Effects of Wood Smoke Constituents on Mucin Gene Expression in Mice and Human Airway Epithelial Cells and on Nasal Epithelia of Subjects with a Susceptibility Gene Variant in Tp53

Abstract

Background: Exposure to wood smoke (WS) increases the risk for chronic bronchitis more than exposure to cigarette smoke (CS), but the underlying mechanisms are unclear.

Objective: The effect of WS and CS on mucous cell hyperplasia in mice and in human primary airway epithelial cells (AECs) was compared with replicate the findings in human cohorts. Responsible WS constituents were identified to better delineate the pathway involved, and the role of a tumor protein p53 (Tp53) gene polymorphism was investigated.

Methods: Mice and primary human AECs were exposed to WS or CS and the signaling receptor and pathway were identified using short hairpin structures, small molecule inhibitors, and Western analyses. Mass spectrometric analysis was used to identify active WS constituents. The role of a gene variant in Tp53 that modifies proline to arginine was examined using nasal brushings from study participants in the Lovelace Smokers Cohort, primary human AECs, and mice with a modified Tp53 gene.

Results: WS at 25-fold lower concentration than CS increased mucin expression more efficiently in mice and in human AECs in a p53 pathway-dependent manner. Study participants who were homozygous for p53 arginine compared with the proline variant showed higher mucin 5AC (MUC5AC) mRNA levels in nasal brushings if they reported WS exposure. The WS constituent, oxalate, increased MUC5AC levels similar to the whole WS extract, especially in primary human AECs homozygous for p53 arginine, and in mice with a modified Tp53 gene. Further, the anion exchange protein, SLC26A9, when reduced, enhanced WS- and oxalate-induced mucin expression.

Discussion: The potency of WS compared with CS in inducing mucin expression may explain the increased risk for chronic bronchitis in participants exposed to WS. Identification of the responsible compounds could help estimate the risk of pollutants in causing chronic bronchitis in susceptible individuals and provide strategies to improve management of lung diseases. https://doi.org/10.1289/EHP9446.

Figure 1.

Evaluation of readouts for mucin production by Alcian blue (AB) staining, mRNA expression, and immunofluorescence in mouse tissues and primary human AECs exposed to cigarette smoke (CS) or wood smoke (WS). (A) AB and hematoxylin and eosin (AB H&E) staining of airways at generation 5 of the left lungs from male mice exposed for 4 and 12 wk to filtered air (FA), CS ($250{\mathrm{mg}/\mathrm{m}}^3$) or WS ($10{\mathrm{mg}/\mathrm{m}}^3$) starting from 8 to 10 wk age. Mucus-positive cells are stained with AB and quantification of the number of mucus cells per millimeter of basal lamina (MCs/mm BL) $n=4/\text{group}$. (B) Immunofluorescent staining showing Muc5ac positivity (shown in red) of left lung sections from mice exposed for 12 wk to FA, WS, and CS (representative images from six images from three mice per group). (C) Muc5ac and SPDEF mRNA levels in the homogenates of the cranial lobe of the right lungs from mice exposed for 4 and 12 wk to FA, WS, and CS. Expression is presented relative to FA control. $n=4$ per group with two repeat qPCR reactions. (D) Primary human AECs differentiated on Transwell cultures in air–liquid interface, treated with WS ($100\text{\hspace{0.17em}ng}/\mathrm{mL}$) and CS ($4\mathrm{\mu }\mathrm{g}/\mathrm{mL}$) extracts for 48 h and MUC5AC and SPDEF mRNAs levels measured by qPCR. $n=5$ per group in each of $N=2$ different experiments. Expression is presented relative to untreated control. (E) Immunofluorescent staining with antibodies to MUC5AC (shown in green) in differentiated human AECs treated with WS ($100\text{\hspace{0.17em}ng}/\mathrm{mL}$) for 48 h. Percentage mucous cells per total epithelial cells quantified from two images/well and four different Transwells. For each figure the $\text{mean}\pm \text{SEM}$ is graphed, difference *, $p<0.05$; **, $p<0.01$; ***, $p<0.001$ with statistical $t$-test. Summary data is provided in Excel Table S1. Note: AEC, airway epithelial cell; DAPI, 4′,6-diamidino-2-phenylindole; HAEC, human airway epithelial cell; MAEC, mouse airway epithelial cell; MUC5AC, mucin 5AC; NT, untreated; qPCR, quantitative polymerase chain reaction; SEM, standard error of the mean; SPDEF, sterile alpha motif pointed domain containing E-twenty-six transcription factor.

