Colin J. Sanderson

GATA-3 has dual regulatory functions in human interleukin-5 transcription.

Interleukin-5 (IL-5) is a T-cell cytokine involved in Type 2 diseases and is commonly described as being coordinately regulated with other Type 2 cytokines, such as IL-4 and IL-13. Considering the unique control of eosinophilia by IL-5, such coordinate regulation would be surprising. In fact, the biological specificity of eosinophilia and its control by IL-5 suggests a unique and independent control of IL-5 regulation. In this report we show the binding of GATA-3 to three sites in the human IL-5 promoter in the human T-cell line PER117. The previously identified −70 site and another site at position −152 are shown to positively regulate IL-5 transcription. More importantly, the site located at −400 acts as a powerful repressor of IL-5 transcription with mutagenesis of this site allowing a high level expression of IL-5 without the activation of other factors normally required for IL-5 expression. Whereas GATA-3 has been proposed to be involved in the regulation of the IL-4/IL-5/IL-13 locus, we show here that it has another function in controlling IL-5 transcription that supports the observed unique biological function of this cytokine.

Role of nuclear factor of activated T cells (NFAT) in the expression of interleukin-5 and other cytokines involved in the regulation of hemopoetic cells.

NFAT (nuclear factor of activated T cells) is a transcription factor that plays a role in the regulation of various cytokines, including those involved in the regulation of hemopoetic cells such as granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin-4 (IL4), interleukin-3 (IL3), interleukin-13 (IL13) and interleukin-5 (IL5). In this report we provide a summary of the various locations in the promoters of each of these cytokines where NFAT has been shown or suggested to bind, and at which sites NFAT has been shown to be involved in transcriptional regulation. We also provide experimental data to show that the binding of NFAT to the nucleotides GAA at positions -113 to -111 of the human IL5 promoter is associated with functional activity in human T cells.

Biological and molecular characteristics of interleukin-5 and its receptor

Interleukin-5 (IL5) is a T cell-derived cytokine involved in the pathogenesis of atopic diseases. It specifically controls the production, the activation and the localization of Eosinophils. The Eosinophils are the major cause of tissue damage resulting in the symptoms of asthma and related allergic disorders. T cells purified from bronchoalveolar lavage and peripheral blood of asthmatics secrete elevated amount of IL5. Therefore IL5 emerges to be an attractive target for the generation of new anti-allergic drugs. Agents which inhibit either the production or the activity of IL5 could be expected to ameliorate the pathological effects of the allergic response. A better understanding of the biology of IL5 and the regulation of its expression is, however, a prerequisite for the development of new therapeutic agents. This review covers the major biological, molecular and structural aspects of IL5 research since the identification of this cytokine ten years ago.

How is expression of the interleukin-5 gene regulated?

Eosinophilia is a uniquely specific phenomenon regulated by interleukin‐5 (IL‐5), suggesting specific control for IL‐5 expression. However in eosinophilia IL‐5 is often co‐expressed with other lymphokines such as IL‐4, indicating that common, as well as independent, control mechanisms must exist. IL‐5 gene expression is regulated at the transcriptional level. The molecular analysis of the IL‐5 promoter region reveals the presence of positive regulatory sites that are common to many lymphokine genes. Results from immunosuppression studies suggest that the key control mechanism of IL‐5 regulation may not depend on specific regulatory factors but on how gene expression is activated.

Analysis of the 5′ and 3′UTRs in the post-transcriptional regulation of the interleukin-5 gene

Post-transcriptional regulation is emerging as an important control point in cytokine gene expression. However, the role that it plays in IL-5 gene expression is unclear with some conflicting reports. Here we investigate the importance of post-transcriptional regulation and the role of the 5 and 3 untranslated regions (UTRs) in mIL-5 gene expression. To do this, IL-5 expression from a panel of cDNA constructs was compared. We found it essential to remove the 5 synthetic oligonucleotide tails, introduced during the cloning of the mIL-5 cDNA, when studying IL-5 expression. The presence of these oligo(G) tails acted as potent inhibitors of translation of both SV40 and SP6 transcripts. Furthermore, the length of the tails was found to be critical to the translational efficiency. Taking this into account, we found no evidence to suggest that IL-5 is regulated at the level of mRNA stability or translation efficiency by either the 5 or 3UTR. These results suggest that post-transcriptional control is not a major factor regulating IL-5 expression.

Transcription factor GATA-3 is involved in repression of promoter activity of the human interleukin-4 gene

GATA-3 was shown to bind to two sites of the IL-4 gene promoter in human T-cell lines PER-117 and Jurkat. A motif located in the region of position-860 and responsible for GATA-3 binding was detected for the first time. Mutation or deletion of this site increased the promoter activity. The findings suggest a direct involvement of GATA-3 in regulation of the human IL-4 gene transcription as a repressor of the promoter activity.

