Cigarette smoke induces growth differentiation factor 15 production in human lung epithelial cells: Implication in mucin over-expression

Abstract

Excessive mucus is a hallmark of chronic obstructive pulmonary disease (COPD). There is an emerging interest in the role of TGF-β signaling in the initiation and progression of COPD. Growth differentiation factor 15 (GDF15) is a divergent member of TGF-β superfamily. However, whether cigarette smoke induces airway epithelial GDF15 production and its functions in the airways have not been revealed. Therefore, we first analyzed GDF15 protein expression in airway epithelium of human COPD smokers versus normal non-smokers. We then examined the regulation and function of GDF15 in human airway epithelial cells in response to cigarette smoke exposure. We found increased GDF15 protein expression in airway epithelium (mainly in ciliated cells) of human COPD smokers compared with normal non-smokers. Furthermore, cigarette smoke exposure consistently up-regulated GDF15 expression in human airway epithelial cells. Moreover, GDF15 was shown to play a critical role in cigarette smoke-induced airway epithelial MUC5AC expression. Lastly, activation of phosphoinositide 3-kinase (PI3K) pathway was largely responsible for GDF15-induced airway epithelial MUC5AC expression. Our findings indicate that human airway epithelial cells can produce GDF15 during cigarette smoke exposure, which subsequently activates PI3K pathway to promote mucin (e.g. MUC5AC) expression. This highlights a novel role of GDF15 in regulating airway mucosal immunity (e.g. mucin) in cigarette smoke-exposed lungs.

Keywords

Airway epithelial cells cigarette smoke GDF15 MUC5AC

Introduction

Airway mucus over-production in chronic obstructive pulmonary disease (COPD) contributes to airway obstruction, accelerated decline of lung function, morbidity and mortality.¹^–⁵ Cigarette smoke is a major risk factor in the pathogenesis of COPD.⁶^–⁹ Mucins are glycoproteins and serve as the major component of mucus. Among 21 identified mucins, MUC5AC appears to be the predominant secreted gel-forming mucin in healthy human airway epithelial cells, and its expression is augmented in smokers and COPD patients.¹⁰^–¹² In addition, cigarette smoke and its components have been shown to induce mucin (e.g., MUC5AC) expression in cultured human airway epithelial cells.¹³^–¹⁶ However, the detailed mechanisms underlying cigarette smoke-induced mucin over-expression remain unclear.

Currently, there is an emerging interest in the role of TGF-β signaling in the initiation and progression of COPD.¹⁷ For example, increased TGF-β1 occurs in airway epithelium and submucosal cells of COPD patients and in cultured cigarette smoke-exposed human primary bronchial epithelial cells, suggesting a role for TGF-β1 in COPD pathogenesis by promoting airway mucus over-production.^18,19 As the TGF-β superfamily consists of more than 40 members,^20,21 it is unlikely that TGF-β1 is fully responsible for mucin over-production in airway epithelium of cigarette smoke-exposed lungs. Nevertheless, whether other TGF-β superfamily members also contribute to mucin over-production in COPD lungs remains largely unknown.

Growth differentiation factor 15 (GDF15), also known as macrophage inhibitory cytokine 1, is a divergent member of the TGF-β superfamily. GDF15 was first cloned from human U937 monocytoid cell line and originally discovered as a factor in immune regulation.²² In a normal condition, GDF15 mRNA is most abundant in liver, placenta and prostate, but weakly expressed in other tissues. However, GDF15 expression is dramatically induced in many tissues or cell types under pathogenic conditions (e.g. acute injury and inflammation). Increased GDF15 may regulate inflammation, apoptosis and cell growth during various diseases, including liver injury,²³ cancers,^24,25 cardiovascular diseases,²⁶^–²⁹ thalassemia,³⁰ and viral infections.^31,32 Additionally, GDF15 has been reported to enhance hypertrophic growth of human ventricular cardiomyocytes via activation of phosphoinositide 3-kinase (PI3K) pathway.³³ GDF15 over-expression in hepatoma cells leads to PI3K/Akt activation in vitro.³²

To date, studies of GDF15 expression and function in the lung are very limited. In situ hybridization in rats has revealed GDF15 mRNA expression in bronchial epithelial cells.³⁴ A recent study has shown extremely lower levels of GDF15 protein in human normal bronchial and alveolar epithelial cells, but much more in vascular endothelial cells of pulmonary arterial hypertension lungs.²⁹ Nevertheless, in our preliminary experiment, we observed that well-differentiated human brushed bronchial epithelial cells highly expressed GDF15 at both mRNA and protein levels, especially upon cigarette smoke exposure. However, whether cigarette smoke induces airway epithelial GDF15 production and its functions in the airways have not been revealed yet. Furthermore, whether GDF15 activates PI3K pathway in human airway epithelial cells remains unknown.

