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Signals and nuclear factors that regulate the expression of interleukin-4 and interleukin- 5 genes in helper T cells  Hyun Jun Lee, MSa, Ikuo Matsuda,

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Presentation on theme: "Signals and nuclear factors that regulate the expression of interleukin-4 and interleukin- 5 genes in helper T cells  Hyun Jun Lee, MSa, Ikuo Matsuda,"— Presentation transcript:

1 Signals and nuclear factors that regulate the expression of interleukin-4 and interleukin- 5 genes in helper T cells  Hyun Jun Lee, MSa, Ikuo Matsuda, MDa, Yoshiyuki Naito, MD, PhDb, Takashi Yokota, PhDa, Naoko Arai, MD, PhDb, Ken-ichi Arai, MD, PhDa  Journal of Allergy and Clinical Immunology  Volume 94, Issue 3, Pages (September 1994) DOI: / (94)90135-X Copyright © 1994 Mosby, Inc. Terms and Conditions

2 FIG. 1 The TH1 and TH2 subsets and their effector functions. Cell-mediated and humoral immunity are mediated by distinct helper T cell subsets. PGE2 may alter the balance of TH1 and TH2 responses. IFN-α, Interferon-α; TGF-β, transforming growth factor β. Journal of Allergy and Clinical Immunology  , DOI: ( / (94)90135-X) Copyright © 1994 Mosby, Inc. Terms and Conditions

3 FIG. 2 Comparison of cis-acting DNA elements of IL-4 and IL-5 genes. Within the promoter regions, IL-4 and IL-5 genes share common sequence motifs. Coordinated and specific expression of the two genes in TH2 cells may be regulated through these elements. Journal of Allergy and Clinical Immunology  , DOI: ( / (94)90135-X) Copyright © 1994 Mosby, Inc. Terms and Conditions

4 FIG. 3 Effects of cAMP on the production of IL-5 and IL-2 in EL-4 cells. A, Northern blot analysis of RNA. EL-4 cells were stimulated with PMA and βt2cAMP, as indicated. At various times after stimulation, total cellular RNA was extracted, and Northern blot analysis of RNA was made with probes specific for IL-2, IL-5, and β-actin cDNA. B, Protein levels of IL-2 and IL-5 in the supernatant of EL-4 cells. Protein levels of IL-2 and IL-5 secreted in the culture supernatants of EL-4 cells used in (A) at 12 hours and 24 hours were measured by ELISA. Journal of Allergy and Clinical Immunology  , DOI: ( / (94)90135-X) Copyright © 1994 Mosby, Inc. Terms and Conditions

5 FIG. 3 Effects of cAMP on the production of IL-5 and IL-2 in EL-4 cells. A, Northern blot analysis of RNA. EL-4 cells were stimulated with PMA and βt2cAMP, as indicated. At various times after stimulation, total cellular RNA was extracted, and Northern blot analysis of RNA was made with probes specific for IL-2, IL-5, and β-actin cDNA. B, Protein levels of IL-2 and IL-5 in the supernatant of EL-4 cells. Protein levels of IL-2 and IL-5 secreted in the culture supernatants of EL-4 cells used in (A) at 12 hours and 24 hours were measured by ELISA. Journal of Allergy and Clinical Immunology  , DOI: ( / (94)90135-X) Copyright © 1994 Mosby, Inc. Terms and Conditions

6 FIG. 4 Effects of cAMP on activities of IL-5 and IL-2 promoters. EL-4 cells were transiently transfected with the IL-5 or IL-2 promoter–chloramphenicol acetyltransferase (CAT) gene fusion construct [pmIL5CAT(1.2) and pmoIL-2CAT, respectively] and control plasmid pUC00CAT. Cells were then stimulated with compounds as indicated, and after 24 hours, CAT activity was measured. (From Lee HJ, et al. J Immunol 1993;151: Copyright 1993, The Journal of Immunology.) Journal of Allergy and Clinical Immunology  , DOI: ( / (94)90135-X) Copyright © 1994 Mosby, Inc. Terms and Conditions

