Endotoxin Upregulates Expression of an Antimicrobial Peptide Gene in Mammalian Airway Epithelial Cells

Tracheal antimicrobial peptide (TAP) is a 38-amino acid peptide isolated from the bovine tracheal mucosa, which has a broad spectrum of antimicrobial activity. It is structurally similar to antimicrobial peptides present in circulating phagocytes and has comparable microbicidal activity.1Diamond G Zasloff M Eck H Tracheal antimicrobial peptide, a cysteine-rich peptide from the mammalian tracheal mucosa: peptide isolation and cloning of a cDNA.PNAS. 1991; 88: 3952-3956Crossref PubMed Scopus (417) Google Scholar We now show by Northern blot analysis that TAP is expressed along the entire length of the airway, from nasal to bronchiolar tissue, and in situ hybridization of airway sections further localizes the message to the columnar epithelial cells lining the trachea. Additional RNA analysis failed to detect TAP message in any tissue other than airway mucosa. These results suggest that TAP expression is limited to the airway epithelium and that this peptide may contribute to host defense of this epithelial surface. We cloned the gene encoding TAP, and sequence analysis indicated that it consisted of two exons, separated by a 1.6 kb intron. We determined the site of transcription initiation by primer extension analysis, and sequence analysis of the 5’ flanking region indicated that there are several putative transcriptional promoter sequences. Of note, an NFκB consensus sequence is found 177 bases upstream from the site of transcriptional initiation. This approximate location of an NFκB site is found in several antimicrobial defense genes of lower eukaryotes and suggests that TAP expression may be similarly modulated by lipopolysaccharide (LPS). To test this possibility, cultured bovine tracheal epithelial cells were exposed to LPS. At concentrations as low as 10 ng/ml, we observed a fivefold to tenfold increase in the TAP message levels by Northern blot analysis. Preliminary analysis of the upstream NFκB site using electrophoretic mobility shift assays indicates that this putative NFκB recognition sequence is specifically bound by a nuclear protein from airway epithelial cells. These results suggest a possible mechanism for pathogen-induced regulation of an antimicrobial gene in mammalian airway epithelial cells.