Figure 2.

Comparison of cigarette smoke (CS) and wood smoke (WS) in modifying the phosphorylation of epidermal growth factor receptor (EGFR) and stability of p53 and determining the involvement of these proteins in WS-induced mucin gene expression. (A) Human AECs were treated with $100\text{\hspace{0.17em}ng}/\mathrm{mL}$ WS or CS extracts for the indicated time (0, 1, 3, 8, 24, or 48 h) and protein levels of phosphorylated ERK1/2 and EGFR were analyzed by Western blotting and normalized to GAPDH and expressed in fold change relative to time point zero. (B) Immunofluorescence staining for phosphorylated EGFR, ERK1/2 in lung tissue sections of mice exposed to WS, FA, or CS for 12 wk. (C) Human AECs treated with inhibitors of ERK1/2 (UO126) or EGFR (AG 1478) 1 h prior to treatment with WS ($100\text{\hspace{0.17em}ng}/\mathrm{mL}$) and/or CS ($100\text{\hspace{0.17em}ng}/\mathrm{mL}$) and Muc5ac and SPDEF quantified by qPCR. Data are from $n=2$ replicates/group in each of $N=3$ experimental repeats. (D) Human AECs treated with WS or CS extracts for the indicated times and proteins analyzed using antibodies to $\mathrm{\gamma }\text{-H}2\text{AX}$ and p53 by Western blotting. (E) Immunofluorescence staining for $\mathrm{\gamma }\text{-H}2\text{AX}$ in lung tissue sections of mice exposed to WS, FA, or CS for 12 wk. (F) Human AECs were treated with Nutlin-3 for 1 h prior to treatment with WS ($100\text{\hspace{0.17em}ng}/\mathrm{mL}$) for 48 h and mRNA levels of MUC5AC and SPDEF measured by qPCR. Data from $n=2$ replicates/group in each of $N=3$ experimental repeats. (G) Differentiated mouse AECs from WT and p53 knockout ($\mathrm{p}{53}^{-/-}$) mice treated with WS ($100\text{\hspace{0.17em}ng}/\mathrm{mL}$) extract for 48 h and mRNA levels of Muc5ac and SPDEF quantified by qPCR. Data from $n=3$ replicates/group in each of $N=3$ experimental repeats. For each figure the $\text{mean}\pm \text{SEM}$ is graphed, difference at *, $p<0.05$; **, $p<0.01$; ***, $p<0.001$ with statistical $t$-test. Figures for Western analyses are representative of at least three individual experiments. Summary data is provided in Excel Table S2. For (D), protein expression is presented relative to time point zero. For (C,F), mRNA expression is presented relative to untreated control. For (G), mRNA expression is presented relative to untreated WT mice. Note: AEC, airway epithelial cell; EGFR, epidermal growth factor receptor; ERK1/2, extracellular signal-regulated kinase 1/2; GADPH, glyceraldehyde-3-phosphate dehydrogenase; $\mathrm{\gamma }\text{-H}2\text{AX}$, histone family member X; HAEC, human airway epithelial cell; KO, knock out; MUC5AC, mucin 5AC; NT, untreated; p53, protein 53; qPCR, quantitative polymerase chain reaction; SEM, standard error of the mean; SPDEF, sterile alpha motif pointed domain containing E-twenty-six transcription factor; WT, wild type.

Figure 3.

