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

Abstract

Objective: The human MUC7 gene encodes a low-molecular-weight mucin glycoprotein that functions in lubrication/protection of epithelial surfaces of the oral cavity and respiratory tract. This study was designed to evaluate the effect of cigarette smoke extract (CSE), cigarette smoke (CS), and Pseudomonas aeruginosa lipopolysaccharide (LPS), either alone or in the combination, on MUC7 expression in vitro and in vivo.

Materials and methods: qRT-PCR was used to determine the levels of mucin gene transcription in the human lung carcinoma cell line NCI-H292 (in vitro) and MUC7 transgenic mouse tissues (in vivo). ELISA was used to assess mucin glycoprotein levels in the cell line, and immunohistochemistry to assess mucins in lung and trachea sections.

Results: In vitro treatment of cells with LPS (10 (microg/ml) or CSE (0.5, 1, 2.5 and 5%) alone, resulted in a statistically significant increase of MUC7 transcripts only with 1%CSE (3.2-fold). The combined CSE/LPS treatment resulted in a synergistic increase of MUC7 with 0.5%CSE/LPS (4.4 fold). MUC7 glycoprotein levels increased only minimally, the highest increase was seen with the 0.5%CSE/LPS combination treatment (1.3-fold). In vivo exposure of MUC7 transgenic mice to CS, LPS or CS/LPS combination resulted in significant increase in MUC7 transcripts only with LPS treatment (in both trachea and lung). Immunohistochemistry indicated variable increase in MUC7 glycoprotein with CS and LPS treatment, both in the trachea and lungs, but CS/LPS exposure appeared to yield the highest increase.

Conclusion: In vitro, CSE and a combination of CSE/LPS treatment upregulated MUC7 gene transcription. In vivo, LPS upregulated MUC7 transcription, and a combination of CS/LPS appeared to increase MUC7 glycoprotein.

Keywords: LPS; MUC7 expression; NCI-H292 cells; cigarette smoke; lungs; trachea..

Fig. (1)

Effect of CSE, LPS and CSE/LPS on mucin gene transcription in vitro, using human NCI-H292 cells. Total RNA was isolated from the control cells and treated cells and reversed transcribed. MUC7 expression (panel A) and MUC5AC expression (panel B) was determined by quantitative Real-Time PCR (qRT-PCR). The level of different mucin mRNA was normalized to GAPDH. Each bar represents the SD from three independent experiments. * indicates p < 0.05. LPS, lipopolysaccharide from P. aeruginosa, used at concentration of 10 µg/ml in all experiments (an optimal dose, determined by the dose-response curve; data not shown). CSE, cigarette smoke extract, used at several different concentrations, either alone or in combination with LPS, as indicated. Relative levels of mucin mRNA are expressed as fold of induction comparing to the control, untreated cells, set arbitrary at a value of 1.

Fig. (2)

Effect of CS, LPS and CS/LPS on mucin gene transcription in vivo, using MUC7 transgenic mice. Total RNA was isolated from the untreated and treated mice tissue and reverse transcribed. Human MUC7, and mouse Muc5ac and Muc10 expression in trachea (panel A) and in lung (panel B) was determined by quantitative Real-Time PCR (qRT-PCR). The level of different mucin mRNA was normalized to mouse Gapdh. Each triangle point represents individual control or experimental mouse (n=5). The horizontal line represents the average expression of the particular mucin in each group of animals. The mice groups are: control (exposed to air), LPS (exposed to lipopolysaccharide), CS (exposed to cigarette smoke) and CS/LPS (exposed to combination of cigarette smoke and lipopolysaccharide). The significant increase in the levels of mucin gene expression in the tissues of treated versus untreated mice, or the p value <0.05 was found in trachea (panel A), for MUC7 with LPS treatment, and for Muc5ac with LPS, CS and CS+LPS; in lung (panel B), only for MUC7 with LPS treatment.

Fig. (3)

Effect of CS, LPS and CS/LPS on mucin production in vivo, using MUC7 transgenic mice determined by immunohistochemistry. A representative results from tissues of several mice are shown. MUC7 and Muc5ac in trachea (panel A), and MUC7 and Muc5ac in lungs (panel B). Fluorescent (upper) and phase contrast (lower) pictures are shown for each tissue sample. For MUC7 mucin, Control(-) and Control(+) were tissue samples from trachea or lung samples from the non-treated nontransgenic mice (not expressing human MUC7) and MUC7 transgenic mice (expressing MUC7), respectively. For Muc5ac mucin control, samples from trachea or lung from the non-treated MUC7 transgenic mice (these express Muc5ac) were used. Pictures (200×) were acquired by a digital camera attached to a Nikon inverted fluorescent microscope. Hematoxylin-eosin staining (not shown) indicated the integrity of the tissue.