In this study, we hypothesized that cigarette smoke can up-regulate GDF15 expression in human airway epithelial cells, which in turn promotes MUC5AC expression via activation of PI3K pathway. First, we analyzed GDF15 protein expression in airway epithelium of human COPD smokers versus normal non-smoker controls. Second, we examined the effects of cigarette smoke on GDF15 and MUC5AC expression in air-liquid interface (ALI) cultures of primary human bronchial epithelial cells. Third, we defined a critical role of epithelial GDF15 in cigarette smoke-induced MUC5AC expression by using the lentivirus-mediated gene knockdown approach. Lastly, we showed the role of PI3K pathway in GDF15-induced human airway epithelial MUC5AC expression.

Materials and methods

Human lung tissues

Lung tissues of normal human non-smoker controls (n = 4) were obtained from de-identified organ donors whose lungs were not suitable for transplantation. Lung tissue sections of human mild to severe COPD smokers (n = 6) were requested from the Lung Tissue Research Consortium (LTRC) of the National Heart, Lung, and Blood Institute (NHLBI).

Air-liquid interface (ALI) cultures of normal human tracheobronchial epithelial cells

Normal human tracheobronchial epithelial cells were obtained from the tracheas and bronchi of de-identified organ donors whose lungs were not suitable for transplantation. Tracheas and bronchi were cut into small pieces and digested with ice-cold DMEM supplemented with 0.2% protease solution and 1 × pen/strep/amphotericin overnight (16 h) at 4°C. The released cells were cultured in collagen-coated 60 mm tissue culture dishes containing bronchial epithelial cell growth medium (BEGM) with supplements (Lonza, Walkersville, MD, USA) at 37°C, 5% CO₂. At 80% confluence, cells were transferred onto collagen-coated 12-well transwell plates at 4 × 10⁴ cells/well (Corning Inc., Corning, NY, USA) in DMEM/BEBM (1:1) with supplements. Culture media were refreshed every other day for 7 d in an immersed culture condition and shifted to ALI culture by reducing the apical medium to 50 µl. On ALI day 10, well-differentiated cells were exposed to air (control) or cigarette smoke for 10 min from research cigarette 2R4F (University of Kentucky, Lexington, KY, USA) using a mainstream smoking chamber specifically designed for cell culture experiments (British American Tobacco Science, Southampton, UK) as previously described.³⁵ After 24 h, MUC5AC and GDF15 mRNA expression, GDF15 protein levels in apical and basolateral supernatants and MUC5AC protein levels in apical supernatants were determined. In experiments designed to characterize co-localization of GDF15 and MUC5AC protein in large airway epithelial cells, well-differentiated cells were exposed to air or cigarette smoke for 10 min on ALI day 10. Twenty-four hours later, cells on transwell membranes were fixed in 10% formalin, embedded in paraffin and processed for immunofluorescent staining.

Submerged cultures of normal human tracheobronchial epithelial cells

To determine if GDF15 activates PI3K pathway in human airway epithelial cells, normal human tracheobronchial epithelial cells at passage 2 were seeded into 12-well cell culture plates at 2 × 10⁵ cells/well in BEGM with supplements at 37°C, 5% CO₂. At 100% confluence, cells were treated with BSA or recombinant GDF15 (rhGDF15, 10 ng/ml, R&D Systems, Minneapolis, MN, USA) for 15, 30, 60 and 120 min. At the end of culture, cells were lysed in RIPA buffer (Pierce, Rockford, IL, USA) supplemented with 1% halt protease and phosphatase inhibitor cocktail (Pierce) for pAkt and total Akt Western blot analysis.

Immunofluorescent staining

Formalin-fixed and paraffin-embedded sections of lung tissues or ALI cultured epithelial cells were deparaffinized and rehydrated, followed by antigen retrieval with microwave boiling in 10 mM citrate buffer (pH 6.0) for 12 min. They were then blocked with 10% normal goat or horse serum (Vector Laboratories, Burlingame, CA, USA), probed with rabbit anti-human GDF15 (1:50; Sigma-Aldrich, St Louis, MO, USA), mouse anti-human acetylated tubulin (1:100; Sigma-Aldrich), mouse anti-human MUC5AC (45M1) (1:50; NeoMarkers, Fremont, CA, USA) or control IgGs (1:50; Vector Laboratories) overnight at 4°C. The slides were incubated with appropriate FITC labeled goat anti-rabbit IgG or Texas red labeled horse anti-mouse IgG (Vector Laboratories) for 2 h at room temperature (20–25°C), and then with DAPI to counterstain the nuclei. After being mounted with DAKO® fluorescent mount medium (Dako, Glostrup, Denmark), the slides were examined under a fluorescent microscope.

