Transcriptional regulation of the human MUC7 mucin gene in airway epithelial cells
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The human MUC7 gene encodes a small mucin glycoprotein that functions in lubricating and protecting epithelial surfaces of the oral cavity, and likely of the respiratory tracts. MUC7 mucin can inhibit bacterial colonization by masking their surface adhesins, thereby mediating the clearance of bacteria. To understand regulation of MUC7 expression, and its underlying mechanisms, this study was designed (i) to investigate the alteration of MUC7 expression in human airway cells in response to stimulations by a bacterial product or a proinflammatory cytokine, and (ii) to characterize the gene promoter structure and function. We have studied the effects of Pseudomonas aeruginosa lipopolysaccharide (LPS) and human tumor necrosis factor-α (TNF-α) on MUC7 gene transcription in three model systems: (i) normal human tracheobronchial epithelial (NHTBE) cells, (ii) a human lung carcinoma cell line (A549), and (iii) MUC7 gene transgenic mice. The levels of MUC7 transcription was assessed using northern blot analysis and real-time PCR. We found that NHTBE cells can express the MUC7 gene only when the cells are differentiating. Both LPS and TNF-α were able to up-regulate MUC7 transcription in NHTBE cells; in A549 cells, TNF-α increased MUC7 transcription, but LPS did not; in transgenic mouse airway tissues (trachea and lung), MUC7 transcription was increased by exposure to LPS. To identify the transcriptional control region, we performed functional analysis of MUC7 gene 5 ' -flanking region. The -2,732/+30 bp region of MUC7 genomic DNA was subcloned into a reporter expression vector. A series of promoter/reporter constructs were generated via sequential deletion of the -2732/+30 bp fragment, and analyzed in A549 cells. Results of the luciferase assay showed that a minimal functional MUC7 promoter is present in the region of -138/+30 bp. This region also revealed the greatest increase in promoter activity in response to TNT-α-stimulation. Two putative AP1 binding elements and one NF-κB binding element are present in the proximal promoter. Further analyses using gel shift assays and mutagenesis demonstrated that the two AP1 elements play an essential role in constitutive expression of the MUC7 gene, and that the NF-κB element is important in response to TNF-α-stimulation. This study brings new insight into understanding MUC7 gene regulation.