CD147 increases mucus secretion induced by cigarette smoke in COPD

Forty-two subjects were recruited for this study, including 14 non-smokers without COPD, 14 smokers without COPD, and 14 smokers with COPD. The inclusion criteria were as follows: All subjects underwent pulmonary nodule resection. A diagnosis of COPD was made according to the guidelines of the Global Initiative for Chronic Obstructive Lung Disease [1]. COPD patients in our study were defined as having a phenotype of chronic bronchitis and had a history of exposure to cigarette smoke. All patients took a pulmonary function test, and forced expiratory volume in 1 s (FEV1)/forced vital capacity (FVC) < 0.70 was used to confirm the airflow limitation to diagnose COPD. All patients were subjected to serum protein electrophoresis tests, and there were no subjects with alpha-1 antitrypsin deficiency. Patients had no history of exposure to occupational dust or chemicals or indoor or outdoor air pollution. The exclusion criteria were as follows: Patients with comorbidities, including interstitial lung disease, heart failure, asthma, and neuromuscular disease, were excluded. None of the subjects had suffered from any respiratory tract infection or received any glucocorticoids or antibiotics during the month preceding the study. All lung samples were obtained from Xiangya Hospital of Central South University (Changsha, China). For each specimen, two tissue blocks (sample size 15–25 mm) were taken from the sub-pleural parenchyma of the lobe obtained in surgery, and they were from at least 5 cm away from the margin of the diseased regions. All participants signed informed consent, and this study was performed with the approval of the Ethics Committee of Xiangya Hospital.

Samples of formalin-fixed lung tissue were embedded in paraffin, sectioned (4 μm) and stained with hematoxylin and eosin (HE) for morphometric measurement or with Alcian blue-periodic acid Schiff stain to assess the mucin glycoproteins in the epithelium. The tissue sections were deparaffinized and stained with mouse anti-human CD147 (1:100, Abcam) and mouse anti-human MUC5AC (1:200, Abcam) primary antibodies overnight at 4 °C; then, the sections were incubated with a horseradish peroxidase (HRP)-conjugated anti-mouse secondary antibody (1:1000, Abcam), and the color was developed using diaminobenzidine. In the negative controls, the primary antibody was substituted with phosphate-buffered saline (PBS). Immunochemistry staining of the lung tissue sections was evaluated at 200-fold magnification in four random fields per section. The mean staining intensity values of CD147 and MUC5AC in the airway epithelium were analyzed using Image-Pro Plus 7.0 software.

CS extract was prepared in a manner similar to that in a previous study [22]. CS extract (100%) was prepared by bubbling smoke from two cigarettes in 10 ml of serum-free RPMI1640 media at a rate of a half cigarette/min. The pH of the CS extract was adjusted to 7.4, the optical density was determined (0.783 ± 0.03), and the CS extract was sterile-filtered through a 0.22-μm filter. The CS extract was always prepared fresh on the day of the experiment.

HBE cells are immortalized human bronchial epithelial cells that were purchased from Fuxiang Biotechnology Co., Ltd. (Shanghai, China). The HBE cells were propagated in RPMI 1640 (Gibco) supplemented with 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin in a 37 °C 5% CO₂ incubator. After serum starvation for 12 h, the cells were treated with CS extract at different concentrations (0, 2, 5, 10, 15, and 20%) for 24 h or with 10% CS extract for different durations (0 h, 6 h, 12 h, 24 h, and 36 h). Before the subsequent tests, a cell counting kit 8 (CCK8, from Engreen) assay was used to evaluate cell proliferation. To investigate the signaling cascade involved in the CS-induced MUC5AC secretion, cells were pretreated with 10 μM SB-3CT (MMP9 inhibitor, Sigma) or 10 μM SB203580 (p38 MAPK inhibitor, Cell Signaling Technology) for 1 h. Following a 24-h incubation in culture medium containing 10% CS, the cells and culture supernatants were harvested for further analysis. HBE cells were transfected with CD147 small interfering RNA (RiboBio) using Lipofectamine 3000 (Invitrogen) to silence CD147; then, the cells were incubated for 2–3 d, and the transfection efficacy was determined. Finally, the cells were treated with CS extract (10%) for 24 h.

