Regulation of Human MUC7 Mucin Gene Expression by Cigarette Smoke Extract or Cigarette Smoke and Pseudomonas aeruginosa Lipopolysaccharide in Human Airway Epithelial Cells and in MUC7 Transgenic Mice

Preparation of Cigarette Smoke Extract

Aqueous cigarette smoke extract (CSE) was used to mimic the effects of cigarette smoke in the experiments with the cell line. Cigarette smoke of Research Cigarettes 1R1 (University of Kentucky) was slowly bubbled through the Phenol Red–free RPMI 1640 medium containing 10 mM HEPES. The suspension (pH adjusted to 7.4) was then filtered through a 0.22 µm pore filter to remove bacteria and large particles. The resulting CSE from one cigarette in 30 ml of the media, considered to be 100%, was then diluted to different concentrations (using the same medium), and then applied to cell cultures within 30 min of preparation (either alone or in combination with LPS).

Cell Culture

The human pulmonary mucoepidermoid carcinoma cell line NCI-H292 was obtained from the ATCC (American Type Culture Collection, Rockville, MD). The cells were grown in RPMI 1640 medium (GIBCO-BRL, Grand Island, NY) with 2 mM glutamine, 4.5 g/L glucose, 10 mM HEPES, 1mM sodium pyruvate; supplemented with 10% fetal bovine serum (GIBCO-BRL), penicillin (100 units/ml), and streptomycin (50 mg/ml), and maintained at 37^oC in a 5% CO₂ incubator. For different treatments, the cells were cultured in a 24-well plate. At about 70% confluency, the cells were exposed to LPS (10 (µg/ml; an optimal dose, based on the dose-response curve determined previously [13]), or CSE (0.5%, 1%, 2.5%, 5%), or a combination of LPS and CSE. After 24 hours, the cells were harvested for RNA and cell lysate preparations, as described below.

In Vivo Experiments with Mice

Animal experiments were approved by the Institutional Animal Care and Use Committee at the State University of New York at Buffalo. MUC7 transgenic mice and non-transgenic mice littermates (produced in our animal facility), both males and females, were used in this experiment. All mice were 8 weeks old and had similar body weights (~25 g). The mice that were exposed to cigarette smoke (CS) were exposed to it for 4 consecutive days, but only during the daylight hours (8 hours, 1 cigarette per hour). The selected time-course of the study was based on the previous study with the rat model of LPS-induced inflammation with concurrent cigarette smoke inhalation [7], except the number of cigarettes per day was reduced to 8 (from 24 in the rat model). A peristaltic pump was put inside a chamber under the fume hood with the mice. The CS was combusted through the pump at a low speed.

The experiment included the following 4 groups with 5 mice in each group. Group 1: Mice exposed to the CS only. Group 2: Mice exposed to CS/LPS. These mice were first exposed to CS for 24 hours, followed by instillation of LPS. Mice were anesthetized by injection of ketamine hydrochloride (80mg/kg) and xylazine hydrochloride (5mg/kg) to circumvent the cough reflex during instillation. The upper portion of trachea was surgically exposed (by making an incision in the skin and separating the thyroid gland overlying muscles and connecting tissues). A microsyringe carring a 25-gauge soft needle filled with 10µl (2.5µg) of LPS solution was inserted into the exposed trachea and the solution was injected into the lumen of mouse trachea. After injection, and a few hours of recovery, the mice were exposed to CS for 3 more days. Group 3: Mice exposed to LPS. These mice were first exposed to air for twenty-four hours, followed by instillation of LPS, and 3 more days of exposure to air. Group 4: Mice exposed to air only. For all groups, on day 5, the mice were sacrificed and tissues (trachea and lungs) were collected for RNA isolation and immunohistochemistry analysis.

RNA Preparation

Total RNA from cultured NCI-H292 cells and animal tissues was prepared using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s specification. The quality and quantity of RNA were determined spectrophotometrically.

