Early growth response-1 is required for CD154 transcription

Abstract

CD154 (CD40 ligand) expression on CD4 T cells is normally tightly controlled, but abnormal or dysregulated expression of CD154 has been well documented in autoimmune diseases, such as systemic lupus erythematosus. Beyond regulation by NFAT proteins, little is known about the transcriptional activation of the CD154 promoter. We identified a species-conserved purine-rich sequence located adjacent to the CD154 transcriptional promoter proximal NFAT site, which binds early growth response (Egr) transcription factors. Gel shift assays and chromatin immunoprecipitation assays reveal that Egr-1, Egr-3, and NFAT1 present in primary human CD4 T cells are capable of binding this combinatorial site in vitro and in vivo, respectively. Multimerization of this NFAT/Egr sequence in the context of a reporter gene demonstrates this sequence is transcriptionally active upon T cell activation in primary human CD4 T cells. Overexpression of Egr-1, but not Egr-3, is capable of augmenting transcription of this reporter gene as well as that of an intact CD154 promoter. Conversely, overexpression of small interfering RNA specific for Egr-1 in primary human CD4 T cells inhibits CD154 expression. Similarly, upon activation, CD154 message is notably decreased in splenic CD4 T cells from Egr-1-deficient mice compared with wild-type controls. Our data demonstrate that Egr-1 is required for CD154 transcription in primary CD4 T cells. This has implications for selective targeting of Egr family members to control abnormal expression of CD154 in autoimmune diseases such as systemic lupus erythematosus.

FIGURE 1

NFAT and Egr cooperate to augment CD154 transcription. Primary human CD4 T cells were transiently transfected (see Materials and Methods) with firefly luciferase reporter plasmids directed by duplications of the hCD154 proximal promoter AP-1/NFAT sites or NFAT/Egr sites (see Materials and Methods for construct design). Cells were then rested (media) or stimulated with phorbol ester and ionomycin (P+I) for 6 h, and cell lysates were analyzed for luciferase activity. Firefly luciferase activity was corrected for transfection efficiency with Renilla luciferase activity from a cotransfected control plasmid. Results are representative of one of three similar experiments. Diagrams depicting the relative positions of the neighboring AP-1, NFAT, and Egr binding sites in the proximal hCD154 promoter, and the combination of transcription factor binding sites in the reporter constructs, are shown at the right.

FIGURE 2

Egr-1 binds the proximal hCD154 promoter in vitro. Nuclear extracts from resting (A, lane 1) or polyclonally activated Jurkat (A) and primary human CD4 T cells (B and C) were incubated with an oligo-nucleotide probe encompassing the hCD154 proximal promoter NFAT and Egr sites. This resulted in the formation of two complexes (arrows) in activated extracts as seen in A (lane 2), B (lane 2), and C (lane 1). Both complexes were partially inhibited by excess self, cold probe (B, lane 3; C, lane 2). The upper complex (C, *) was specifically inhibited by an anti-Egr-1 Ab (A, lane 4; C, lane 3) but not by Abs to Egr-2 (A, lane 5; C, lane 4), Egr-3 (A, lane 6; C, lane 5), or STAT1 (A, lane 7). Anti-NFAT1 also inhibited upper complex formation in Jurkat extracts (A, lane 2) but primarily blocked lower complex formation in primary CD4 T cell extracts (B, lane 4).

FIGURE 3

Egr-1 and NFAT1 bind the proximal hCD154 promoter in vivo. Primary human CD4 T cells were activated for 2 h with phorbol ester and ionomycin and underwent chromatin immunoprecipitation with iso-type control and anti-Egr-1, -Egr-2, -Egr-3, and -NFAT1 Abs as described in Materials and Methods. A, Immunoprecipitated DNA was amplified and quantified by real-time PCR using primers and a probe specific to the proximal hCD154 promoter (see Materials and Methods). Samples were analyzed in triplicate for the test (red, green, and yellow curves) and control (pink, blue, and purple curves) Abs. B, Fold differences in binding relative to control Abs were calculated (see Materials and Methods), and averages and SEs of the means from five independent experiments are depicted for each test transcription factor analyzed.

FIGURE 4

Egr-1 augments CD154 transcription. Primary human CD4 T cells were transiently transfected with a firefly luciferase reporter plasmid driven by the proximal hCD154 promoter (10). A, The cells were cotransfected with expression plasmids for Egr-1, Egr-2, Egr-3, or control (empty vector). After a brief rest, the cells were activated for 6 h with medium alone or phorbol ester and ionomycin (P+I). Cell lysates were analyzed in duplicate for luciferase activity and corrected for transfection efficiency with a cotransfected Renilla luciferase control plasmid. Results are representative of one of five similar experiments. B, Primary CD4 T cells were transfected with expression plasmids for Egr-1, NFAT1, Egr-1+NFAT1, or controls (empty vectors). The cells were activated for 6 h with P+I, and cell lysates were analyzed for luciferase activity. Results are representative of one of four similar experiments.

FIGURE 5

Inhibition of endogenous Egr-1 lowers CD154 surface expression. Primary human CD4 T cells were transiently transfected with increasing amounts of expression plasmids for Egr-1 siRNA (no. 590; see Materials and Methods) or scrambled sequence control. Cells were then activated for 2 h with phorbol ester and ionomycin and analyzed for CD154 surface expression (A) by flow cytometry or Egr-1 mRNA (B) by real-time RT-PCR. Results are representative of one of six similar experiments. The black filled area under the curve represents isotype control Ab staining. The unfilled curves depict CD154 Ab staining in the presence of 7.5 μg of Egr-1 siRNA (bottom) or scrambled control (top) expression plasmids. The mean fluorescence intensities (MFI) of CD154 expression are noted.

FIGURE 6

Egr-1 is required for CD154 message induction. T cell transgenic primary murine CD4 T cells from Egr-1 WT (+/+) or knockout (−/−) mice were rested (−) or stimulated (+) in vitro for 5 h with the appropriate peptide/MHC combination (see Materials and Methods). mRNA was isolated and analyzed by Northern blot with a probe specific to murine CD154 cDNA. The blot was then stripped and probed for the GAPDH housekeeping gene as a loading control. Results are representative of one of two similar experiments.