Regulatory Mechanisms of Human T Cell IL–5 Synthesis: Differential Roles of the Proximal Promoter–Binding Proteins in IL–5 Gene Transcription

Abstract

Background: IL–5 is crucially involved in eosinophilic inflammation. We have previously reported that IL–5 synthesis by atopic and nonatopic asthmatics is significantly enhanced compared to control subjects. T cell IL–5 synthesis is regulated at the transcriptional level. The proximal human IL–5 promoter (–62 to –46) homologous to the conserved lymphokine element 0 (CLE0) is essential for activation–induced gene transcription by Th cells. Methods: Luciferase reporter analysis and gel shift analysis were performed. Results: The CLE0 homologous element is the overlapping binding site for a constitutive and an inducible binding factor. Site–directed mutagenesis successfully differentiated the two bindings, and revealed that the transcriptional induction was ascribed to the inducible binding, while the constitutive binding was rather inhibitory. A mutant element which lost the constitutive binding, but retained the inducible binding, exerted 3 times more transcriptional activity compared to the wild–type element. In contrast, another mutant element which lost the inducible binding and retained the constitutive binding exhibited no transcriptional induction. Gel shift analysis was performed to clarify that the inducible binding was more prominent and the constitutive binding was less in IL–5 producing Th clones compared to IL–5–nonproducing clones. Conclusion: The ratio of the inducible/constitutive binding to the CLE0 homologous element may determine the capacity of human Th cells to transcribe the IL–5 gene.

Transcriptional Regulation of IL-5 Gene by Nontransformed Human T Cells Through the Proximal Promoter Element.

“…Significant induction of luciferase activity was not detected by transfecting IL-5 low-and nonproducing Con A blast lymphocytes with these three constructs (data not shown), consistent with the lower transcriptional induction of PIL-5(-5 1 l )Luc compared with IL-5 high-producing Con A blast lymphocytes ( Table 2). The results are consistent with our previous findings obtained using human T cell hybridomas that transcribe IL-5 gene upon activation (33), and further extend that the transcriptional regulation through the proximal promoter element is essential for the nontransformed humanT cells which might work in asthmatic patients in vivo.…”

“…Upon stimulation, the inducible band with a faster mobility became detectable (lane 2). The CLE0mt6oligonucleotide competed out the faster migrating complex (lane 4), whereas the CLE0mt7 oligonucleotide competedout the slower migrating complex (lane 6), thus confirming these mutagenesis effects previously reported by us (33). The proximal promoter binding complexes were next characterized among IL-5 high-, low-, and non-producing Con A blast lymphocytes.…”

“…To elucidate the role of two complexes binding to the proximal promoter element of human IL-5 gene, 3xCLE0-tk-Luc, 3xCLE0mt6-tk-Luc, and 3xCLE0mt7-tk-Luc constructs were transfected into the IL-5 high-producing Con A blast lymphocytes, and the resultant transcriptional induction was analyzed. As previously reported, CLE0mt6 oligonucleotide retains the inducible binding (AP-1) and loses the constitutive binding (Oct-1), whereas CLEmt?oligonucleotide loses the inducible binding and retains the constitutive binding (33). A representative result of three independent experiments is shown in Fig.…”

“…The constitutive band was identified as Oct-1 molecule, whereas the inducible band comprised ofAP-1 family proteins. Serial 2 bp mutagenesis introduced in this element successfully differentiated the two binding complexes (33). The mutant element which lost constitutive binding but retained inducible binding exerted 3 times more transcriptional activity compared to the wild type element.…”

Transcriptional regulation of the IL-5 gene in peripheral T cells of asthmatic patients

“…A variety of transcription factors and elements have also been reported to control the human IL‐5 gene. However, in most of these studies human and murine cell lines have been used [21–28]. These transformed cell lines have been standard materials for the study of transcriptional regulation, whereas they may not represent the intracellular and nuclear events that occur physiologically in T cells of allergic subjects.…”