Reverse Transcription

Reverse transcriptions were carried out using Super Script™ III Reverse Transcriptase (Invitrogen, Carlsbad, CA) following the manufacturer’s instructions. Briefly, first-strand cDNA was synthesized in a total volume of 20 µl. Reactions contained total RNA (2 µg), random hexamer primers (0.25 µg), dNTPs (0.5 mM), rRNasin (200 units), SuperScript™ III Reverse Transcriptase (200 units) and 5 x first strand buffer (4 µl). The reactions were incubated at 65^oC for 5 mins, followed by 1 min incubation on ice, 10 min at 25^oC, and 50 mins at 50^oC. Reactions were then inactivated at 70^oC for 15 mins.

Real-Time PCR

Quantitative real-time PCR was performed using Applied Biosystems 7500 Real Time PCR System (Foster City, CA) following the TaqMan Gene Expression Assay protocol (Applied Biosystems). TaqMan primers and probes for the human genes MUC7, MUC5AC and GAPDH (internal control), and the mice genes Muc5ac, Muc10 and Gapdh (internal control) were Pre-Developed (cataloged) by Applied Biosystems. The use of the human GAPDH and mouse Gapdh as an internal control was based on our previous findings that showed these to be optimal in our system over actin or tubulin internal controls. Each 50-μL reaction (in 96-well set-up) contained the following components: 20x TaqMan Gene Expression Assay mix (2.5 μL), cDNA template + H₂O (22.5 μL), 2x TaqMan Universal PCR Master Mix (25 μL). The TaqMan Gene Expression Assay mix contains FAM^TM dye-labeled TaqMan probe and 2 unlabeled PCR primers (the primers were designed to span intron/exon boundaries to verify that PCR products were amplified from the cDNA). To optimize the reaction, we amplified serially diluted cDNA templates that were reverse transcribed from total RNA, and plotted the log of the abundance of target gene products against Ct (threshold cycle). PCR amplification was carried out under the control of SDS software (Sequence Detection Systems). The data was analyzed with Relative Quantification Study Document. The relative quantity of MUC7 or MUC5AC mRNA was normalized to human GAPDH, and Muc5ac or Muc10 mRNA to mice Gapdh expression. The relative quantifications were calculated using ∆ ∆ CT method.

Cell Lysate Preparation and ELISA Assays

NCI-H292 cell lysates were prepared by dissolving the cells in radioimmunoprecipitation assay lysis buffer (Millipore Co, Billerica, MA). ELISA was performed with a Protein Detector ELISA kit (Kirkegaad and Perry Laboratory Inc, Gaithersburg, MD) following the manufacturer’s instructions. The primary MUC7 antibody (rabbit antisera) was prepared by Bio-Synthesis (Lewisville, TX); it is specific to a 15-mer peptide derived from MUC7 mucin glycoprotein (aa 39-53). The primary MUC5AC/Muc5ac antibody was purchased from AnaSpec, Inc. (San Jose, CA). Its reactivity includes human, mouse and rat. The secondary antibody was goat anti-rabbit IgG-alkaline phosphatase conjugate. The lysates (60µl) plus coating buffer (40µl, included in the kit) were pipetted into 96-well microtiter plates. Human saliva (60µl) plus coating buffer (40µl) was used as a positive control, and 100µl coating buffer was used as a negative control. The plate was read at 630 nm using ELISA Microplate reader with 630 filter.

Immunohistochemistry

Prior to immunohistochemistry, each histological sample was incubated in 0.5 M sucrose in PBS overnight and cryosectioned (7µm). Immediately after cryosectioning, tissue sections were washed three times in PBS (10 min at RT) and incubated with a blocking agent (1% BSA, 10% goat serum, 0.1% Triton X-100 in PBS) for 2hrs at RT. Then, they were incubated for 1 hr at RT with monoclonal mouse anti-MUC7 antibody [14] and MUC5AC/Muc5ac monoclonal antibody (AnaSpec, Inc., Catalog# 53684; species reactivity includes human, mouse and rat). Both antibodies were diluted 1:1000 in the blocking agent. After washing three times with PBS (10 min RT), tissue sections were incubated for 2.5 hrs at RT with a fluorescent secondary goat anti-mouse Ig-G (Alexa Fluor 488, Molecular Probes, USA) diluted in 1% BSA, 10% goat serum in PBS (1:150). The sections were then washed three times in PBS (10 min each at RT), air dried for 10 min at RT and covered with a cover glass using an aqueous mounting medium (Immu-Mount, Thermo-Shandon, USA). Fluorescent pictures (200×) were acquired by a digital camera (70 ms exposure time; SPOT RT-KE Diagnostic instruments Inc.), attached to a Nikon inverted fluorescent microscope (Nikon Eclipse TE2000-U).