GDF15 gene knockdown in human lung epithelial cell line

Human lung mucoepidermoid carcinoma derived NCI-H292 cells (clone CRL-1848) were from the American Type Culture Collection (Manassas, VA, USA). A vesicular stomatitis virus envelope glycoprotein (VSV-G) pseudotyping approach was utilized to transduce human GDF15 short hairpin RNA (shRNA) encoded in a lentiviral vector (pLL3.7) to NCI-H292 cells. Antiparallel pairs of oligonucleotides that encode shRNA of GDF15 were ordered from the IDT laboratories (San Diego, CA, USA) and the sequences are: Sense: 5′-TGCATCCCTGTGAACAGTAATTCAA-GAGATTACTGTTCACAGGGATGCTTTTTTC-3′; Anti-sense: 5′-TCGAG-AAAAAAGCATCCCTGT-GAACAGTAATCTCTTGAATTACTGTTCACAGGGATGCA-3′. Human GDF15 shRNA encoded in pLL3.7 was generated as previously described.^36,37 Briefly, NCI-H292 cells at passage 8 were cultured in 6-well culture plates (1 × 10⁵ cells/well) in RPMI1640 containing 10% FBS without antibiotics until about 60% confluence when they were transduced with either pLL3.7-sh firefly luciferase (shLUC, an irrelevant gene control) or pLL3.7-GDF15 (shGDF15) once daily for three consecutive days. Forty-eight hours after the last transduction, cells were transferred onto collagen-coated 12-well transwell plates at 4 × 10⁴ cells/well in RPMI1640 containing 10% FBS with antibiotics. Culture media were refreshed every other day until cells were 100% confluence and the apical medium was reduced to 50 µl for additional 5 d. Cells were then exposed to air or cigarette smoke for 10 min as described above. After 24 h, MUC5AC and GDF15 mRNA expression, GDF15 protein levels in basolateral supernatants and MUC5AC protein levels in apical supernatants were determined.

NCI-H292 cells in submerged cultures

NCI-H292 cells were regularly maintained in RPMI1640 containing 10% FBS with antibiotics. To reveal if GDF15 directly induces MUC5AC expression in human lung epithelial cells, NCI-H292 cells at passage 8–15 were seeded at the concentration of 1 × 10⁵ per well in 12-well cell culture plates in RPMI1640 containing 0.1% FBS. After overnight growth, cells were treated with BSA or rhGDF15 (1, 3, and 10 ng/ml) in serum-free medium for 24 h. GDF15 concentration was chosen from our calculation of GDF15 protein levels in epithelial lining fluid (ELF) of cigarette smoke-exposed human airways in our preliminary experiment. Specifically, we measured GDF15 levels in bronchoalveolar lavage fluid of normal smoker controls and current COPD smokers, and converted them to 1.2 to 7.4 ng/ml in ELF based on the 100- to 200-fold dilution factor of ELF with a standard lavage procedure.^38,39 At the end of culture, cells were lysed in TRIzol for RNA extraction to determine MUC5AC mRNA expression.

To determine if GDF15 activates PI3K pathway in human lung epithelial cells, NCI-H292 cells at passage 8–15 were seeded at the concentration of 5 × 10⁵ per well in 6-well cell culture plates in RPMI1640 containing 0.1% FBS. After overnight growth, cells were treated with BSA or rhGDF15 (10 ng/ml) in serum-free medium for 15, 30, 60 and 120 min. At the end of culture, cells were lysed in RIPA buffer (Pierce) supplemented with 1% halt protease and phosphatase inhibitor cocktail (Pierce) for pAkt and total Akt Western blot analysis.

To demonstrate if PI3K pathway is involved in GDF15-induced MUC5AC expression in human lung epithelial cells, NCI-H292 cells at passage 8–15 were seeded at the concentration of 1 × 10⁵ per well in 12-well cell culture plates in RPMI1640 containing 0.1% FBS. After overnight growth, cells were treated with a selective PI3K inhibitor wortmannin (1 µM, Cell Signaling, Danvers, MA, USA) for 1 h prior to the BSA or rhGDF15 (10 ng/ml) treatment in serum-free medium. After an additional 24 h, cells were lysed in TRIzol for RNA extraction to determine MUC5AC mRNA expression.

Western blot analysis

Equal amount of protein samples were separated on 10% Precise™ Protein Gels (Pierce), transferred onto nitrocellulose membranes, probed with pAkt rabbit antibody or Akt rabbit antibody (Cell Signaling), followed with HRP-linked secondary Abs and Pierce® ECL western blotting substrate.

ELISA

GDF15 protein levels in cell culture supernatants were determined by using human GDF15 DuoSet ELISA Development kit (R&D Systems) as per the manufacturer’s instruction. The human MUC5AC ELISA kit was purchased from the ELISA Tech (Aurora, CO, USA). The 1:100 diluted apical supernatant from ALI cultures of normal human brushed bronchial epithelial cells after IL-13 stimulation was used as a positive control reference, and its MUC5AC level was defined as 100 arbitrary units (AUs). The relative MUC5AC protein levels in our cell culture samples were calculated by normalizing their OD values to the positive control reference, and expressed as AUs.

