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 that are produced either by epithelial cells lining the respiratory, digestive, and urogenital tracts or by granulocytes and macrophages. A growing body of evidence has implicated these peptides in host defense, particularly mucosal innate immunity. We previously reported the cloning of the full-length cDNA for a porcine beta-defensin (pBD-1), which was found to be expressed throughout the airway and oral mucosa. Here, we provide the structural organization of the pBD-1 gene, showing that the entire gene spans approximately 1.9 kilobases with two short exons separated by a 1.5-kilobase intron. Fluorescence in situ hybridization mapped the pBD-1 gene to porcine chromosome 15q14-q15. 1 within a region of conserved synteny to the chromosomal locations of human and mouse alpha- and beta-defensins. We also provide several independent lines of evidence showing that the pBD-1 gene is expressed constitutively during inflammation and infection, despite its resemblance to many inducible epithelial beta-defensins in amino acid sequence, genomic structure, and sites of expression. First, stimulation of primary porcine tongue epithelial cells with lipopolysaccharide, tumor necrosis factor-alpha, and interleukin (IL)-1beta failed to up-regulate the expression of pBD-1 mRNA. Second, pBD-1 gene expression was not enhanced in either digestive or respiratory mucosa of pigs following a 2-day infection with Salmonella typhimurium or Actinobacillus pleuropneumoniae. Last, direct transfection of the pBD-1 gene promoter into NIH/3T3 cells showed no difference in reporter gene activity in response to stimulation by lipopolysaccharide and IL-1beta. The constitutive expression of pBD-1 in airway and oral mucosa, which is consistent with a lack of consensus binding sites for nuclear factor-kappaB or 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.
Zhang
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Department of Anatomy and Physiology, Kansas State University, Manhattan, Kansas 66506, USA.
Hiraiwa
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Yasue
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Wu
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Ross
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CR
Troyer
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Blecha
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D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001424:Bacterial Infections / Q000378:metabolism*; D001483:Base Sequence; D002478:Cells, Cultured; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D023082:Defensins; D004847:Epithelial Cells / Q000378:metabolism; D015870:Gene Expression; D007249:Inflammation / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009092:Mucous Membrane / Q000382:microbiology; D011401:Promoter Regions, Genetic; D011506:Proteins / Q000235:genetics*; D013552:Swine
D023082:Defensins; D011506:Proteins