7 FIG. 5 Effects of cAMP agonist on the IL-5 promoter activity. EL-4 cells were transfected with the IL-5 promoter–luciferase gene fusion construct [pmIL5Luc(1.2)]. Twenty-four hours after transfection, cells were incubated with the indicated compounds in the presence or absence of PMA for 16 hours, and luciferase activity was measured. Journal of Allergy and Clinical Immunology  , DOI: ( / (94)90135-X) Copyright © 1994 Mosby, Inc. Terms and Conditions

8 FIG. 6 Effects of cAMP and PKA on IL-5 and IL-2 promoter activities. The IL-5 or IL-2 promoter–luciferase gene fusion construct [pmIL5Luc(1.2) and pIL2Luc, respectively] were cotransfected with either an expression plasmid pcDL-SRα296 containing no insert (A) or SRα-PKA containing cDNA encoding the catalytic subunit of PKA (B). (From Lee HJ, et al. J Immunol 1993;151: Copyright 1993, The Journal of Immunology.) Journal of Allergy and Clinical Immunology  , DOI: ( / (94)90135-X) Copyright © 1994 Mosby, Inc. Terms and Conditions

9 FIG. 7 Competitive inhibition of NF-κB binding by the P sequence. EMSAs were performed as described previously.31 Unlabeled oligonucleotides (100-fold) were used as competitors. Journal of Allergy and Clinical Immunology  , DOI: ( / (94)90135-X) Copyright © 1994 Mosby, Inc. Terms and Conditions

10 FIG. 8 Binding to the P sequence of recombinant P65 prepared by a baculovirus expression system (rP65). (rP65 was kindly provided by Dr. Garry Nolan.) EMSAs were performed as described previously.31, 35 A, Binding of rP65. B, Competitive inhibition of rP65 binding to the P sequence. Unlabeled oligonucleotides (20-fold excess) were used as a competitors. Journal of Allergy and Clinical Immunology  , DOI: ( / (94)90135-X) Copyright © 1994 Mosby, Inc. Terms and Conditions

11 FIG. 9 Contransfection of expression plasmids of NF-κB activates transcription through the P sequence. Transient transfection into Jurkat cells was performed with the diethylaminoethyl cellulose–dextran method as described by Matsuda et al.31 The construction of expression plasmids for NF-κB has been described.31 pGMTATA(3P), pGMTATA(3P02), and pGMTATA(3P10) were constructed by inserting the 3P, 3P02, ande 3P10 oligonucleotides,31 respectively, into the unique Bgl II site of pmGMLuc-30.22, 23 Journal of Allergy and Clinical Immunology  , DOI: ( / (94)90135-X) Copyright © 1994 Mosby, Inc. Terms and Conditions

12 FIG. 10 Sequence comparison of the P sequence, the NF-κB binding site, and the NF-AT binding site. The P sequence is underlined. (Modified from Matsuda I, et al. Biochem Biophys Res Comm 1994;199:439-46, by permission.) Journal of Allergy and Clinical Immunology  , DOI: ( / (94)90135-X) Copyright © 1994 Mosby, Inc. Terms and Conditions

13 FIG. 11 Model for coordinated and differential expression of cytokine genes by NF-AT–like factors. NF-AT is composed of AP1 and non-AP1 components, which are regulated by PMA and Ca2+ signals, respectively. Because NF-AT–like sequences are found in the regulatory regions of various cytokine genes, they are coordinately regulated through NF-AT. The optimal signal for induction is specific to each gene. It is likely that different sets of the NF-AT complex are formed by the combinations of different NF-AT–like factors and their target sequences. TCR, T-cell receptor. Journal of Allergy and Clinical Immunology  , DOI: ( / (94)90135-X) Copyright © 1994 Mosby, Inc. Terms and Conditions

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