figures 2B and 2D). However, the effects of NRG1α on MUC5B gene and protein expression were less than those of NRG1β1. At 5 nM, NRG1α and -1β1 caused 3.3- and 7.5-fold increases in MUC5B protein, respectively. MUC5B protein induced by NRG1β1 treatment of HBEC cultures was analyzed by immunohistochemistry over a time course at ALI. After treatment of HBECs for 7 days at ALI, expression of MUC5B increased significantly over the untreated controls, and continued to increase up to 14 days at ALI (Figure 2F). As NRG1β1 had the most pronounced effects on MUC5AC and MUC5B, subsequent studies focused on the NRG1β1 isoform.

ErbB Receptors Involved in NRG1β1-Induced Goblet Cell Formation

The ErbB receptors expressed in HBECs were analyzed by RT-PCR and Western blot. The ErbB receptor family members, EGFR and ErbB2–4, mRNAs were found to be expressed in HBECs from three independent donors (Figure 3A). Similarly, protein for all four ErbB receptors was found expressed in HBECs from two independent donors (Figure 3B).

To establish if the ErbB receptor(s) are involved in NRG1β1-induced goblet cell formation, anti-ErbB receptor antibodies were evaluated for their inhibitory effects on NRG1β1-induced MUC5AC and MUC5B protein expression. Previous studies in other cell systems have indicated that NRG1β1 signals through ErbB2/ErbB3 or ErbB2/ErbB4 heterodimers (32), so the studies focused on the ErbB2–4 receptors. An antibody against the ErbB2 receptor tested at 5 μg/ml significantly inhibited NRG1β1-induced MUC5AC protein to almost vehicle-treated levels (Figure 4A). The anti-ErbB2 receptor antibody also significantly reduced NRG1β1-induced MUC5B protein by 50% compared with vehicle-treated controls (Figure 4B). Similarly, an anti-ErbB3 receptor–neutralizing antibody tested at 5 μg/ml caused a significant reduction in NRG1β1-induced MUC5AC protein to vehicle levels (Figure 4C), and it significantly reduced NRG1β1-induced MUC5B protein by 50% (Figure 4D). In contrast, an anti-ErbB4 receptor–neutralizing antibody had no effect on either MUC5AC or MUC5B protein (Figures 4E and 4F). Representative histology images of antibody-treated cells are shown in Figure E1 in the online supplement. The EGFR receptor inhibitor, AG1478, had no effect on NRG1β1-induced MUC5AC or MUC5B expression (data not shown), whereas an EGFR/ErbB2 receptor inhibitor, AEE788 (28), at a concentration of 10 μM, significantly inhibited NRG1β1-induced MUC5AC and MUC5B protein (Figures 4G and 4H), consistent with the effects of the anti-ErbB2 receptor antibody. In addition, NRG1β1 treatment of HBECs resulted in phosphorylation of the ErbB2 receptor (Figure 3C), and this phosphorylation was inhibited by pretreatment of the cells with the EGFR/ErbB2 receptor inhibitor, AEE788 (Figure 3D).

Role of p38MAPK and ERK1/2 in NRG1β1-Induced Goblet Cell Formation

The signaling pathways leading to NRG1β1-induced goblet cell formation in vitro were investigated using inhibitors of p38MAPK and ERK1/2. The role of p38MAPK in NRG1β1-induced goblet cell formation was examined using the p38MAPK inhibitors, SB203580 and SB202190. Both inhibitors caused a significant reduction in NRG1β1-induced MUC5AC protein at concentrations between 0.1 and 10 μM, as determined by immunohistochemistry (Figures 5A and 5C). Similarly, SB203580 and SB202190, at concentrations between 0.1 and 10 μM, caused a dose-dependent, significant reduction in NRG1β1-induced MUC5B protein in HBEC cultures (Figures 5B and 5D). Representative images of HBECs treated with inhibitors are shown in Figure E2. Interestingly, both p38MAPK inhibitors also inhibited constitutive MUC5B protein expression in the HBEC cultures. The ERK1/2 inhibitor, SL327, was used to examine the role of the ERK1/2 kinases in NRG1β1-induced goblet cell formation. Treatment of differentiated HBECs with SL327 (0.1–10 μM) caused a significant decrease in MUC5AC protein in the cultures (Figure 5E). Likewise, SL327 inhibited NRG1β1-induced MUCB protein expression, but the effect was only significant at the highest concentration of inhibitor tested (10 μM) (Figure 5F). Changes in phosphorylation of ERK1/2 in response to NRG1β1 treatment of HBECs were analyzed by Western blot. Treatment of HBECs with NRG1β1 over 5–15 minutes resulted in phosphorylation of ERK1/2 (Figure 5G), and this was inhibited by pretreatment of the cells with the EGFR/ErbB2 receptor inhibitor, AEE788 (Figure 5H).