Quantitative real-time RT-PCR

TaqMan® Gene Expression Assay for human GDF15 (Hs03986124_s1) was obtained from Applied Biosystems (Foster City, CA, USA). The following primers and probe for human MUC5AC cDNA (Genbank accession number, AF015521) were used. Forward, 5′-CGTGTTGTCACCGAGAACGT-3′; Reverse, 5′-ATCTTGATGGCCTTGGAGCA-3′; Probe, 5′-CTGCGGCACCACAGGGACCA-3′. Quantitative real-time RT-PCR was performed on the CFX96™ Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). Housekeeping gene GAPDH was evaluated as an internal positive control. The comparative cycle of threshold (ΔΔCt) method was used to demonstrate the relative mRNA levels of target genes.

Statistical analysis

Data are presented as mean ± SEM. One-way analysis of variance (ANOVA) was used for multiple comparisons, and a Turkey’s post hoc test was applied where appropriate. Student’s t test was used when only two groups were compared. A P value of <0.05 was considered significant.

Results

Airway epithelial cells of COPD smokers express increased GDF15 protein

To examine GDF15 protein expression in human airway epithelium, lung tissue sections from normal human non-smoker controls and COPD smokers were subjected to GDF15 and acetylated tubulin immunofluorescent staining. Because only small airways were available in surgically resected distal lungs from COPD smokers, we compared GDF15 expression in small airways between normal non-smokers and COPD smokers. No staining was seen in tissues incubated with the control IgG (Figure 1A). In normal human non-smoker lungs (Figure 1C), small airway epithelial cells exhibited very weak staining. Smooth muscle cells were also weakly positive as previously reported.²⁹ However, increased GDF15 protein staining was seen mainly in ciliated cells of small airway epithelium of COPD smoker lung tissue, as demonstrated by dual immunofluorescent staining (Figure 1E).

Figure 1.

Airway epithelial cells of COPD smokers express increased GDF15 protein. Representative immunofluorescent (IF) images of GDF15 (green in E) and acetylated tubulin (red in C) and corresponding bright field images in lung sections of normal human non-smoker controls (A–D) and COPD smokers (E and F). Original magnification, 400×.

Cigarette smoke exposure induces GDF15 and MUC5AC expression in cultured human primary airway epithelial cells

In the current study, well-differentiated normal human tracheobronchial epithelial cells were exposed to whole cigarette smoke, which enables us to determine the effects of all cigarette smoke components (e.g. particulates, gas and vapor) on epithelial functions. As shown in Figure 2A, cigarette smoke exposure significantly increased GDF15 expression at both mRNA (upper panel) and protein (middle panel and lower panel) levels.

Figure 2.

Cigarette smoke exposure induces GDF15 and MUC5AC expression in cultured human primary airway epithelial cells. ALI cultures of normal human tracheobronchial epithelial cells were exposed to air or whole cigarette smoke for 10 min. After 24 h, samples were collected to examine GDF15 and MUC5AC expression. (A) GDF15 expression at mRNA (upper panel) and protein levels in apical and basolateral supernatants (middle panel and lower panel); (B) MUC5AC expression at mRNA (upper panel) and protein levels in apical supernatants (lower panel). Data are presented as means ± SEM (n = 5). ASN, apical supernatants; AUs, arbitrary units; BSN, basolateral supernatants; CS, cigarette smoke.

Previous studies have reported that upon exposure to cigarette smoke extract (CSE), MUC5AC mRNA and protein expression was increased in cultured human airway epithelial cells.^12,16,40 In the current study, we tested if whole cigarette smoke exposure was able to induce lung epithelial MUC5AC expression in vitro. As shown in Figure 2B, cigarette smoke exposure significantly increased MUC5AC expression at both mRNA (upper panel) and protein (lower panel) levels in well-differentiated human primary airway epithelial cells.

To characterize the cellular source of GDF15 and its co-localization with MUC5AC protein in large airway epithelial cells, ALI cultured normal human tracheobronchial epithelial cells with air or cigarette smoke exposure were processed for immunofluorescent staining. As shown in Figure 3, we found that ciliated cells produced GDF15 upon cigarette smoke exposure, which supported our data in COPD lung tissues. GDF15 did not co-localize with MUC5AC protein (i.e. goblet cells).

Figure 3.

Ciliated cells are the major source of GDF15 upon cigarette smoke exposure. ALI cultures of normal human tracheobronchial epithelial cells were exposed to air or whole cigarette smoke for 10 min. After 24 h, cells were processed for immunofluorescent staining. Right panel: representative immunofluorescent (IF) images of GDF15 (green, C, E, G and I), acetylated tubulin (red, C and E) or MUC5AC (red, G and I) compared with IgG control (A). Left panel: corresponding bright field images (B, D, F, H and J) are shown to illustrate epithelial structure under the ALI cultures. Original magnification, 400×. CS, cigarette smoke.