Fractionation of wood smoke (WS) extract using high-performance liquid chromatography (HPLC) and identification of WS constituents that induce mucin gene expression using GC-MS. Fractionation of WS and identification of WS. (A) SPDEF promotor construct in PGL3 basic vector transfected into HEK-293 cells 1 d prior to treatment with HPLC fractions of WS or left untreated (NT). SPDEF promotor activity in the cell lysates measured by luminometer. Data from $n=2$ replicates/group in each of $N=3$ experimental repeats. (B) Identified compounds in the active HPLC fractions, F2 and F15, by tandem mass spectrometry. (C) SPDEF promoter luciferase activity after treatment with WS extract or sugars found in fraction 2. Cells were treated with $100\text{nM}$ of all sugars except for $\text{maltitol-}\mathrm{\beta }$ at $10\text{nM}$. Data from $n=2$ replicates/group in each of $N=3$ experimental repeats. (D) SPDEF promoter luciferase activity after treatment with WS extract or combinations of sugars found in fraction 2. Data from $n=2$ replicates/group in each of $N=3$ experimental repeats. (E) mRNA expression of MUC5AC or SPDEF from primary human AECs differentiated on Transwell membranes and treated with oxalate $100\text{nM}$ for 48 h. Data from $n=2$ replicates/group in each of $N=3$ experimental repeats, and (F) MUC5AC and SPDEF mRNA levels quantified by qPCR from mouse AECs exposed to $100\text{nM}$ oxalate or left untreated for 48 h. Data from $n=2$ replicates/group in each of $N=3$ experimental repeats. (G) Human AECs were treated with $100\text{\hspace{0.17em}ng}/\mathrm{mL}$ oxalate for indicated times (0, 1, 3, 8, 24, or 48) and p53 and $\mathrm{\gamma }\text{-H}2\text{AX}$ protein levels measured by Western blotting. Image is representative of $N=3$ experimental repeats. For each figure the $\text{mean}\pm \text{SEM}$ is graphed, difference at *, $p<0.05$; **, $p<0.01$; ***, $p<0.001$ with statistical $t$-test. For (A,C–F), luciferase activity and mRNA expression are graphed relative to the untreated control. For (G), protein expression is presented relative to time point zero. Summary data is provided in Excel Table S3. Note: AEC, airway epithelial cell; GC-MS, gas chromatography–mass spectrometry; $\mathrm{\gamma }\text{-H}2\text{AX}$, gamma histone family member X; HAEC, human airway epithelial cell; Muc5ac, mucin 5ac; NT, untreated; p53, protein 53; qPCR, quantitative polymerase chain reaction; SEM, standard error of the mean; SPDEF, sterile alpha motif pointed domain containing E-twenty-six transcription factor.

Figure 4.

Identification of the receptor involved in for wood smoke (WS)- and oxalate-induced SPDEF promotor activity. (A) HEK293T cells transfected with SPDEF promotor luciferase construct were treated for 1 h with inhibitors of sodium/glucose cotransporter 1 (Phlorizin) and the ATP-sensitive potassium channels (glyburide) or cystic fibrosis transmembrane conductance regulator (GlyH-101), and solute carrier family members (SLCs) [niflumic acid (NA)] ($30\mathrm{\mu }\mathrm{M}$) prior to treatment with WS ($100\text{\hspace{0.17em}ng}/\mathrm{mL}$) for 24 h and SPDEF promoter luciferase activity measured by Luminometer 48 h later. Data from $n=2$ replicates/group in each of $N=3$ experimental repeats. Data graphed relative to nontreated control (NT) (B) HEK-293T cells were transfected with the SPDEF promotor construct and 24 h later treated with WS or oxalate and cells were treated with NA (1, 10, and $30\mathrm{\mu }\mathrm{M}$) during this time. The luciferase activity was measured 24 h later. Data from $n=2$ replicates/group in each of $N=3$ experimental repeats. Data graphed relative to NT. (C) SPDEF promoter luciferase activity in HEK293T cells that were infected with retroviruses with mock or SLC26A9 shRNA vectors. Cells were transfected 1 d prior to WS extract or oxalate or NT before SPDEF luciferase activity measurement with luminometer. Data graphed relative to NT. Data from $n=3$ replicates/group in each of $N=4$ experimental repeats. (D) Primary human AECs differentiated on Transwell membranes and infected with shCTRL or shSLC26A9 expression constructs and treated with WS extract or oxalate or nontreated for 48 h. Representative images of Transwell membrane immunostained for MUC5AC (shown in green). MUC5AC and SPDEF mRNA levels quantified by qPCR. Data from $n=2$ replicates/group in each of $N=2$ experimental repeats. Data graphed relative to NT. Fold differences for gene expression levels of qRT-PCR were calculated using the $2^{-\mathrm{\Delta }\mathrm{\Delta }\text{Ct}}$ method, normalizing all $2^{-\mathrm{\Delta }\mathrm{\Delta }\text{Ct}}$ to nontreated controls and calculating the mean fold differences for the nontreated, the WS -, and oxalate-treated groups. For each figure the $\text{mean}\pm \text{SEM}$ is graphed, difference at *, $p<0.05$; **, $p<0.01$; ***, $p<0.001$ with statistical $t$-test. Summary data is provided in Excel Table S4. Note: AEC, airway epithelial cell; ATP, adenosine triphosphate; MUC5AC, mucin 5AC; SEM, standard error of the mean; SPDEF, sterile alpha motif pointed domain containing E-twenty-six transcription factor; qRT-PCR, quantitative real-time polymerase chain reaction; shCTRL, short hairpin control; SLC26A9, solute carrier family 26, member 9; WSE, wood smoke extract.

