Positive feedback of the amphiregulin-EGFR-ERK pathway mediates PM2.5 from wood smoke-induced MUC5AC expression in epithelial cells.

Female Sprague-Dawley rat, wood smoke inhalation model

Healthy animals; readouts taken from 4 um para-sagittal sections of the right lung and from whole-lung RNA, n = 5 rats per group

Primary human bronchial/tracheal epithelial ALI culture

NCI-H292 human airway epithelial cell line

NCI-H292 human airway epithelial cell line

NCI-H292 human airway epithelial cell line

NCI-H292 human airway epithelial cell line

NCI-H292 human airway epithelial cell line

NCI-H292 human airway epithelial cell line

NCI-H292 human airway epithelial cell line

Airway mucous cell metaplasia and mucin expression in rats after 3 months of wood smoke

AB-PAS staining, MUC5AC/MUC5B immunohistochemistry and whole-lung real-time PCR in Sprague-Dawley rats exposed to wood smoke or clean air for three months (n = 5 rats/group). Wood smoke induced severe airway mucous cell metaplasia and highly elevated MUC5AC; MUC5B also rose but not significantly. Quantification is reported only in figure panels, so no numeric values are recorded here.

Exposure conditions

Outputs

Evidence

MUC5AC mRNA and protein in primary human airway epithelial ALI cultures after 24 h WSPM2.5

Differentiated primary human bronchial/tracheal epithelial cells at air-liquid interface treated apically with WSPM2.5 (8 or 40 ug/ml) for 24 h. MUC5AC mRNA by real-time PCR and MUC5AC protein in culture supernatants by ELISA, both expressed as fold change over untreated cells; the control record anchors that scale at 1.0.

Exposure conditions

Outputs

Evidence

MUC5AC mRNA and protein in NCI-H292 cells after 24 h WSPM2.5

NCI-H292 cells treated with non-cytotoxic WSPM2.5 concentrations (0.5-24 ug/ml) for 24 h. At 8 ug/ml, MUC5AC mRNA rose 40.26-fold and MUC5AC protein in the supernatant 4.65-fold over untreated cells. Induction was also time dependent, rising continuously across the 36 h experiment. The control record anchors the fold-change scale at 1.0.

Exposure conditions

Outputs

Evidence

EGFR and ERK phosphorylation in NCI-H292 cells after WSPM2.5

Western blot time course in NCI-H292 cells stimulated with WSPM2.5 8 ug/ml. EGFR phosphorylation rose continuously, first significant at 60 min. ERK phosphorylation was biphasic, starting within 5 min with a second wave from 8 h to 24 h. AG1478 abolished ERK activation, placing EGFR upstream of ERK. Densitometry is reported only in figure panels, so no numeric values are recorded.

Exposure conditions

Outputs

Evidence

Ligand dependence of WSPM2.5-induced EGFR activation in NCI-H292 cells

NCI-H292 cells pretreated for 1 h with an EGFR-neutralizing antibody (0.5, 1 or 2 ug/ml), which prevents ligand binding to EGFR, or with a mouse control IgG, then stimulated with WSPM2.5 8 ug/ml. The antibody significantly reduced both EGFR phosphorylation (60 min) and MUC5AC expression (24 h), showing that EGFR activation by WSPM2.5 is ligand dependent. Results are reported as figure panels, so no numeric values are recorded.

Exposure conditions

Outputs

Evidence

Identification of amphiregulin as the EGFR ligand driving WSPM2.5-induced signaling

NCI-H292 cells pretreated 1 h with neutralizing antibodies against AR, TGF-alpha or HB-EGF, then stimulated with WSPM2.5 (60 min for p-EGFR, 5 min for p-ERK, 24 h for MUC5AC mRNA). AR neutralization suppressed most of the WSPM2.5-induced EGFR and ERK phosphorylation and MUC5AC expression, while TGF-alpha and HB-EGF blockade gave only non-significant partial reductions. Recombinant AR (300-1,800 pg/ml) induced MUC5AC dose-dependently. Results are reported as figure panels, so no numeric values are recorded.

Exposure conditions

Outputs

Evidence

Amphiregulin mRNA and protein in NCI-H292 cells over 36 h of WSPM2.5

NCI-H292 cells exposed to WSPM2.5 8 ug/ml for 2-36 h. AR mRNA by real-time PCR was elevated at 2 h (2.91-fold) and significantly increased at 24 h; AR protein released into the medium (ELISA) was significantly higher at 24 and 36 h. The control record anchors the fold-change scale at 1.0; the 24 and 36 h protein values are reported only in figure panels.

Exposure conditions

Outputs

Evidence

HB-EGF mRNA in NCI-H292 cells after 36 h of WSPM2.5

HB-EGF mRNA was significantly higher after 36 h of WSPM2.5 8 ug/ml (3.38-fold), but HB-EGF protein release measured by ELISA remained unchanged. The control record anchors the fold-change scale at 1.0.

Exposure conditions

Outputs

Evidence

EGFR-ERK dependence and de novo synthesis of amphiregulin in NCI-H292 cells

Pretreatment with EGFR-neutralizing antibody (0.5 ug/ml), AG1478 (10 uM) or PD98059 (5 uM) significantly reduced WSPM2.5-induced AR mRNA expression and AR release, and exogenous AR (1,800 pg/ml) increased AR mRNA in an AG1478- and PD98059-sensitive way. Actinomycin D and cycloheximide (10 ug/ml) inhibited AR secretion and attenuated MUC5AC mRNA, showing de novo AR synthesis is required. This closes the positive feedback loop between AR release and EGFR-ERK activation. Results are reported as figure panels, so no numeric values are recorded.