GDF15 gene knockdown inhibits cigarette smoke-induced MUC5AC expression in human lung epithelial cells

Human lung epithelial NCI-H292 cells are a well-characterized and widely used cell model system of mucin production.⁴¹ In our preliminary experiment, we validated that NCI-H292 cells responded to cigarette smoke exposure regarding GDF15 expression in a similar manner to well-differentiated normal human primary airway epithelial cells. However, the baseline of GDF15 protein in NCI-H292 cells is much less (only about 10%) than that in well-differentiated normal human primary airway epithelial cells. Thus, in the present study, NCI-H292 cells served as a model system where GDF15 gene can be knocked-down to define its role in cigarette smoke-induced MUC5AC expression. Indeed, compared with the control shLUC vector, shGDF15 lentiviral vector resulted in a significant reduction of GDF15 expression in the absence or presence of cigarette smoke exposure (Figure 4A). Importantly, the reduction of epithelial GDF15 expression accordingly attenuated cigarette smoke-induced MUC5AC expression at both mRNA (upper panel) and protein (lower panel) levels in NCI-H292 cells (Figure 4B).

Figure 4.

GDF15 gene knockdown inhibits cigarette smoke-induced MUC5AC expression in human lung epithelial cells. NCI-H292 cells were transduced with either pLL3.7-sh firefly luciferase (shLUC) or pLL3.7-GDF15 (shGDF15) and then exposed to air or whole cigarette smoke for 10 min. After 24 h, samples were collected to examine GDF15 and MUC5AC expression. (A) GDF15 expression at mRNA (upper panel) and protein levels in basolatera supernatants (lower panel); (B) MUC5AC expression at mRNA (upper panel) and protein levels in apical supernatants (lower panel). Data are presented as means ± SEM (n = 3). AUs, arbitrary units; CS, cigarette smoke.

GDF15-induced airway epithelial MUC5AC expression is partially dependent on PI3K/Akt signaling pathway

The mechanisms underlying GDF15 modulation of airway epithelial mucin production have not been investigated. In our preliminary experiment, compared with human primary airway epithelial cells, NCI-H292 cells, owing to thier low basal GDF15 expression, were more sensitive to rhGDF15 protein treatment (∼10 ng/ml) regarding MUC5AC induction. This feature allows us to study epithelial GDF15 function in cigarette smoke-exposed human airways by using GDF15 under the physiological concentration range (1–10 ng/ml). Thus, in the present study, we utilized NCI-H292 cells as a model system to disclose molecular mechanisms involved in GDF15-mediated MUC5AC induction in human airway epithelial cells.

First, to reveal if GDF15 directly induces epithelial MUC5AC expression, NCI-H292 cells were treated with BSA or rhGDF15 at indicated physiological doses. As shown in Figure 5A, GDF15 protein dose-dependently increased MUC5AC mRNA expression. Second, to demonstrate if GDF15 activates PI3K/Akt signaling pathway in human lung epithelial cells, NCI-H292 cells and normal human tracheobronchial epithelial cells under submerged cultures were treated with BSA or rhGDF15 at different time-points to determine Akt activation. GDF15 protein time-dependently induced phosphorylation of Akt in NCI-H292 cells (Figure 5B, upper panel). GDF15-induced PI3K/Akt activation was similarly observed in normal human tracheobronchial epithelial cells under submerged cultures, but with a much higher GDF15 dose than that in NCI-H292 cells (Figure 5B, lower panel). Third, to further prove the involvement of PI3K/Akt activation in GDF15-indcued MUC5AC expression, NCI-H292 cells were treated with BSA or rhGDF15 in the absence or presence of a selective PI3K inhibitor wortmannin. Pretreatment of wortmannin significantly attenuated GDF15-induced epithelial MUC5AC mRNA expression in NCI-H292 cells (Figure 5C). However, MUC5AC protein was undetectable in cell culture supernatants of the submerged NCI-H292 cell cultures at such a low cell density. Together, our data suggest that GDF15 may activate PI3K/Akt signaling pathway to promote MUC5AC expression in human lung epithelial cells.

Figure 5.

GDF15-induced airway epithelial MUC5AC expression is partially dependent on PI3K signaling pathway. (A) NCI-H292 cells were treated with BSA or recombinant human GDF15 (rhGDF15) at indicated physiological doses. After 24 h, MUC5AC mRNA expression was determined. Data are normalized to the BSA control of each experiment and presented as means ± SEM (n = 5); (B) NCI-H292 cells (upper panel) or normal human tracheobronchial epithelial cells under submerged cultures (lower panel) were treated with BSA or rhGDF15 at the indicated dose at different time-points and Akt activation was determined. Western blot pictures of pAkt and total Akt are representative of three independent experiments; (C) NCI-H292 cells were treated with BSA or rhGDF15 (10 ng/ml) in the absence or presence of a selective PI3K inhibitor wortmannin (Wort, 1 µM). After 24 h, MUC5AC mRNA expression was determined. Data are normalized to the DMSO + BSA control of each experiment and presented as means ± SEM (n = 5).

