Relationship Between LPS-induced Airway Mucin Expression And PTEN In NCI-H292 Cell Line

Background Surface of the airway is coated by mucus, a protective barrier against toxins and pathogens, which clears particles and infectious agents from the airways via mucociliary clearance, an important component of the innate immune system of the lungs. However, mucus and mucins are overproduced in the airways of patients with chronic airway diseases such as asthma, chronic obstructive pulmonary disease (COPD), or cystic fibrosis (CF). Excessive mucus greatly contributes to airway obstruction and increases morbidity and mortality in such diseases. Mucus is composed of water, ions, proteins, lipids, and glycoproteins. Mucin glycoproteins are a major macromolecular constituent of normal mucus. Mucins are classified by their MUC protein backbone, which is encoded by a MUC gene. In patients and healthy individuals, secretory mucins MUC5AC, MUC2, MUC5B are the three major airway mucins.Mucus overproduction is associated with a variety of reactions to bacterial and viral infections and with inflammatory factors. Lipopolysaccharides (LPS) are characteristic components of the cell wall of Gram negative bacteria. LPS can induce MUC5AC, MUC2 gene expression and mucin protein production by upregulating the epidermal growth factor receptor (EGFR) mRNA expression and inducing activation of EGFR-specific tyrosine phosphorylation. Activation of EGFR causing subsequent downstream signaling MAPK cascade activation, then induced MUC gene expression. Although MUC5B mucin is present at high levels in mucus from healthy and diseased airways, the effct of LPS on MUC5B mRNA expression is still elusive. Our study willthen investigate this issue.Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is firstly discovered in 1997.The PTEN gene constitutes a novel candidate tumor suppressor and exists as a 55-kDa protein. Besides the firmly established standpoint that PTEN has a lipid phosphatase activity ,it also has a weak protein phosphatase activity. Expression of PTEN has opposite effects, such as blocking downstream signaling of EGFR including the Ras/MEK/MAPK cascade and PI3K/Akt signaling. Recently, reports show that PTEN has a negative effect on smoking solution-induced MUC5AC synthesis. Delivery of smoking solution to NCI-H292 cells increased MUC5AC protein expression with decreased PTEN expression. It is known that cigarette smoking and LPS shares the common way of EGFR-MAPK cascade to upregulate MUC5AC mRNA expression. SO we presumed that PTEN might also play an important role in regulating airway mucin production induced by LPS.SO we examine the effect of PA-LPS on mucin production and PTEN expression in airway epithelium using NCI-H292 cells, in order to investigate the relationship between LPS-induced airway mucin expression and PTEN.Objective The primary objective of this study was to observe the effect of PA-LPS on the major three airway mucin MUC5AC, MUC5B, MUC2 and PTEN production, in order to investigate the relationship between MUC5AC, MUC5B, MUC2 expression and PTEN.Methods The NCI-H292 airway epithelial cells (human pulmonary mucoepidermoid carcinoma cell line) were seeded at a density of 7×10~4 cells/ml into 6 well plates. Cells were maintained in the RPMI 1640 medium supplemented with 10% fetal calf serum, penicillinb and streptomycin at 37℃ in a humidified atmosphere of 95% air and 5% CO2.When confluent, the cells were cultured with RPMI 1640 medium whithout fetal calf serum for 24 h. Then cells were incubated for 24h with PA-LPS solution. For the controls, cells were incubated with the same dose normal saline for the same time periods. Then total RNA was collected at 24h after the PA-LPS delivery. The expression of MUC5AC mRNA , MUC5B mRNA, MUC2 mRNA and PTENmRNA in the cells were measured by reverse trancription-polymerase chain reaction(RT-PCR). The protein of MUC5AC , MUC2 and PTEN were examined by immunocytochemistry. Results1. The expression of MUC5AC mRNA , MUC5B mRNA and MUC2 mRNA in cellsCompared with control group, the expression of MUC5AC mRNA , MUC5B mRNA and MUC2 mRNA in cells increased remarkablely in LPS delivery group.2. The expression of PTEN mRNA in cellsCompared with control group,the expression of PTEN mRNA in cells decreased remarkablely in LPS delivery group.3. The protein of MUC5AC and MUC2 measured by immunocytochemistry in cells3.1 The result of immunocytochemistry for MUC5AC showed significant cytosol staining in LPS delivery group cells, only a very small proportion cells in control group displayed cytosol staining. Compared with control group,the expression of MUC5AC protein increased remarkablely in LPS delivery group.3.2 Immunocytochemistry for MUC2 showed significant cytosol staining in LPS delivery group cells, only a very small proportion cells in control group displayed cytosol staining. The expression of MUC2 protein increased remarkablely in LPS delivery group.4. The protein of PTEN measured by immunocytochemistry in cellsImmunocytochemistry for PTEN showed cytosol staining in contral group cells, rarely plasma membrane and nucleus staining is been seen. Compared with control group, only a very small proportion cells in LPS group displayed cytosol staining. The expression of PTEN protein were decreased remarkablely in LPS delivery group.5. The correlative analysis of MUC5AC mRNA, MUC5B mRNA, MUC2mRNA and PTEN mRNA in cellsThe expression of MUC5AC mRNA in cells was closely correlated with PTENmRNA expression. However, there was no significant correlation between MUC5B mRNA, MUC2 mRNA and PTEN mRNA expression.Conclusions1. The expression of MUC5AC mRNA ,MUC5B mRNA ,MUC2 mRNA and protein of MUC5AC, MUC2 was up-regulated significantly by PA-LPS.2. The expression of PTEN mRNA and PTEN protein was down-regulated significantly by PA-LPS with the increase of mucin.3.There was close correlation between LPS-induced airway mucin expression and PTEN. PTEN might play an important role in LPS-induced mucin over-production.