title: Nuclear factor kappa B plays a pivotal role in polyinosinic-polycytidylic acid-induced expression of human β-defensin 2 in intestinal epithelial cells.
abstract: 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-κB)-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-κB binding sites. A luciferase assay revealed the importance of the proximal NF-κB binding site for poly I:C-induced expression of hBD-2. Among NF-κB 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-κB plays an indispensable role in poly I:C induced hBD-2 expression in HT-29 cells.
authors.affiliation: Departments of Pathology, Nihon University School of Dentistry, Nihon University, Tokyo, Japan.
authors.forename: D
authors.initials: D
authors.lastname: Omagari
authors.forename: N
authors.initials: N
authors.lastname: Takenouchi-Ohkubo
authors.forename: S
authors.initials: S
authors.lastname: Endo
authors.forename: T
authors.initials: T
authors.lastname: Ishigami
authors.forename: A
authors.initials: A
authors.lastname: Sawada
authors.forename: I
authors.initials: I
authors.lastname: Moro
authors.forename: M
authors.initials: M
authors.lastname: Asano
authors.forename: K
authors.initials: K
authors.lastname: Komiyama
mesh_terms: D020121:5' Untranslated Regions / Q000235:genetics; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D020397:Goblet Cells / Q000276:immunology / Q000378:metabolism* / Q000473:pathology; D019073:HT29 Cells; D006801:Humans; D018928:Immunity, Mucosal; D007413:Intestinal Mucosa / Q000473:pathology; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D010766:Phosphorylation; D011070:Poly I-C / Q000276:immunology*; D011485:Protein Binding / Q000235:genetics; D012367:RNA, Viral / Q000276:immunology; D051196:Toll-Like Receptor 3 / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D014108:Tosylphenylalanyl Chloromethyl Ketone / Q000494:pharmacology; D014777:Virus Diseases / Q000276:immunology*; D023083:beta-Defensins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
chemical_list: D020121:5' Untranslated Regions; D000911:Antibodies, Monoclonal; D016328:NF-kappa B; D012367:RNA, Viral; D051196:Toll-Like Receptor 3; D023083:beta-Defensins; D014108:Tosylphenylalanyl Chloromethyl Ketone; D011070:Poly I-C