Role of PI3K in NRG1β1-Induced Goblet Cell Formation

To investigate the role of PI3K in NRG1β1-induced goblet cell formation in HBECs, the PI3K inhibitor, LY294002, was tested. At a concentration of LY294002 of 10 μM, a significant reduction in NRG1β1-induced MUC5AC and MUC5B protein was observed (Figures 6A and 6B). The effects of NRG1β1 on AKT phosphorylation in HBECs were examined using an anti-AKT (Ser 473) antibody by Western blot. Phosphorylation of AKT was detected after 30 minutes, and could still be detected after 2 hours of stimulation (Figure 6C). The phosphorylation of AKT was reduced by the PI3K inhibitor, LY294002, at 10 μM (Figure 6D).

NRG1β1, MUC5AC, and MUC5B are Increased in a Mouse Model of Goblet Cell Formation

We analyzed the expression of MUC5AC and MUC5B in a mouse OVA challenge model, which is associated with increases in mucin-containing goblet cells in the airways (31). In mouse lungs, a significant increase in both MUC5AC and MUC5B mRNA was detected (Figures 7A and 7B). A 57-fold increase in MUC5AC gene expression was observed after OVA challenge compared with saline controls, and MUC5B gene expression increased threefold after OVA challenge. NRG1β1 protein was measured in BAL fluid from OVA-treated mice, and was found to be significantly increased compared with saline-treated controls (Figure 7C). A ninefold increase in NRG1β1 was detected in BAL fluid from OVA-challenged mice compared with saline-challenged control mice.

Mucins MUC5AC and MUC5B are major components of airway mucus secretions, and contribute to the viscoelastic properties of the mucus. Although MUC5AC is the predominant mucin in asthma, MUC5B is reported to be the major mucin in subjects with COPD (12, 20, 21). We have shown, for the first time, that NRG1β1 induces MUC5AC and MUC5B protein expression in HBEC cultures, both markers of airway goblet cells. In contrast to the results with the well-characterized mediator of goblet cell formation, IL-13, which only induced expression of MUC5AC in HBECs, NRG1β1 was able to induce expression of both MUC5AC and MUC5B.

Interestingly, an increase in NRG1 protein has been reported in bronchial biopsies of subjects with COPD, although no association with mucus was examined (33). To address the role of NRG1β1 in goblet cell formation in vivo, we analyzed its expression in a mouse OVA model of goblet cell formation (31). In this model, goblet cells appear rapidly after single or multiple OVA challenge (31, 34). We analyzed the changes in mucin gene expression in mouse lungs after OVA challenges, and found a statistically significant increase in both mucins, consistent with previously published data (3537). Interestingly, NRG1β1 protein was also found elevated in the BAL fluid of OVA-challenged mice. Currently, there are no good antibodies available to neutralize the activity of mouse NRG1β1 to establish a direct link between increased NRG1β1 in mouse airways and increased numbers of goblet cells. NRG1β1 has been shown to be secreted from lung fibroblasts (38), suggesting that these cells may provide a source of NRG1β1, which could stimulate goblet cell formation in the lung.

NRG1β1 is a member of the NRG growth factor family, which comprises four members: NRG1–4 (39). At least 15 different isoforms of NRG1 exist as a result of alternative splicing (32). Two of these isoforms, NRG1α and -1β1, differ in the C-terminal portion of the EGF-like domain (40). In this study, we found that NRG1β1, but not NRG1α, induced the expression of MUC5AC in HBECs grown at ALI, whereas both isoforms stimulated expression of MUC5B, with NRG1β1 being the most potent. This result is perhaps not surprising, as NRG1β1 has been reported to bind to its receptor, the ErbB3 receptor, with 100-fold higher affinity than NRG1α (41). During the course of the current study, NRG1α was reported to stimulate differentiation of human airway epithelia, and cause an increase in goblet cell number in human airway epithelial cell cultures, as assessed by Periodic Acid Schiff (PAS) staining for mucins (42). However, the effects of NRG1α on the mucins, MUC5AC and MUC5B, were not reported, and the effects of NRG1β1 were not studied.

NRGs are signaling proteins that mediate multiple cell–cell interactions via the receptor tyrosine kinases of the ErbB family. Analysis of expression of the ErbB family of receptors in HBECs indicated the presence of mRNA and protein for all four family members, EGFR and and ErbB2–4. This is consistent with immunohistochemical data indicating expression of EGFR, ErbB2, and ErbB3 in the human bronchial epithelium (4345). Other studies have detected no or weak expression of the ErbB4 receptor in the human bronchial epithelium by immunohistochemistry, which is in contrast to our data. We have detected expression of ErbB4 receptor mRNA and protein in cultured HBECs. It is not clear why our results differ from previously published data, but could reflect differences in HBEC donors analyzed and techniques used to analyze expression.

