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Development of an in vitro test system measuring transcriptional downregulatory activities on IL-13
Jeong June Choi1, Bo-Kyung Park, Sunyoung Park
1Laboratory of Pathology, College of Oriental Medicine, Daejeon University, Daejeon 300-716, Korea.
Abstract:
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.
Insights
Researchers developed a novel in vitro system to screen natural products for asthma drug candidates by measuring Interleukin-13 (IL-13) transcriptional activity. This method efficiently identified compounds that downregulate IL-13, aiding in the discovery of new antiasthmatic agents.
Area of Science:
- Biotechnology
- Pharmacology
- Immunology
Background:
- Interleukin-13 (IL-13) is a key mediator in bronchial asthma pathogenesis.
- Targeting IL-13 offers a potential therapeutic strategy for asthma treatment.
Purpose of the Study:
- To develop and validate an in vitro screening system for identifying natural products that downregulate IL-13 transcription.
- To assess the system's efficacy in selecting potential antiasthmatic drug candidates.
Main Methods:
- Cloning the IL-13 promoter region into a luciferase reporter plasmid (pGL4.14-IL-13).
- Establishing a stable cell line (BF1 clone) expressing the reporter plasmid.
- Screening 354 natural product extracts for their ability to inhibit IL-13 promoter activity.
Main Results:
- The system demonstrated significant luciferase activity induction upon stimulation, which was downregulated by cyclosporin A.
- An extract from Morus bombycis (TBRC 156) and fisetin (TBRC 353) inhibited IL-13 promoter activity and mRNA expression.
- Fisetin also suppressed IL-13 protein secretion, while myricetin (TBRC 354) showed no effect.
Conclusions:
- The developed in vitro system effectively discriminates effects on IL-13 expression.
- This system is suitable as a simple, time-saving primary screening method for antiasthmatic agents targeting IL-13 transcription.
