Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Development of an In Vitro Test System Measuring Transcriptional Downregulatory Activities on IL-13

  • Choi, Jeong-June (Laboratory of Pathology, College of Oriental Medicine, Daejeon University) ;
  • Park, Bo-Kyung (Laboratory of Pathology, College of Oriental Medicine, Daejeon University) ;
  • Park, Sun-Young (Laboratory of Pathology, College of Oriental Medicine, Daejeon University) ;
  • Yun, Chi-Young (Traditional Medicine Bio-Research Center, Daejeon University) ;
  • Kim, Dong-Hee (Laboratory of Pathology, College of Oriental Medicine, Daejeon University) ;
  • Kim, Jin-Sook (Korea Institute of Oriental Medicine) ;
  • Hwang, Eun-Sook (College of Pharmacy, Division of Life and Pharmaceutical Science, Center for Cell Signaling and Drug Discovery Research, Ewha Womans University) ;
  • Jin, Mi-Rim (Laboratory of Pathology, College of Oriental Medicine, Daejeon University)
  • Received : 2008.06.01
  • Accepted : 2008.07.28
  • Published : 2009.03.31
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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 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.

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References

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