Cell culture supernatants were collected and used to assay the total protein concentrations. MUC5AC protein levels were measured following protocols provided by the ELISA kit manufacturer (Sangon, Shanghai). Protein was extracted as soon as possible after specimen collection. The standard was diluted; then, the samples, standards and blank were added to the wells of the plate and incubated for 1 h at 37 °C. The liquid was discarded; then, the plate was washed 5 times and patted dry. Chromogenic reaction reagent was added and incubated in the dark for 15 min at 37 °C. Finally, stop solution was added, and the absorbance at 450 nm was measured within 10 min.

Total RNA was extracted from the HBE cells in each group using TRIZOL (Invitrogen). cDNA was generated using a Transcriptor First Strand cDNA Kit (Roche) in accordance with the manufacturer’s protocol. Quantitative RT-PCR was performed using a StepOnePlus PCR system (Applied Biosystems) and gene expression assays. The PCR primers (Sangon) used for MUC5AC were 5’-TACTCGCTCGAGGGCAACA-3′ (forward) and 5’-TGCAGTGCAGGGTCACATTC-3′ (reverse); those for CD147 were 5’-GACTGGGCCTGGTACAAGATCAC-3′ (forward) and 5′- GCCTCCATGTTCAGGTTCTCAA-3′ (reverse); and those for GADPH were 5′-TGTGTCCGTCGTGGATCTGA-3′ (forward) and 5′-CCTGCTTCACCACCTTCTTGAT-3′ (reverse).

Cells were lysed in RIPA lysis buffer with protease and phosphatase inhibitors (Roche). Equal amounts of cell lysates from the protein samples were resolved by 10 and 12% SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes. These membranes were incubated with 5% skimmed milk or 5% BSA in PBST at room temperature for 1 h; after rinsing 3 times, the membranes were incubated overnight at 4 °C with the following specific primary antibodies: mouse anti-human CD147 (at 1:1000, Abcam), rabbit anti-human MMP9, rabbit anti-human p38 MAPK and rabbit anti-human phosphorylated p38 MAPK (at 1:1000, Cell Signaling Technology Company). This step was then followed by incubation with horseradish peroxidase (HRP)-conjugated goat anti-mouse and anti-rabbit secondary antibodies (1:5000, Jackson) for 1 h at room temperature. The blots were visualized with an enhanced chemiluminescence kit (ECL plus). The intensity of each band was measured by using Quantity One software. The relative protein expression levels were determined by normalization to that of β-actin.

Forty-two subjects were recruited for this study and divided into three groups: non-smokers (14 subjects), smokers without COPD (14 subjects) and smokers with COPD (14 subjects). The primary clinical and lung function data for these subjects in the study are listed in Table 1. In the smokers with COPD group, 12 subjects were GOLD 2 stage, and 2 were GOLD 3 stage. There was no difference in age or sex among all subjects. Cigarette smoke history was not significantly different between smokers without COPD and smokers with COPD (p > 0.05). The number of smoking years was higher in smokers with COPD than in smokers without COPD (p < 0.05). FEV1 (% predicted) and the FEV1/FVC ratio were significantly lower in smokers with COPD than in non-smokers and smokers without COPD (p < 0.05).

To determine the inflammation response and mucus secretion in the lung specimens, HE was used to stain areas with inflammation, and AB-PAS was used to stain mucin glycoproteins. The HE staining results revealed that the airway epithelial cells in smokers with COPD had epithelial integrity destruction and inflammatory cell infiltration. The airway epithelial cells in non-smokers without COPD were well-arranged, with complete basement membranes and less inflammatory cell infiltration, and the pathological features of the cells in smokers without COPD were in between those in non-smokers and smokers with COPD (Fig. 1 a, b, c). There were 54% AB-PAS-positive areas in the smokers with COPD group; the non-smokers without COPD group had 5% AB-PAS-positive areas, and the percentage of AB-PAS-positive areas in the smokers without COPD group (21.3%) was in between them (Fig. 1 d, e, f, g).

The average optical density was used to determine the expression of CD147 and MUC5AC. IHC demonstrated that the average optical density of CD147 in smokers without COPD was more than two-fold that in smokers without COPD (p < 0.05); there was more CD147 expression in smokers without COPD than in non-smokers without COPD (p < 0.05). The average optical density of MUC5AC was higher in smokers with COPD than in smokers without COPD (p < 0.05), and the mean density in smokers without COPD was higher than that in non-smokers without COPD (p < 0.05) (Fig. 2). Consistent with previous data, mucin concentrations were higher in smokers with COPD than in those who had never smoked [2]. What is more, we found that CD147 was also higher in smokers with or without COPD than in non-smokers.