Exposure conditions

Outputs

Evidence

Change in MUC5AC mRNA, 8 ug/ml WSPM2.5 vs untreated, primary ALI cultures

Change in MUC5AC mRNA, 40 ug/ml WSPM2.5 vs untreated, primary ALI cultures

Change in MUC5AC protein, 8 ug/ml WSPM2.5 vs untreated, primary ALI cultures

Change in MUC5AC protein, 40 ug/ml WSPM2.5 vs untreated, primary ALI cultures

Change in MUC5AC mRNA, 8 ug/ml WSPM2.5 vs untreated, NCI-H292 cells

Change in MUC5AC protein, 8 ug/ml WSPM2.5 vs untreated, NCI-H292 cells

Change in AR mRNA, 2 h WSPM2.5 vs untreated, NCI-H292 cells

Change in HB-EGF mRNA, 36 h WSPM2.5 vs untreated, NCI-H292 cells

EGFR activation leads to mucin hypersecretion

Rat whole-body wood smoke exposure

A total of 40 g of China fir sawdust was smouldered and the smoke channeled through a piston pump into the exposure room. Rats exposed 1 h four times per day, five days per week, for three months. Exposure-room averages: CO 90.15 ppm, O2 20.94%, PM2.5 20.4 mg/m3. Control rats breathed clean room air. Approved by the Local Ethics Committee of Guangzhou Medical University.

Equipment: Wood smoke exposure room; Piston pump; China fir sawdust

WSPM2.5 collection and resuspension

PM2.5 collected from China fir burned in a conventional Chinese wood stove (April 23-May 6, 2015) with a high-volume sampler (TE-6070, Tisch) fitted with a PM2.5 selective-inlet head (1.13 m3/min), onto glass fibre membrane filters (1.6 um pore, 406 cm2). Filters soaked in DMSO, filtered through a 5 um needle filter, pooled; 40 mg/ml stock stored at -20 C and sonicated 3 x 25 s before use. Controls prepared from clean filters containing 0.1% DMSO.

Equipment: High-volume sampler TE-6070 (Tisch, USA) with PM2.5 selective-inlet head; Glass fibre membrane filters (1.6 um pore size); Sonicator

AB-PAS and MUC5AC/MUC5B immunohistochemistry on rat lung

Right lung inflated at 25 cm H2O, fixed in 4% paraformaldehyde for 24 h, paraffin-embedded and cut into 4 um para-sagittal sections (n = 5 rats/group). Sections stained with Alcian blue/periodic acid Schiff for mucus and immunostained for MUC5AC (1:50) and MUC5B (1:200) with an immunohistochemistry detection kit (Gene Tech, Shanghai). Quantified with Image-Pro Plus 6.0.

Real-time quantitative RT-PCR

TRIzol RNA extraction; 500 ng RNA reverse transcribed in 10 ul with the PrimeScript RT Reagent Kit (Takara). 2 ul cDNA amplified in 25 ul with SYBR Premix Ex Taq on an MXP3000 QPCR system (Stratagene): 95 C 30 s; 40 cycles of 95 C 5 s and 60 C 30 s; 95 C 1 min; melt 60-95 C. Relative amounts calculated by the 2^-ddCt method.

MUC5AC ELISA on culture supernatants

50 ul of sample incubated with 50 ul bicarbonate-carbonate buffer for 2 h at 37 C, blocked with 2% BSA for 1 h, incubated with mouse anti-MUC5AC (1:200) for 2 h at 37 C, then HRP-conjugated anti-mouse IgG (1:5000) for 1 h. Developed with TMB, stopped with 2 M H2SO4, absorbance read at 450 nm. Standard curve from serially diluted bovine submaxillary gland mucin. Data expressed as the ratio of the observed value to the control group value.

ELISA for EGFR ligands AR, TGF-alpha and HB-EGF

Commercial ELISA kits for AR, TGF-alpha and HB-EGF (R&D Systems) used on cell culture medium per the manufacturer's instructions; optical density measured at 450 nm.

Western blot for phospho-EGFR and phospho-ERK

RIPA lysis with protease and phosphatase inhibitor cocktails, 30 min on ice; centrifuged 12,000 x g, 4 C, 20 min. Protein quantified by BCA. 30 ug protein per lane on 10% SDS-PAGE, transferred to PVDF, blocked in 5% milk, probed 16 h at 4 C with anti-phospho-ERK1/2 (1:1000) or anti-phospho-EGFR (1:10000), then HRP-conjugated secondary IgG and SuperSignal West Femto substrate. Membranes stripped and re-probed with anti-ERK (1:2000), anti-EGFR (1:100,000) and anti-GAPDH (1:1000) for normalization.

MUC5AC immunofluorescence on differentiated ALI cultures

Differentiated bronchial/tracheal epithelial cells fixed in 4% paraformaldehyde 20 min, permeabilized with 0.5% Triton X-100, blocked with 3% BSA 1 h, incubated overnight at 4 C with mouse monoclonal anti-MUC5AC (1:100), then Cy3-conjugated anti-mouse IgG (1:200) for 1 h; nuclei stained with DAPI 5 min. Inserts excised, mounted and imaged by confocal microscopy; MUC5AC signal expressed as the ratio of Cy3 to total DAPI fluorescence (Image-Pro Plus 6.0).