Figure 5.

Evaluation of mucin gene expression by p53 genotype in nasal brushings of people exposed to wood smoke (WS) and the role of oxalate in mice and airway epithelial cells (AECs). (A) MUC5AC mRNA in nasal brushings from nonexposed (NE) and WS-exposed participants. The ratios of fold differences from WS-exposed to the average of NE are shown ($N=60$ ratios for $\mathrm{p}{53}^{\text{ArgArg}}$ and $\mathrm{p}{53}^{\text{ProPro}}$ and $N=30$ ratios for $\mathrm{p}{53}^{\text{ArgArg}}$). (A,B): Expression of MUC5AC and SPDEF mRNA relative fold levels to $\mathrm{p}{53}^{\text{ProPro}}$ in differentiated human AECs obtained from humans homozygous for the p53 arginine and p53 proline variants exposed to either (B) $100\text{\hspace{0.17em}ng}/\mathrm{mL}$ WS or (C) $10\text{\hspace{0.17em}ng}/\mathrm{mL}$ oxalate for 48 h. Data from $n=2$ replicates/group in each of $N=3$ experimental repeats, the $\text{mean}\pm \text{SEM}$ is graphed, *, different from p53 proline ($p<0.05$) with statistical $t$-test. (D) Differentiated mouse AECs from wild-type (WT) or $\mathrm{p}{53}^{\text{AxxA}}$ mice treated with WS ($100\text{\hspace{0.17em}ng}/\mathrm{mL}$) extract for 48 h and Muc5ac and SPDEF mRNA relative fold levels to WT, quantified by qPCR. Data from $n=2$ replicates/group in each of $N=2$ experimental repeats, the $\text{mean}\pm \text{SEM}$ is graphed, *, different from WT ($p<0.05$) with statistical $t$-test. (E) Muc5ac and SPDEF mRNA levels in the right cranial lobes from female mice exposed to WS for 1 d at 8–10 wk of age. Data from $n=3$ replicates/group (F) Mucus cells per millimeter of basal lamina (BL) in the left lung sections of 21 WT and 28 $\mathrm{p}{53}^{\text{AXXA}}$ mice 1 d after instillation with oxalate (6 male and 6 female WT and 8 male and 8 female $\mathrm{p}{53}^{\text{AXXA}}$ mice) or PBS (4 male and 5 female WT and 6 male and 6 female $\mathrm{p}{53}^{\text{AXXA}}$ mice) at 8–10 wk of age. Data from $n=3$ replicates/group in each of $N=3$ experimental repeats, the $\text{mean}\pm \text{SEM}$ is graphed, *, significantly different from PBS instilled group ($p<0.05$) with statistical $t$-test. Representative images of airways from these mice shows mucous cells stained with Alcian blue. Summary data is found in Excel Table S5. Note: HAECs, human airway epithelial cells; MAECs, mouse airway epithelial cells; MUC5AC, mucin 5AC; PBS, phosphate-buffered saline; Oxa, oxalate; SEM, standard error of the mean; SPDEF, sterile alpha motif pointed domain containing E-twenty-six transcription factor; qPCR, quantitative polymerase chain reaction; WSE, wood smoke exposed.