Cell proliferation was detected by CCK8 assay. The cytotoxic effect of CS extract on HBE cells occurred in a time- and concentration-dependent manner. Incubation with 1–2% CS extract for 6–36 h had no effect on cell proliferation, and HBE cell proliferation decreased with increasing CS extract concentrations and increasing incubation times (Fig. 3). 15% CS extract treated group has very few cells due to the high concentration of CS.

HBE cells were treated with different concentration of CS extract for 24 h. CS extract (0–10%) induced MUC5AC and CD147 expression in a concentration-dependent manner at the mRNA and protein levels after 24 h of incubation. The mRNA expression level of MUC5AC was increased by approximately 1.5- and 2.6-fold following incubation with 5 and 10% CS, respectively (p < 0.05). Stimulation with 0, 5 and 10% CS induced averages of 6 ng/ml, 43 ng/ml and 161 ng/ml MUC5AC protein secretion in the culture supernatant (Fig. 4). The mRNA expression of CD147 was increased by approximately 1.3- and 24-fold after 24 h of incubation with 5 and 10% CS (p < 0.05). Stimulation with 5 and 10% CS induced 1.3- and 1.8-fold increases in CD147 protein expression (p < 0.05) (Fig. 4). However, the expression of MUC5AC and CD147 in HBE cells was lower after treatment with 15% CS extract than after treatment with 10% CS extract (Fig. 4). As shown in Fig. 3, cell viability was highly affected because the cell number decreased. These data suggested that in vitro CS extract induces MUC5AC and CD147 production, which was in agreement with the lung specimen results.

To functionally connect CD147 to MUC5AC secretion, we depleted CD147 using small interfering RNA transduced with Lipofectamine 3000. We established three CD147 siRNAs to transfect HBE cells; all siRNA silenced CD147 expression, and the one inducing the greatest silencing was chosen for the subsequent experiments (Fig. 5). HBE cells were transfected with CD147 siRNA and then incubated with 10% CS extract for 24 h. CD147 knockdown by siRNA resulted in an approximately 1-fold reduction in the MUC5AC mRNA and protein expression levels compared to siRNA control treatment (p < 0.05) (Fig. 5).

To further explore how CD147 regulates MUC5AC production, we used signaling molecule inhibitors to search for the possible signaling pathway. HBE cells were pretreated with 10 μM SB-3CT (MMP9 inhibitor) or 10 μM SB203580 (p38 MAPK inhibitor) for 1 h prior to CS extract incubation.

Pre-treatment with SB-3CT or SB203580 markedly abrogated the increase in MUC5AC mRNA expression and protein production induced by CS (p < 0.05 vs. untreated group) (Fig. 6).

HBE cell exposure to CS led to significant increases in the levels of MMP9 and phosphorylated p38 MAPK (p-p38 MAPK) (p < 0.05 vs. untreated group); MMP9 expression and p-p38 MAPK were markedly inhibited by SB203580 (p < 0.05 vs. CS-treated group), and SB-3CT attenuated MMP9 expression (p < 0.05 vs. CS treated group) but did not affect p-p38 MAPK (p > 0.05 vs. CS-treated group) (Fig. 6). In this cell model, we verified that CS induced MUC5AC expression through the p38 MAPK/MMP9 signaling pathway.

Western blot analysis revealed that transfection with CD147 siRNA prior to incubation with CS markedly reduced MUC5AC secretion and MMP9 and p38 MAPK expression (p < 0.05, vs. CS-treated group), and pretreatment with SB-3CT or SB203580 caused additional reductions in MMP9 and p-p38 MAPK (p < 0.05, vs. CD147 siRNA + CS treatment group) (Fig. 6).

Considering the non-specific inhibition of SB-3CT and SB203580, we also detected the expression of MMP2, which can be inhibited by SB-3CT, and the expression of phosphorylated Akt, which can be inhibited by SB203580; we found that although CS extract increased the expression of MMP2 and phosphorylation of Akt, CD147 had no effect on their expression (Additional file 1: Figure S1). Together, these data suggested that CD147 induced MUC5AC via the p38 MAPK/MMP9 signaling pathway.