Discussion

For the first time, we studied the expression and function of lung epithelial-derived GDF15 in response to cigarette smoke exposure. We found increased GDF15 protein expression in airway epithelium of human COPD smokers compared with normal non-smoker controls. Furthermore, cigarette smoke exposure consistently up-regulated GDF15 expression in human airway epithelial cells. Moreover, GDF15 was shown to play a critical role in cigarette smoke-induced MUC5AC expression in human airway epithelial cells. Lastly, activation of PI3K/Akt signaling pathway was largely responsible for GDF15-induced airway epithelial MUC5AC expression.

To date, whether cigarette smoke exposure up-regulates GDF15 expression in the lung, especially in airway epithelium, is undefined. In the current study, we have clearly demonstrated that airway epithelial cells, especially ciliated cells of COPD patients with smoking history expressed higher levels of GDF15 protein than those in normal non-smokers. However, we have not determined if other types of cells (e.g. Clara cells) also increase GDF15 in response to cigarette smoke exposure. Moreover, we have disclosed that cigarette smoke exposure up-regulated GDF15 expression in well-differentiated human primary airway epithelial cells (mainly in ciliated cells), as well as human lung epithelial NCI-H292 cells. These results will significantly advance our knowledge of GDF15 expression and regulation in the lung.

In contrast to the extensive studies of other TGF-β family members (e.g. TGF-β1 and 2) in regulating airway mucosal immunity, such as mucin production, the function of epithelial GDF15 in the airways has not been investigated. In the present study, we have elucidated that GDF15 acted as an important mediator in cigarette smoke-induced MUC5AC expression by human airway epithelial cells. First, knockdown of GDF15 protein by using lentivirus-mediated RNA interference in NCI-H292 cells consistently attenuated MUC5AC expression after cigarette smoke exposure. However, we realize that our lentiviral GDF15 shRNA-mediated gene knockdown efficiency was low (<45%) in NCI-H292 cells. Future studies will utilize GDF15 knockout mouse tracheal epithelial cells to further define the role of GDF15 in airway mucin (e.g. MUC5AC) regulation. Second, recombinant GDF15 protein dose-dependently induced MUC5AC expression in NCI-H292 cells. To our knowledge, this is the first report of airway mucin gene regulation by this novel mechanism.

The TGF-β family proteins bind to distinct type I and type II serine/threonine kinase receptors and exert their biological functions mainly through the small mother against decapentaplegic (Smad) signaling pathway.⁴² However, GDF15-initiated cell signaling pathways are poorly understood. For example, whether GDF15 activates Smad-dependent pathway remains controversial. Xu et al. have shown that an acute treatment with recombinant GDF15 protein in neonatal cardiomyocytes resulted in a prominent induction of Smad2/3 phosphorylation and a mild induction of Smad1/5/8 phosphorylation.⁴³ However, when Si et al. examined the intracellular signaling pathways in human hepatoma cell line stably over-expressing human GDF15, the classical intracellular Smad cascade was not activated by GDF15.³² This suggests that Smad signaling pathways in response to GDF15 stimulation could be cell-type specific. However, several recent studies have consistently shown a significant phosphorylation of Akt owing to GDF15 over-expression or stimulation in a variety of cell types, including human cardiomyocytes and pulmonary microvascular endothelial cells.^29,32,43 Previous publications have demonstrated that cigarette smoke induces MUC5AC expression partly through the PI3K/Akt signaling pathway in human airway epithelial cells.^44,45 However, whether cigarette smoke activates the PI3K/Akt pathway directly or indirectly through cigarette smoke-induced mediators remains elusive. In the present work, we have clearly demonstrated that GDF15 promoted PI3K/Akt phosphorylation in human lung epithelial NCI-H292 cells for the first time. In addition, GDF15-induced PI3K/Akt activation was similarly observed in normal human tracheobronchial epithelial cells under submerged cultures. Furthermore, activation of PI3K/Akt signaling pathway was required for MUC5AC induction by GDF15 in NCI-H292 cells. Together, these results suggest that cigarette smoke may indirectly activate PI3K/Akt pathway through GDF15 to promote mucin (e.g. MUC5AC) expression in human airway epithelial cells. However, whether GDF15 induces Smad cascade activation to promote mucin production in the context of cigarette smoke exposure deserves future studies.

Conclusions

Our findings indicate that human airway epithelial cells are able to produce GDF15 during cigarette smoke exposure, which subsequently activates the PI3K/Akt signaling pathway to promote mucin (e.g. MUC5AC) expression. This highlights a novel role of GDF15 in regulating airway mucosal immunity (e.g. mucin) in cigarette smoke-exposed lungs, which may contribute to COPD pathogenesis. Targeting epithelial GDF15 might be promising for preventing or treating mucin over-production in cigarette smoke-exposed lungs.

