Cloning and characterization of the gene for a new epithelial beta-defensin - Genomic structure, chromosomal localization, and evidence for its constitutive expression

Mammalian beta-defensins are endogenous cysteine-rich peptide antibiotics t hat are produced either by epithelial cells lining the respiratory, digesti ve, and urogenital tracts or by granulocytes and macrophages. A growing bod y of evidence has implicated these peptides in host defense, particularly m ucosal innate immunity. We previously reported the cloning of the full-leng th cDNA for a porcine beta-defensin (pBD-1), which was found to be expresse d throughout the airway and oral mucosa. Here, we provide the structural or ganization of the pBD-1 gene, showing that the entire gene spans similar to 1.9 kilobases with two short exons separated by a 1.5-kilobase intron. Flu orescence in situ hybridization mapped the pBD-1 gene to porcine chromosome 15q14-q15.1 within a region of conserved synteny to the chromosomal locati ons of human and mouse alpha- and beta-defensins. We also provide several i ndependent lines of evidence showing that the pBD-1 gene is expressed const itutively during inflammation and infection, despite its resemblance to man y inducible epithelial beta-defensins in amino acid sequence, genomic struc ture, and sites of expression. First, stimulation of primary porcine tongue epithelial cells with lipopolysaccharide, tumor necrosis factor-alpha, and interleukin (IL)-1 beta failed to up-regulate the expression of pBD-1 mRNA , Second, pBD-1 gene expression was not enhanced in either digestive or res piratory mucosa of pigs following a a-day infection with Salmonella typhimu rium or Actinobacillus pleuropneumoniae. Last, direct transfection of the p BD-1 gene promoter into NIH/3T3 cells showed no difference in reporter gene activity in response to stimulation by lipopolysaccharide and IL-1 beta, T he constitutive expression of pBD-1 in airway and oral mucosa, which is con sistent with a lack of consensus binding sites for nuclear factor-kappa B o r NF-IL-6 in its promoter region, suggests that it may play a surveillance role in maintaining the steady state of microflora on mucosal surfaces.