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. 2000 Jan;68(1):113-9.
doi: 10.1128/IAI.68.1.113-119.2000.

"VSports注册入口" Transcriptional regulation of beta-defensin gene expression in tracheal epithelial cells

G Diamond  1 , V Kaiser, J Rhodes, J P Russell, C L Bevins
Affiliations

Transcriptional regulation of beta-defensin gene expression in tracheal epithelial cells

G Diamond et al. Infect Immun. 2000 Jan.

Abstract (V体育ios版)

Innate immunity provides an ever-present or rapidly inducible initial defense against microbial infection VSports手机版. Among the effector molecules of this defense in many species are broad-spectrum antimicrobial peptides. Tracheal antimicrobial peptide (TAP) was the first discovered member of the beta-defensin family of mammalian antimicrobial peptides. TAP is expressed in the ciliated epithelium of the bovine trachea, and its mRNA levels are dramatically increased upon stimulation with bacteria or bacterial lipopolysaccharide (LPS). We report here that this induction by LPS is regulated at the level of transcription. Furthermore, the transfection of reporter gene constructs into tracheal epithelial cells indicates that DNA sequences in the 5' flanking region of the TAP gene, within 324 nucleotides of the transcription start site, are responsible in part for mediating gene induction. This region includes consensus binding sites for NF-kappaB and nuclear factor interleukin-6 (NF IL-6) transcription factors. Gel mobility shift assays indicate that LPS induces NF-kappaB binding activity in the nuclei of these cells, while NF IL-6 binding activity is constitutively present. The gene encoding human beta-defensin 2, a human homologue of TAP with similar inducible expression patterns in the airway, was cloned and found to have conserved NF-kappaB and NF IL-6 consensus binding sites in its 5' flanking region. Previous studies of antimicrobial peptides from insects indicated that their induction by infectious microbes and microbial products also occurs via activation of NF-kappaB-like and NF IL-6-like transcription factors. Together, these observations indicate that a strategy for the induction of peptide-based antimicrobial innate immunity is conserved among evolutionarily diverse organisms. .

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Figures

FIG. 1
FIG. 1
Induction of TAP mRNA levels in TEC by LPS and other inflammatory mediators. (A) Concentration dependence of LPS on TAP mRNA levels in bovine TEC. Total RNA was isolated from primary cultures of TEC (2 × 105 cells/culture) after incubation for 16 h with no additions (lane 1) or in the presence of purified LPS from Pseudomonas aeruginosa (1 ng/ml [lane 2], 10 ng/ml [lane 3], or 100 ng/ml [lane 4]). Probes for Northern blot analysis were TAP48a (TAP) and α-tubulin cDNA (Tubulin). (B) Effect of My4, an anti-CD14 blocking monoclonal antibody, on LPS-inducible changes in TAP mRNA levels. Cultures of TEC were incubated without LPS (lane 1) or with LPS (100 ng/ml) (lanes 2 to 4) for 16 h. The anti-CD14 (αCD14) mouse monoclonal antibody My4 (500 ng/ml) (lane 3) or an equivalent concentration of immunoglobulin G2b (IgG2b), an isotype-specific control antibody (lane 4), was coincubated as indicated. Northern blot analysis was as described for panel A. (C) Effects of inflammatory cytokines and bacterial products on TAP mRNA levels. RNA was isolated from TEC after incubation for 16 h with no additions (lane 1) or in the presence of TNF-α (100 ng/ml) (lane 2), IL-1β (100 ng/ml) (lane 3), IL-6 (500 U/ml) (lane 4), LPS (100 ng/ml) (lane 5), lipoteichoic acid (LTA) (10 μg/ml) (lane 6), or muramyl dipeptide (MDP) (100 μg/ml) (lane 7). Northern blot analysis was as described for panel A. (D) Nuclear run-on transcription of TAP in TEC. TEC were untreated (Control) or treated with bacterial LPS (100 ng/ml) for 16 h. Nuclei from 5 × 106 cells were harvested and used for nuclear run-on analysis as described in Materials and Methods. Nylon filter blots containing cDNA encoding TAP and tubulin were hybridized under high-stringency conditions with 32P-labeled primary RNA transcripts. Specific hybridization of RNA was assessed by autoradiography and quantitated by phosphorimager analysis.
FIG. 2
FIG. 2
Promoter analysis of the upstream region of the TAP gene. A map of the putative promoter region of the TAP gene is shown at the top. Segments of the 5′ flanking region of the TAP gene were ligated into a promoterless luciferase reporter expression vector. Plasmid constructs containing the putative NF-κB and NF IL-6 sites are shown to the left, along with the control vector containing no promoter. Plasmid constructs were transfected into cultured primary TEC, followed by stimulation with LPS. The promoter activity for each plasmid is exhibited as relative luminescence of each transfection, normalized to cotransfected β-galactosidase, and is shown to the right of the respective plasmid.
FIG. 3
FIG. 3
Mobility shift analysis of NF-κB in bovine TEC nuclear extracts. A 32P-labeled, double-stranded oligonucleotide probe containing the NF-κB sequence flanking the TAP gene (ds-TAP/NF32) was incubated with nuclear extracts from bovine TEC treated with LPS (100 ng/ml) or untreated. Specificity of binding is shown by competition with unlabeled specific (ds-TAP/NF32) and nonspecific (ds-TAP/NFmut32) double-stranded oligonucleotides, as described in Materials and Methods. The composition of the NF-κB complex was analyzed by including either anti-p50 or anti-p65 antibodies in the binding reaction. Inclusion of the anti-p50 antibody causes retarded migration of the binding complex, whereas anti-p65 antibody decreases the signal intensity of the binding complex.
FIG. 4
FIG. 4
Structural organization of the HBD-2 gene and other selected antimicrobial peptide genes. The HBD-2 gene was cloned from a human genomic bacteriophage library. The sequence of 4.8 kb of DNA encompassing the gene was determined from each complementary strand. Intron-exon boundaries were defined based on comparison with the published cDNA sequence. Putative enhancer binding sites were identified by using MacVector software. The organization of the HBD-2 gene was compared with those of the TAP gene (9) and two inducible antimicrobial peptide genes from insects, acidic attacin (46) and diptericin (28).
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VSports手机版 - References

    1. Akira S, Isshiki H, Sugita T, Tanabe O, Kinoshita S, Nishio Y, Nakajima T, Hirano T, Kishimoto T. A nuclear factor for IL-6 expression (NF-IL6) is a member of a c/EBP family. EMBO J. 1990;9:1897–1906. - PMC (VSports最新版本) - PubMed
    1. Ausubel F M, et al., editors. Current protocols in molecular biology. Vol. 1. New York, N.Y: Wiley; 1994.
    1. Bals R, Wang X, Wu Z, Freeman T, Bafna V, Zasloff M, Wilson J M. Human beta-defensin 2 is a salt-sensitive peptide antibiotic expressed in human lung. J Clin Invest. 1998;102:874–880. - PMC - PubMed
    1. Betts J C, Cheshire J K, Akira S, Kishimoto T, Woo P. The role of NF-κB and NF-IL6 transactivating factors in the synergistic activation of human serum amyloid A gene expression by interleukin-1 and interleukin-6. J Biol Chem. 1993;268:25624–25631. - PubMed
    1. Boman H G. Gene-encoded peptide antibiotics and the concept of innate immunity: an update review. Scand J Immunol. 1998;48:15–25. - PubMed

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