Footnotes

Funding

This study was supported by the Flight Attendant Medical and Research Institute (FAMRI) Clinical Innovator Award (grant number 072148) to HWC and National Jewish Health Basic Science Section Micro-grant to QW.

Acknowledgements

The authors thank Jyoti Thaikoottathil and Sean Smith for their technical assistance in cell culture.

Conflict of interest

None of the authors has a ﬁnancial or personal relationship with a commercial entity that has an interest in the subject of this manuscript.

References

Speizer

Fay

Dockery

Ferris

Jr . Chronic obstructive pulmonary disease mortality in six U.S. cities. Am Rev Respir Dis 1989; 140: S49–S55.

Lange

Nyboe

Appleyard

. Relation of ventilatory impairment and of chronic mucus hypersecretion to mortality from obstructive lung disease and from all causes. Thorax 1990; 45: 579–585.

Vestbo

Prescott

Lange

. Association of chronic mucus hypersecretion with FEV1 decline and chronic obstructive pulmonary disease morbidity. Copenhagen City Heart Study Group. Am J Respir Crit Care Med 1996; 153: 1530–1535.

Vestbo

. Epidemiological studies in mucus hypersecretion. Novartis Found Symp 2002; 248: 3–12.

Hogg

Chu

Utokaparch

. The nature of small-airway obstruction in chronic obstructive pulmonary disease. N Engl J Med 2004; 350: 2645–2653.

Carp

Janoff

. Possible mechanisms of emphysema in smokers. In vitro suppression of serum elastase-inhibitory capacity by fresh cigarette smoke and its prevention by antioxidants. Am Rev Respir Dis 1978; 118: 617–621.

Carp

Miller

Hoidal

Janoff

. Potential mechanism of emphysema: alpha 1-proteinase inhibitor recovered from lungs of cigarette smokers contains oxidized methionine and has decreased elastase inhibitory capacity. Proc Natl Acad Sci USA 1982; 79: 2041–2045.

MacNee

Wiggs

Belzberg

Hogg

. The effect of cigarette smoking on neutrophil kinetics in human lungs. N Engl J Med 1989; 321: 924–928.

Rahman

Morrison

Donaldson

MacNee

. Systemic oxidative stress in asthma, COPD, and smokers. Am J Respir Crit Care Med 1996; 154: 1055–1060.

10.

Ordonez

Khashayar

Wong

. Mild and moderate asthma is associated with airway goblet cell hyperplasia and abnormalities in mucin gene expression. Am J Respir Crit Care Med 2001; 163: 517–523.

11.

Wang

Feng

Wen

. Increased expression of human calcium-activated chloride channel 1 is correlated with mucus overproduction in the airways of Chinese patients with chronic obstructive pulmonary disease. Chin Med J (Engl) 2007; 120: 1051–1057.

12.

Cortijo

Mata

Milara

. Aclidinium inhibits cholinergic and tobacco smoke-induced MUC5AC in human airways. Eur Respir J 2011; 37: 244–254.

13.

Takeyama

Jung

Shim

. Activation of epidermal growth factor receptors is responsible for mucin synthesis induced by cigarette smoke. Am J Physiol Lung Cell Mol Physiol 2001; 280: L165–L172.

14.

Shao

Nakanaga

Nadel

. Cigarette smoke induces MUC5AC mucin overproduction via tumor necrosis factor-alpha-converting enzyme in human airway epithelial (NCI-H292) cells. Am J Physiol Lung Cell Mol Physiol 2004; 287: L420–L427.

15.

Phillips

Kluss

Richter

Massey

. Exposure of bronchial epithelial cells to whole cigarette smoke: assessment of cellular responses. Altern Lab Anim 2005; 33: 239–248.

16.

Glader

Moller

Lilja

. Cigarette smoke extract modulates respiratory defence mechanisms through effects on T-cells and airway epithelial cells. Respir Med 2006; 100: 818–827.

17.

Konigshoff

Kneidinger

Eickelberg

. TGF-beta signaling in COPD: deciphering genetic and cellular susceptibilities for future therapeutic regimen. Swiss Med Wkly 2009; 139: 554–563.

18.

Chung

. Inflammatory mediators in chronic obstructive pulmonary disease. Curr Drug Targets Inflamm Allergy 2005; 4: 619–625.

19.

Baraldo

Bazzan

Turato

. Decreased expression of TGF-beta type II receptor in bronchial glands of smokers with COPD. Thorax 2005; 60: 998–1002.

20.

Kim

. Transforming growth factor-beta : biology and clinical relevance. J Biochem Mol Biol 2005; 38: 1–8.

21.

ten Dijke

Hill

. New insights into TGF-beta-Smad signalling. Trends Biochem Sci 2004; 29: 265–273.

22.