NRG1 binds to ErbB3 or ErbB4 receptors of the tyrosine kinase family, which form heterodimers with the ErbB2 receptor (32). The ErbB3 receptor lacks tyrosine kinase activity, but dimerization with ErbB2 results in the formation of an active heterodimer, which can mediate downstream signals (46). Through the use of antibodies against the ErbB2, ErbB3, and ErbB4 family members, we have shown that NRG1β1-induced MUC5AC and MUC5B expression is inhibited by antibodies against ErbB2 and ErbB3 receptors. In addition, an EGFR/ErbB2 receptor inhibitor also significantly inhibited NRG1β1-induced MUC5AC and MUC5B expression, supporting a role for the ErbB2 receptor in NRG1β1-induced goblet cell formation. In contrast, an antibody against the ErbB4 receptor failed to block NRG1β1-induced mucin production. The anti-ErbB4 receptor antibody used in this study has previously been used to study the role of the ErbB4 receptor growth stimulation of ovarian cancer cells (47, 48). Additionally, The ErbB2 receptor was shown to be phosphorylated in response to NRG1β1 treatment of HBECs, and this could be inhibited with an EGFR/ErbB2 receptor inhibitor. Thus, our studies suggest that an ErbB2/ErbB3 receptor heterodimer, rather than an ErbB2/ErbB4 heterodimer, is involved in NRG1β1-induced MUC5AC and MUC5B expression.

The signaling pathways involved in NRG1β1-mediated goblet cell formation were studied using p38MAPK, ERK1/2, and PI3K inhibitors. Both inhibitors of p38MAPK and ERK1/2 kinases blocked NRG1β1-induced MUC5AC and MUC5B protein expression, implicating these kinases in NRG1β1 signaling in HBECs. NRG1β1 was also shown to induce phosphorylation of AKT in HBECs, and this could be inhibited with the PI3K inhibitor, LY294002. Similarly, LY294002 inhibited NRG1β1-induced MUCAC and MUC5B expression, implicating the involvement of the PI3K/AKT pathway in NRG1β1-mediated airway goblet cell formation. The PI3K pathway has also been linked with NRG1-induced branching morphogenesis in the developing lung (49), and, in breast cancer cells, NRG1β1-induced activation of the PI3K and p38MAPK pathways has been demonstrated (5052).

NRG1 isoforms have been reported to have other effects on the lung epithelium. The NRG1β1 isoform has been shown to induce proliferation of lung epithelial cell lines, NCI-H520 and NCI-H441 (53). Additionally, NRG1 isoforms have been linked with lung development and lung repair processes. A role for NRG1 and the ErbB2 and ErbB3 receptors in human lung development has been suggested through immunohistochemical and functional studies on fetal lung tissue (54). NRG1β1 was shown to be secreted from fetal lung fibroblasts and to stimulate type II cell surfactant synthesis, and is therefore proposed to control fetal lung maturation through mesenchymal–epithelial interactions (38). More recently, the NRG1α isoform has been suggested to play a role in epithelial wound repair and remodeling in the airways (55). In the latter study, the ErbB2 receptor was shown to be expressed on the basolateral surface of differentiated epithelial cells, and NRG1α ligand expressed at the apical surface so ligand receptor interactions do not take place until an epithelial injury has occurred.

In summary, we have demonstrated that NRG1β1 regulates the expression of the airway goblet cell mucins, MUC5AC and MUC5B. This was shown to occur through ErbB2 and ErbB3 receptors in HBECs, and also involved activation of p38MAPK, ERK1/2, and PI3K pathways. We have also demonstrated that NRG1β1 protein is increased in the airways in a mouse model of airway goblet cell formation. However, a direct link between NRG1β1, goblet cell formation and mucus production in vivo remains to be established. Mucus overexpression in respiratory diseases contributes to the underlying pathology, and, in particular, the mucins, MUC5AC and MUC5B, are major components of airway mucus in COPD and asthma. Therefore, an increase in our understanding of the mediators and mechanisms regulating goblet cell formation will help to identify new therapeutic targets for respiratory diseases. Consequently, NRG1β1 may represent a potential therapeutic target for respiratory diseases, such as asthma and COPD, where mucus hypersecretion plays a role.

The authors are grateful to Phil Kemp, Nicolas Piot, and Debbie Bayley for their technical assistance.

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Correspondence and requests for reprints should be addressed to Carol E. Jones, Ph.D., Novartis Institutes for Biomedical Research, Respiratory Disease Area, Wimblehurst Road, Horsham, West Sussex RH12 5AB, UK. E-mail:

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