Distal regulatory elements play an important role in regulation of the human IL-5 gene

Eosinophil infiltration of the lung is a feature of both allergic and nonallergic asthma, and IL‐5 is the key cytokine regulating the production and activation of these cells. Despite many studies focusing on the IL‐5 promoter in both humans and mice there is as yet no clear picture of how the IL‐5 gene is regulated. The aim of this study was to determine if distal regulatory elements contribute to appropriate regulation of the human IL‐5 (hIL‐5) gene. Activity of the –507/+44 hIL‐5 promoter was compared to expression of the endogenous IL‐5 gene in PER‐117 T cells. The IL‐5 promoter was not sufficient to reproduce a physiological pattern of IL‐5 expression. Further, functional analysis of the 5′ and 3′ intergenic regions revealed a number of novel regulatory elements. We have identified a conserved enhancer located approximately 6.2u2004kb upstream of the hIL‐5 gene. This region contains two potential GATA‐3‐binding sites and increases expression from the hIL‐5 promoter by up to ninefold.

The conserved lymphokine element 0 is a powerful activator and target for corticosteroid inhibition in human interleukin-5 transcription

The role of eosinophilia in allergic disorders indicates hIL-5 as a potential target for therapy. The conservation of hIL-5 gene proximal elements suggests they are important in controlling expression. Corticosteroids are important in the treatment of allergy, and are powerful inhibitors of IL-5 expression. This study aimed at understanding the role of hIL-5 conserved proximal elements, and elucidating the target of corticosteroid activity, in hIL-5 gene expression. Methods used include transient transfection of PBMC and PER117 cells with hIL-5 deletion constructs, EMSA, Western Blotting, and RT-PCR. The conserved proximal CLE0/TATA elements driving a reporter gene gave similar or higher expression than a 500 bp promoter in primary human T cells and a T-cell line. Two and three copies of IL-5 CLE0 upstream of the silent IL-4 minimal promoter gave 30–45 fold increases in expression in forward orientation, but little activity in reverse orientation. Consequently, CLE0 is a powerful activator but not a classical enhancer. Deletion analysis identified CLE0 as the key element in the inhibition of IL-5 reporter constructs by dexamethasone, and RT-PCR analysis indicated that GILZ expression correlated with dexamethasone-induced inhibition of IL-5. Ectopic expression of GILZ, confirmed by western blotting, gave a 90% inhibition of promoter constructs in absence of dexamethasone. CLE0 is a powerful activator sufficient for the inducible expression of IL-5, and functions when moved upstream in a heterologous promoter. CLE0 is also the main target for IL-5 inhibition by dexamethasone, and we present evidence consistent with a role of GILZ in this.

Binding of Octamer Factors to a Novel 3′-Positive Regulatory Element in the Mouse Interleukin-5 Gene

The development of eosinophilia is regulated by interleukin (IL)-5. The biological specificity of eosinophilia suggests a tight and independent regulation of IL-5 expression. A number of regulatory regions in the 5′-end of the IL-5 gene have been described; many of them are involved in the regulation of other genes, and it is not clear how the specific expression of IL-5 is regulated. In this study, we report the finding of a novel 3′-regulatory element. Data base analysis of a 2-kilobase fragment of the 3′-end of the mouse IL-5 gene revealed the presence of a 40-base pair-long repetitive sequence that consists of four direct repeats of ATGAATGA distributed in a symmetrical manner. This sequence, named mouse downstream regulatory element-1 (mDRE1), was shown to be protected in DNase I footprinting assays in vitro. Electrophoretic mobility shift assays using specific antibodies identified the transcription factors Oct-1 and Oct-2 as responsible for the formation of the specific complexes with mDRE1 and nuclear extracts from both EL4 and primary T-cells. Competition electrophoretic mobility shift assays with oligonucleotides containing different numbers of ATGAATGA repeats showed that Oct-1 and Oct-2 bind to different motifs in the mDRE1 sequence. Deletion of mDRE1 from a 9.5-kilobase IL-5 gene construct significantly decreased the expression of the luciferase reporter gene, suggesting that it plays a positive role in the expression of the IL-5 gene.

Identification of residues involved in binding of IL5 to βcom using βIL3 and βcom chimeras

In mice there are two forms of the beta chain used in the IL3 receptor system, βcom and βIL3. βcom is used by the IL3, IL5 and GM‐CSF receptors whereas βIL3 is only used in the IL3 receptor. In this work an assay was developed to identify residues of βIL3 that restrict IL5 activity. It was found that such residues reside within the 2nd CRM of the molecule. Furthermore, when residues in the βIL3 B′‐C′ loop were replaced with βcom sequence a form of βIL3 was produced that was able to respond to IL5. This region is also responsible for IL3 binding to βIL3 in the absence of alpha chain. It is therefore an important structural motif of βcom and βIL3 responsible for both ligand interaction and specificity.