Statistical Analysis

Statistical analyses of real-time PCR and ELISA results were performed upon comparisons made between the treated group and the control group using the Student’s t-test. A p value of less than 0.05 was considered statistically significant. Data is expressed as mean ± standard error (SE).

In Vitro Studies

Effects of LPS, CSE and CSE/LPS on MUC7 (and MUC5AC) Gene Transcription

NCI-H292 cells were selected for these experiments since it was reported in the literature that these cells express LPS receptors [15, 16]. Quantitative real time RT-PCR was used to assess the steady-state MUC7 gene transcription. Expression of human MUC5AC, the high molecular weight respiratory mucin, shown previously to be upregulated by CSE, was also assessed for comparative purposes [7]. The results are shown in Fig. (1).

For MUC7 (Fig. 1A), a statistically significant level of induction (p value less than 0.05) was found with the 1% CSE treatment (3.2-fold, left panel) and with the 0.5% CSE plus LPS combination treatment (4.4-fold, right panel). The p value less than 0.05 was also observed for 1% CSE compared with 0.5% CSE (left panel). For MUC5AC gene (Fig. 1B), the cell treatment with LPS alone resulted in the statistically significant increase of MUC5AC transcripts (6 fold, left panel). The treatment with CSE alone resulted in some increase of MUC5AC transcripts with each concentration of CSE used, but none of the increases were statistically significant. On the other hand, the combination of 1% CSE with LPS showed a statistically significant increase of MUC5AC transcripts (11 fold, right panel). The treatment with 2.5% CSE and LPS resulted up to 23 fold increase in MUC5AC transcripts, but due to the large SD, there is no statistically significant difference compared to the untreated cells. The p value less than 0.05 was also observed for 1% CSE plus LPS group compared with 0.5% CSE plus LPS group.

In Vivo Studies

In Vitro Studies

The treatment of the NCI-H292 cells with P. aeruginosa LPS did not lead to increase of MUC7 transcription despite the fact that, as reported, these cells express TLR4 [15, 16] through which the bacterial LPS activate cells. In our previous study, we showed that MUC7 transcription was also not increased by LPS treatment of the A549 lung carcinoma cell line, but it was increased in the primary normal human tracheobronchial epithelial (NHTBE) cells [13]. We attributed the lack of MUC7 induction by the LPS in the A549 cells to the lack of the LPS receptors, TLR4 (as determined by flow cytometric analysis by another group) [18], and the MUC7 induction in the primary cells to the presence of TLR4. However, it should be noted that the more recent studies using RT-PCR showed that A549 cells do express TLR4 [16, 19]. Thus it appears that these human airway epithelial cell lines, although extensively used for in vitro studies, have a weakness, i.e. expressing variable levels of TLR4 receptor. MUC5AC transcription in the NCI-H292 cells, used in this study, increased significantly upon the LPS treatment (6-fold), and this finding is consistent with the previous studies using the same cell line [7, 20].

The treatment of NCI-H292 cells with different doses of CSE resulted in a statistically significant increase in the level of MUC7 transcription only with the 1% dose (3.2 fold). For MUC5AC, a moderate increase was seen with the most CSE doses used, but none of the increases were statistically significant. The previous studies indicated that MUC5AC was upregulated by CSE in NCI-H292 cells [7] and further that cigarette smoke causes mucin overproduction via EGF receptor (EGFR) activation in airway epithelial cells in vitro and in vivo [21, 22].