Bootcov

Bauskin

Valenzuela

. MIC-1, a novel macrophage inhibitory cytokine, is a divergent member of the TGF-beta superfamily. Proc Natl Acad Sci USA 1997; 94: 11514–11519.

23.

Hsiao

Koniaris

Zimmers-Koniaris

. Characterization of growth-differentiation factor 15, a transforming growth factor beta superfamily member induced following liver injury. Mol Cell Biol 2000; 20: 3742–3751.

24.

Welsh

Sapinoso

. Analysis of gene expression identifies candidate markers and pharmacological targets in prostate cancer. Cancer Res 2001; 61: 5974–5978.

25.

Boyle

Pedley

Martyn

. Macrophage inhibitory cytokine-1 is overexpressed in malignant melanoma and is associated with tumorigenicity. J Invest Dermatol 2009; 129: 383–391.

26.

Brown

Breit

Buring

. Concentration in plasma of macrophage inhibitory cytokine-1 and risk of cardiovascular events in women: a nested case-control study. Lancet 2002; 359: 2159–2163.

27.

Wollert

Kempf

Peter

. Prognostic value of growth-differentiation factor-15 in patients with non-ST-elevation acute coronary syndrome. Circulation 2007; 115: 962–971.

28.

Wollert

Kempf

Lagerqvist

. Growth differentiation factor 15 for risk stratification and selection of an invasive treatment strategy in non ST-elevation acute coronary syndrome. Circulation 2007; 116: 1540–1548.

29.

Nickel

Jonigk

Kempf

. GDF-15 is abundantly expressed in plexiform lesions in patients with pulmonary arte-rial hypertension and affects proliferation and apoptosis of pulmonary endothelial cells. Respir Res 2011; 12: 62–62.

30.

Tanno

Bhanu

Oneal

. High levels of GDF15 in thalassemia suppress expression of the iron regulatory protein hepcidin. Nat Med 2007; 13: 1096–1101.

31.

Wan

Miao

Quraishi

Kennedy

Creek

Pirisi

. Gene expression changes during HPV-mediated carcinogenesis: a comparison between an in vitro cell model and cervical cancer. Int J Cancer 2008; 123: 32–40.

32.

Liu

Cheng

. Growth differentiation factor 15 is induced by hepatitis C virus infection and regulates hepatocellular carcinoma-related genes. PLoS One 2011; 6: e19967–e19967.

33.

Heger

Schiegnitz

von Waldthausen

. Growth differentiation factor 15 acts anti-apoptotic and pro-hypertrophic in adult cardiomyocytes. J Cell Physiol 2010; 224: 120–126.

34.

Bottner

Laaff

Schechinger

. Characterization of the rat, mouse, and human genes of growth/differentiation factor-15/macrophage inhibiting cytokine-1 (GDF-15/MIC-1). Gene 1999; 237: 105–111.

35.

Zhang

Case

Bowler

. Cigarette smoke modulates PGE(2) and host defence against Moraxella catarrhalis infection in human airway epithelial cells. Respirology 2011; 16: 508–516.

36.

Chu

Thaikoottathil

Rino

. Function and regulation of SPLUNC1 protein in Mycoplasma infection and allergic inflammation. J Immunol 2007; 179: 3995–4002.

37.

Refaeli

Field

Turner

. The protooncogene MYC can break B cell tolerance. Proc Natl Acad Sci USA 2005; 102: 4097–4102.

38.

Rennard

Basset

Lecossier

. Estimation of volume of epithelial lining fluid recovered by lavage using urea as marker of dilution. J Appl Physiol 1986; 60: 532–538.

39.

Schmidt

Luboeinski

Markart

. Alveolar antioxidant status in patients with acute respiratory distress syndrome. Eur Respir J 2004; 24: 994–999.

40.

Baginski

Dabbagh

Satjawatcharaphong

Swinney

. Cigarette smoke synergistically enhances respiratory mucin induction by proinflammatory stimuli. Am J Respir Cell Mol Biol 2006; 35: 165–174.

41.

Rose

Voynow

. Respiratory tract mucin genes and mucin glycoproteins in health and disease. Physiol Rev 2006; 86: 245–278.

42.

Ago

Sadoshima

. GDF15, a cardioprotective TGF-beta superfamily protein. Circ Res 2006; 98: 294–297.

43.

Kimball

Lorenz

. GDF15/MIC-1 functions as a protective and antihypertrophic factor released from the myocardium in association with SMAD protein activation. Circ Res 2006; 98: 342–350.

44.

Lee

Kang

Hur

. Peroxisome proliferator-activated receptor-gamma inhibits cigarette smoke solution-induced mucin production in human airway epithelial (NCI-H292) cells. Am J Physiol Lung Cell Mol Physiol 2006; 291: L84–L90.

45.

Kolosov

. Regulation of cigarette smoke-mediated mucin expression by hypoxia-inducible factor-1alpha via epidermal growth factor receptor-mediated signaling pathways. J Appl Toxicol 2011 (epub ahead of print).