title: HT-29 and Caco-2 reporter cell lines for functional studies of nuclear factor kappa B activation.
abstract: The NF-κB is a transcription factor which plays a key role in regulating biological processes. In response to signals, NF-κB activation occurs via phosphorylation of its inhibitor, which dissociates from the NF-κB dimer allowing the translocation to the nucleus, inducing gene expression. NF-κB activation has direct screening applications for drug discovery for several therapeutic indications. Thus, pathway-specific reporter cell systems appear as useful tools to screen and unravel the mode of action of probiotics and natural and synthetic compounds. Here, we describe the generation, characterization, and validation of human epithelial reporter cell lines for functional studies of NF-κB activation by different pro- and anti-inflammatory agents. Caco-2 and HT-29 cells were transfected with a pNF-κB-hrGFP plasmid which contains the GFP gene under the control of NF-κB binding elements. Three proinflammatory cytokines (TNF-α, IL-1β, and LPS) were able to activate the reporter systems in a dose-response manner, which corresponds to the activation of the NF-κB signaling pathway. Finally, the reporter cell lines were validated using lactic acid bacteria and a natural compound. We have established robust Caco-2-NF-κB-hrGFP and HT-29-NF-κB-hrGFP reporter cell lines which represent a valuable tool for primary screening and identification of bacterial strains and compounds with a potential therapeutic interest.
authors: Cell Biology Unit, Institut Pasteur de Montevideo, Mataojo 2020, 11400 Montevideo, Uruguay.
authors: Giuliana
authors: G
authors: Mastropietro
authors: Cell Biology Unit, Institut Pasteur de Montevideo, Mataojo 2020, 11400 Montevideo, Uruguay.
authors: Inés
authors: I
authors: Tiscornia
authors: Cell Biology Unit, Institut Pasteur de Montevideo, Mataojo 2020, 11400 Montevideo, Uruguay.
authors: Karen
authors: K
authors: Perelmuter
authors: Cell Biology Unit, Institut Pasteur de Montevideo, Mataojo 2020, 11400 Montevideo, Uruguay.
authors: Soledad
authors: S
authors: Astrada
authors: Cell Biology Unit, Institut Pasteur de Montevideo, Mataojo 2020, 11400 Montevideo, Uruguay.
authors: Mariela
authors: M
authors: Bollati-Fogolín
mesh_terms: D018938:Caco-2 Cells; D016764:Cell Polarity; D005455:Fluorescent Antibody Technique; D019073:HT29 Cells; D006801:Humans; D016328:NF-kappa B / Q000502:physiology*; D051197:Toll-Like Receptor 4 / Q000032:analysis; D051996:Transcription Factor RelA / Q000032:analysis
chemical_list: D016328:NF-kappa B; D051197:Toll-Like Receptor 4; D051996:Transcription Factor RelA