Development of an In Vitro Test System Measuring Transcriptional Downregulatory Activities on IL-13

• Published in: Journal of microbiology and biotechnology Vol. 19; no. 3; pp. 331 - 337
• Main Authors: Choi, J.J., Daejeon University, Daejeon, Republic of Korea, Park, B.K., Daejeon University, Daejeon, Republic of Korea, Park, S.Y., Daejeon University, Daejeon, Republic of Korea, Yun, C.Y., Daejeon University, Daejeon, Republic of Korea, Kim, D.H., Daejeon University, Daejeon, Republic of Korea, Kim, J.S., Korea Institute of Oriental Medicine, Daejeon, Republic of Korea, Hwang, E.S., Ewha Womans University, Seoul, Republic of Korea, Jin, M.R., Daejeon University, Daejeon, Republic of Korea
• Format: Journal Article
• Language: English
• Published: Seoul Korean Society for Applied Microbiology 01.03.2009; 한국미생물·생명공학회
• Subjects: Animals; Asthma - genetics; Asthma - metabolism; Biological and medical sciences; Biotechnology; Carcinogens - pharmacology; Cell Line, Tumor; Cyclosporine - pharmacology; Down-Regulation; Drug Evaluation, Preclinical - methods; Fundamental and applied biological sciences. Psychology; Genes, Reporter; Humans; IL-13; Immunosuppressive Agents - pharmacology; Interleukin-13 - biosynthesis; Interleukin-13 - genetics; INTERLEUKINE; INTERLEUKINS; INTERLEUQUINAS; Ionomycin - pharmacology; Ionophores - pharmacology; luciferase assay; Mice; natural therapeutic candidates for allergic disease; pGL4.14-IL-13 expressing stable cell lines; Rats; Tetradecanoylphorbol Acetate - pharmacology; Transcription, Genetic - drug effects; 생물학; Drug; Allergy; Disease; Transcription; natural therapeutic candidates for allergic disease; Enzyme; Luciferase; IL-13; In vitro; Cell line; luciferase assay; Oxidoreductases; pGL4.14-IL-13 expressing stable cell lines
• ISSN: 1017-7825
• DOI: 10.4014/jmb.0806.358

Interleukin-13 (IL-13) has been proposed as a therapeutic target for bronchial asthma as it plays crucial roles in the pathogenesis of the disease. We developed an in vitro test system measuring transcriptional downregulatory activities on IL-13 as a primary screening method to select drug candidates from natural products. The promoter region of IL-13 (-2,048 to +1) was cloned into the upstream of a luciferase gene in the plasmid pGL4.14 containing the hygromycin resistance gene as a selection marker, generating pGL4.14-IL-13. The EL-4 thymoma and RBL-2H3 mast cells transiently expressing this plasmid highly produced the luciferase activities by responding to PI (PMA and ionomycin) stimulation up to 8-fold and 13-fold compared with the control, respectively, whereas cyclosporin A, a well-known antiasthmatic agent, significantly downregulated the activities. The BF1 clone of RBL-2H3 cells constitutively expressing pGL4.14-IL-13 was established by selecting surviving cells under a constant lethal dose of hygromycin treatment. The feasibility of this system was evaluated by measuring the downregulatory activities of 354 natural products on the IL-13 promoter using the BF1 clone. An extract from Morus bombycis (named TBRC 156) significantly inhibited PI-induced luciferase activities and IL-13 mRNA expression, but not the protein expression. Fisetin (named TBRC 353) inhibited not only PI-induced luciferase activities and mRNA expression, but also the IL-13 protein secretion, whereas myricetin (named TBRC 354) could not suppress the IL-13 expression at all. Our data indicated that this in vitro test system is able to discriminate the effects on IL-13 expression, and furthermore, that it might be suitable as a simple and time-saving primary screening system to select antiasthmatic agents by measuring transcriptional activities of the IL-13 promoter.