Regarding the question whether the MUC7 gene transcription can be enhanced by the LPS/CSE combination treatment in NCI-H292 cell line, we found that MUC7 mRNA was statistically significantly increased only with the 0.5% CSE/LPS treatment. In fact, the MUC7 transcript was increased by 4.4 fold, which represents a synergistic increase. However, 1% CSE/LPS treatment led to no MUC7 transcription increase, and in fact the level was even lower than for the untreated, control cells, indicating that this combination lead to antagonistic effect, eliminating the 3.2-fold increase achieved with 1% CSE. A suppression of MUC7 expression may not be due to potential toxicity of the combined stimuli, because stimulation of cells with 1% CSE/LPS treatment yielded 11-fold transcription increase of MUC5AC. This latter finding also represents a synergistic effect and it is in an agreement with the previous study, which demonstrated that co-stimulation with 1% CSE/LPS synergistically increased MUC5AC mucin transcription (15-fold) in NCI-H292 cells [7]. The underlying mechanisms of synergy between the CSE and LPS are unclear, but the authors of the above study speculated that CSE acts at multiple levels to synergistically enhance mucin production. We have noticed a recent study that reported that CSE induced expression of TLR4 and LPS binding in another epithelial cell line, namely 16-HBE [23], and thus it is tempting to speculate that this could be one possible explanation for the observed synergy between the CSE and LPS treatment of NCI-H292 cells and the transcription of MUC7 and MUC5AC.

The CS, LPS, and the combination of CS/LPS treatment of the NCI-H292 cell line resulted only in a small increase in both MUC7 glycoprotein (maximum 1.3 fold with the 0.5% CSE/LPS treatment) and MUC5AC glycoprotein production (maximum 1.5 fold with the 1% CSE/LPS treatment; data not shown). Note, however, that both of these highest increases in glycoprotein production were achieved with the same combination of CSE/LPS treatment that yielded the synergistic increase of the MUC7 and MUC5AC transcripts, indicating some degree of correlation between transcription and glycoprotein production. Other studies, using the same cell line, reported elevation of MUC5AC glycoprotein upon CSE treatment, which appeared to increase in a dose dependent manner, from 1.3-2.5 fold [24] and from 2-3 fold [7], and with the combination of CSE/LPS treatment, 4-fold induction was seen [7].

In Vivo Studies

We chose to use CS rather than CSE for in vivo experiments in order to resemble the smoke exposure experience by smokers. Even though intranasal administration of CSE in vivo maybe more convenient, the study by Elliot et al. [25] showed that its resemblance to the CS exposure experience is not equivalent. CSE resulted in a heavier exposure to cigarette smoke components over the same treatment length.

Measuring induction of human MUC7 mRNA after treatment of MUC7 transgenic mice with LPS, CS or combination of LPS/CS showed statistically significant increase in MUC7 mRNA in both trachea and lung with LPS treatment only (Fig. 2). The two-fold increase of MUC7 mRNA with the LPS treatment is consistent with our previous in vivo study [13]. Mouse Muc5ac transcript was found to statistically increase in trachea (but not in lungs) with all three treatments (LPS, CS, LPS/CS), compared to the control animals. On the other hand, the study [7] using a rat model showed no induction of rat muc5AC with LPS or CS alone, in either trachea or lungs; the induction was seen only with the combination of LPS/CS, in both trachea and lungs.

The levels of MUC7 or Muc5a glycoprotein reactivity/ production in the animal tissues using immunohistochemistry in our study, and the level of total mucin content in the tracheobronchial lavage fluid measured by enzyme-linked lectin assay by the investigators using the rat model [7], are hard to compare. Also, our immunohistochemistry results are not quantitative. Nevertheless, the exposure of animals to a combination of LPS/CS treatment seems to result in increased levels of MUC7 and Muc5ac mucin reactivity/production in our mouse model, and increased the total mucin levels in the rat model, as compared to the animals treated with CS or LPS alone.

The role of MUC7 and its regulation in different stages of different diseases is still not understood. MUC7 is a small, soluble, non-gel-forming mucin. Its main function is thought to be clearance of various bacteria and thus inhibition of bacterial colonization. It has been shown to interact in vitro with many oral and respiratory microorganisms, including P. aeruginosa [26]. Thus, the increased production of MUC7 soluble mucin, seen upon exposure of mice to P. aeruginosa LPS/CS exposure shown in vivo by immunohistochemistry in this study, may reduce the viscosity of the mucus caused by overproduction of the large, gel forming mucins in the respiratory track. This increase in the soluble MUC7 may facilitate the mucocilliary clearance, and thus have a protective role in the respiratory track by maintaining an appropriate balance between detrimental and beneficial outcomes.