Nf-kB is Involved in Pseudomonas Aeruginosa-mediated Transcriptional Regulation of the Human Beta-defensin 2 Gene in Human Corneal Epithelial Cells

Abstract

Abstract: : Purpose: As previously reported, bacterial culture supernatant from gram-negative P. aeruginosa up-regulate hBD-2 mRNA expression in human corneal epithelial cells (HCE). The lipopolysaccharide portion of gram-negative bacteria has also been shown to activate NF-kB in hepatocytes. We investigated whether up-regulation of hBD-2 mRNA by P. aeruginosa supernatant involved activation of NF-kB. Methods: SV-40 trasformed HCE cells were transiently transfected with an hBD-2 promoter construct containing either NF-kB binding or NF-kB mutated site subcloned into a luciferase reporter plasmid pGL3-Basic. Cells were stimulated with P. aeruginosa strain PAO1 culture supernatant for 4 hrs at 37₀C in 5% CO₂ before luciferase assay was performed. Results: Stimulation with bacterial supernatant did not enhance luciferase activity in HCE cells transfected with the control luciferase reporter plasmid. Stimulation increased the luciferase activity 5-fold in cells transfected with plasmid containing NF-kB binding site. This increase was lost with mutation of NF-kB binding site. Conclusion: Data from this study provide evidence that NF-kB is involved in P. aeruginosa induced hBD-2 transcriptional regulation in HCE. Further studies are underway to define the signaling pathway that leads to NF-kB traslocation.

Keywords: 372 cornea: epithelium • 531 Pseudomonas • 605 transcription factors