Development of an in vitro test system measuring transcriptional downregulatory activities on IL-13.
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 wellknown 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.
Laboratory of Pathology, College of Oriental Medicine, Daejeon University, Daejeon 300-716, Korea.
Jeong June
JJ
Choi
Bo-Kyung
BK
Park
Sunyoung
S
Park
Chi-Young
CY
Yun
Dong Hee
DH
Kim
Jin Sook
JS
Kim
Eun Sook
ES
Hwang
Mirim
M
Jin
D000818:Animals; D001249:Asthma / Q000235:genetics / Q000378:metabolism; D002273:Carcinogens / Q000494:pharmacology; D045744:Cell Line, Tumor; D016572:Cyclosporine / Q000494:pharmacology; D015536:Down-Regulation; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D017930:Genes, Reporter; D006801:Humans; D007166:Immunosuppressive Agents / Q000494:pharmacology; D018793:Interleukin-13 / Q000096:biosynthesis / Q000235:genetics; D015759:Ionomycin / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D051379:Mice; D051381:Rats; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects*
D002273:Carcinogens; D007166:Immunosuppressive Agents; D018793:Interleukin-13; D007476:Ionophores; D015759:Ionomycin; D016572:Cyclosporine; D013755:Tetradecanoylphorbol Acetate