Specific Activation of Human Interleukin-5 Depends on de Novo Synthesis of an AP-1 Complex

Gretchen T.F Schwenger,Chee Choy Kok,Estri Arthaningtyas,Marc A Thomas,Colin J Sanderson,Viatcheslav A Mordvinov

doi:10.1074/jbc.m207414200

Abstract

It is clear from the biology of eosinophilia that a specific regulatory mechanism must exist. Because interleukin-5 (IL5) is the key regulatory cytokine, it follows that a gene-specific control of IL5 expression must exist that differs even from closely related cytokines such as IL4. Two features of IL5 induction make it unique compared with other cytokines; first, induction by cyclic adenosine monophosphate (cAMP), which inhibits other T-cell-derived cytokines, and second, sensitivity to protein synthesis inhibitors, which have no effect on other cytokines. This study has utilized the activation of different transcription factors by different stimuli in a human T-cell line to study the role of conserved lymphokine element 0 (CLE0) in the specific induction of IL5. In unstimulated cells the ubiquitous Oct-1 binds to CLE0. Stimulation induces de novo synthesis of the AP-1 members JunD and Fra-2, which bind to CLE0. The amount of IL5 produced correlates with the production of the AP-1 complex, suggesting a key role in IL5 expression. The formation of the AP-1 complex is essential, but the rate-limiting step is the synthesis of AP-1, especially Fra-2. This provides an explanation for the sensitivity of IL5 to protein synthesis inhibitors and a mechanism for the specific induction of IL5 compared with other cytokines.

Highlights

Primarily mediated by T-lymphocytes, which produce IL5 after activation [5]
This study has utilized the activation of different transcription factors by different stimuli in a human T-cell line to study the role of conserved lymphokine element 0 (CLE0) in the specific induction of IL5
CHX Inhibits IL5 but Not IL4 messenger RNA (mRNA) Synthesis—To investigate whether the activation of IL5 transcription in PER-117 cells was sensitive to inhibitors of protein synthesis, cells were treated with CHX

Summary

EXPERIMENTAL PROCEDURES

Cell Culture, Stimulation of Cells, and IL5 Measurements—PER-117 cells [29] were grown in RPMI 1640 medium supplemented with 7.5% fetal calf serum (Trace), 100 mM Eagle’s nonessential amino acid solution (Invitrogen), 1 mM sodium pyruvate (Invitrogen), 2 mM L-glutamine (Sigma), 75 mM monothioglycerol (Sigma), and 10 mM Hepes, pH 7.3 (Invitrogen)). The PCR mixture contained 1 ␮l of cDNA, 1.0 ␮M each primer, 0.5 mM MgCl2 (2.5 mM for IL4), 0.2 mM deoxynucleotide triphosphates, and 1 unit Thermus thermophilus DNA polymerase (Promega) in Mg2ϩ-free reaction buffer. Protein-DNA binding reactions, and polyacrylamide gel electrophoresis were performed as described [33]. Antisense Co-transfection Experiments—The pCR2.1hIL5p plasmid was used to isolate a 553-bp hIL5 promoter fragment. 10 ␮g of both antisense construct and hIL5 reporter construct DNA were electroporated at 960 microfarads and 280 V into 107 PER-117 cells in 400 ␮l of growth media using the Bio-Rad gene pulser. After an additional incubation period of 16 h, the cells were harvested and resuspended in 100 ␮l of reaction buffer containing 50 mM Tris-HCl pH 7.8, 15 mM MgSO4, 33.3 mM dithiothreitol, 0.1 mM EDTA, 250 ␮M lithium-CoA (Sigma), 500 ␮M sodium luciferin (Molecular Probes), and 0.5% Triton X-100. Luciferase activity was measured in a Victor 1420 multilabel reader (Wallac, Finland)

RESULTS

DISCUSSION

Methods