Abstract
The early growth response gene product Egr-1 has been shown to have great impact on growth, proliferation, and differentiation in a wide variety of cells, including T cells. In this study, we show that Egr-1 is rapidly induced upon T cell stimulation and is expressed predominantly in T helper type 2 (Th2) compared with type 1 (Th1) cells. We further investigate the role of Egr-1 in regulation of the Th2 cytokine interleukin-4 (IL-4) expression. IL-4 is a key Th2 cytokine that regulates humoral immunity and also causes allergic inflammation. Regulation of IL-4 gene transcription in Th2 cells has been shown to be controlled by multiple T cell receptor (TCR)-induced transcription factors. However, only a few transcription factors were shown to be selectively induced in differentiated Th2 cells in response to TCR stimulation. Chromatin immunoprecipitation analysis demonstrates that Egr-1 binds to the IL-4 promoter in vivo upon T cell stimulation. Ectopic expression of Egr-1 enhances endogenous IL-4 mRNA expression and elevates IL-4 promoter activity. We also show that Egr-1, nuclear factor of activated T cell, and NF-kappaB cooperatively bind to an NFAT/NF-kappaB-overlapping IL-4 enhancer element and activate the IL-4 promoter synergistically. Furthermore, we show that antisense oligonucleotides that knock down Egr-1 expression attenuate IL-4 transcription. Our study provides the first evidence that Egr-1 protein is differentially expressed in Th1 and Th2 cells and is involved in the acute phase of the IL-4 transcription in response to TCR stimulation.
Highlights
Interleukin (IL)2-4 plays a pivotal role in the differentiation of T helper type 2 (Th2) cells that secrete IL-4, IL-5, and IL-13 and in the development of humoral immunity [1,2,3]
We show that early growth response protein (Egr)-1 is rapidly induced upon T cell stimulation and is expressed predominantly in T helper type 2 (Th2) compared with type 1 (Th1) cells
We show that Egr-1, nuclear factor of activated T cell, and NF-B cooperatively bind to an NFAT/NF-B-overlapping IL-4 enhancer element and activate the IL-4 promoter synergistically
Summary
Cell Lines and Cell Culture—The cell lines used in this study were the human T cell leukemia cell line Jurkat, the mouse Th1 clones Cl29 and B10BI, and the mouse Th2 clones D10G4.1 and L1/1. After removing the cell debris by centrifugation at 13,000 rpm for 30 min, equal amounts of proteins were separated by SDS-PAGE, blotted onto a nitrocellulose membrane (Amersham Biosciences), and blocked with 5% bovine serum albumin in 0.05% Tween 20/phosphate-buffered saline. The sequences of primers and fluorescent-labeled probes for TaqMan quantitative real time PCR are as follows. The DNA-binding reactions between protein and 32P-labeled probe were performed on ice for 30 min. DNA Transfection— 4 ϫ 106/ml Jurkat T cells in RPMI 1640 medium, 8 g of the reporter plasmid, and 2– 6 g of each indicated expression plasmid were electroporated using a Bio-Rad Gene Pulser set at 960 microfarads, 240 V. The transfection efficiency was more than 80% as controlled by transfection with an expression vector containing the green fluorescent protein (pCMVEGFPspectrin) and assessed by fluorescence-activated cell sorter.
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