Transcriptional regulation of the IL-5 gene in peripheral T cells of asthmatic patients.
Mechanisms that underlie the regulation of IL-5 gene expression in human peripheral T cells remain incompletely defined because of the low efficiency of transfection of plasmid constructs into non-transformed T cells. To elucidate the cellular and molecular mechanisms of IL-5 production, concanavalin A (ConA)-stimulated blastocytes derived from peripheral blood lymphocytes of asthmatic patients were employed in this study. Transcriptional activity of the synthetic human IL-5 promoter in ConA-stimulated blastocytes correlated with the production of IL-5. Deletion analysis of the reporter gene showed that the cis-regulatory element located at - 119 to - 80 is critical for inducible IL-5 promoter activity. Electrophoretic mobility shift assay revealed that an oligonucleotide probe corresponding to the element (- 119 to - 90) gave two specific bands. The slower migrating band was absolutely dependent on stimulation and was composed of a co-operative complex of the transcription factors, nuclear factor of activated T cells (NFAT) and activating protein-1 (AP-1). The faster migrating band was also inducible and was identified as AP-1-less NFAT. Mutation of either the NFAT or AP-1 element abrogated the slower migrating band and at the same time abolished transcriptional activity of the human IL-5 promoter/enhancer gene. Cyclosporin A equivalently suppressed DNA-binding activity of the composite NFAT/AP-1 site, promoter activity and protein production of IL-5. In conclusion, these data suggests that the composite NFAT/AP-1 binding element (- 115 to - 100) plays a crucial role in IL-5 synthesis by peripheral T cells of asthmatic patients.
Ogawa
K
K
Clinical Research Center for Allergy and Rheumatology, National Sagamihara Hospital, Sagamihara, Kanagawa, Japan. k-ogawa@tanabe.co.jp
Kaminuma
O
O
Okudaira
H
H
Kikkawa
H
H
Ikezawa
K
K
Sakurai
N
N
Mori
A
A
D000328:Adult; D001249:Asthma / Q000235:genetics / Q000276:immunology*; D002478:Cells, Cultured; D003208:Concanavalin A / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D005786:Gene Expression Regulation; D006801:Humans; D015848:Interleukin-5 / Q000235:genetics*; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D011401:Promoter Regions, Genetic; D013601:T-Lymphocytes / Q000276:immunology*; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; D015848:Interleukin-5; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D018808:Transcription Factor AP-1; D014157:Transcription Factors; D003208:Concanavalin A