Nuclear factor kappa B plays a pivotal role in polyinosinic-polycytidylic acid-induced expression of human β-defensin 2 in intestinal epithelial cells

P>Intestinal epithelial cells (IECs) play an important role in protecting the intestinal surface from invading pathogens by producing effector molecules. IECs are one of the major sources of human beta-defensin 2 (hBD-2), and can produce it in response to a variety of stimuli. Although IECs express Toll-like receptor 3 (TLR-3) and can respond to its ligand, double-stranded RNA (dsRNA), hBD-2 expression in response to dsRNA has not been elucidated. In the present study, using an artificial analogue of dsRNA, polyinosinic-polycytidylic acid (poly I:C), we investigated whether the human IEC line, HT-29, can produce hBD-2 in response to poly I:C. HT-29 cells can express hBD-2 mRNA only when stimulated with poly I:C. The induction of hBD-2 mRNA expression was observed at 3 h after stimulation and peaked at 12 h of post-stimulation. Pre-incubation of the cells with nuclear factor kappa B (NF-kappa B)-specific inhibitor, l-1-4'-tosylamino-phenylethyl-chloromethyl ketone (TPCK) and isohelenine abolished the expression of hBD-2. Detection of the poly I:C signal by TLR-3 on the surface of HT-29 cells was revealed by pre-incubating the cells with anti-TLR-3 antibody. The 5'-regulatory region of the hBD-2 gene contains two NF-kappa B binding sites. A luciferase assay revealed the importance of the proximal NF-kappa B binding site for poly I:C-induced expression of hBD-2. Among NF-kappa B subunits, p65 and p50 were activated by poly I:C stimulation and accumulated in the nucleus. Activation of the p65 subunit was investigated further by determining its phosphorylation status, which revealed that poly I:C stimulation resulted in prolonged phosphorylation of p65. These results indicate clearly that NF-kappa B plays an indispensable role in poly I:C induced hBD-2 expression in HT-29 cells.