Induction of the early growth response (Egr) family of transcription factors during thymic selection

Abstract

There is little known about the regulation of gene expression during TCR-mediated differentiation of immature CD4+8+ (double positive) thymocytes into mature T cells. Using the DPK CD4+8+ thymocyte precursor cell line, we demonstrate that the early growth response-1 gene (Erg-1), encoding a zinc finger transcription factor, is rapidly upregulated after TCR stimulation. We also report that Egr-1 is expressed by a subset of normal double positive thymocytes in the thymic cortex, as well by a majority of medullary single positive thymocytes. Expression of Egr-1 is dramatically reduced in the thymus of major histocompatibility complex knockout mice, but can be induced by anti-CD3 antibody stimulation of isolated thymocytes from these animals. These and other data suggest that high level expression of Egr-1 in the thymus is a consequence of selection. A similar pattern of expression is found for family members Egr-2 and Egr-3. Using the DPK cell line, we also demonstrate that expression of Egr-1, 2, and 3 is dependent upon ras activation, as is the initiation of differentiation to a single positive cell. In contrast, the calcineurin inhibitor cyclosporin A, which inhibits DPK cell differentiation as well as positive selection, inhibits expression of Egr-2 and Egr-3, but not Egr-1. The identification of the Egr family in this context represents the first report of a link between the two known signaling pathways involved in positive selection and downstream transcriptional regulators.

Figure 1

The Egr-1 gene is rapidly induced after TCR-mediated activation of the DPK double positive cell line. (A) Total RNA isolated from DPK cells cultured with immobilized anti-CD3ε mAb for the indicated times (shown in hours), was subjected to RT-PCR analysis using Egr-1 or CD4 primers. (B) Electrophoretic mobility shift assay using nuclear lysates prepared from DPK cells 8 h after activation by immobilized antiCD3ε mAb. Probes contained a single Egr-1 binding site (left) or overlapping Egr-1 and Sp1 sites (right). (C) DPK cells were cultured with DCEK-ICAM fibroblast antigen presenting cells and 1 μm pigeon cytochrome c peptide for the indicated times. Total RNA was isolated and subjected to a competitive RT-PCR assay (see Materials and Methods). Note the different scales for Egr-1 and CD4 mRNA expression.

Figure 2

DPK cell differentiation and Egr-2,3 mRNA induction is cyclosporin A sensitive, while Egr-1 mRNA induction is cyclosporin A resistant. (A, B) DPK cells were cultured with DCEK-ICAM fibroblast antigen presenting cells and 2 μM pigeon cytochrome c peptide in the presence or absence of 100 ng/ml cyclosporin A, or appropriate dilution of solvent (DMSO) as indicated. Cells were harvested and stained for CD69 after 1 or 3 d in culture (A) or stained for CD4 and CD8 after 3 d in culture (B). (C) RT-PCR analysis of total RNA derived from DPK cells activated for 6 h with immobilized anti-CD3ε mAb in the presence or absence of 300 ng/ml cyclosporin A, using Egr-1, Egr-2 (Krox-20), CD4, CD69 or Egr-3 primers. (*) Also shown for the indicated samples is the relative level of Egr-1 cDNA normalized to expression of CD4 cDNA as determined by competitive RT-PCR assay. The identity of the lower major band in Egr-3 RT-PCR was verified by sequencing.

Figure 3

Expression of a dominant negative mutant of p21ras blocks DPK cell differentiation. (A) RT-PCR assay of N-ras expression in DPK cells or thymocytes derived from wild-type or MHC-deficient mice. (B) Cell lysates from DPK cells and four independent lines that express Ha-ras N17 were analyzed by Western blot and probed with anti-ras antibody. Endogenous p21ras is not visible in this exposure. (C) DPK or 17N4 cells were cultured with DCEK-ICAM fibroblast antigen presenting cells in the presence (bold lines) or absence (thin lines) of 2 μM pigeon cytochrome c peptide. After 3 d of culture, cells were collected, stained with mAb to CD69 and analyzed by flow cytometry. (D) DPK or 17N4 cells were cultured as in (C) except that cells were harvested on day 1 or 3 as indicated and stained with anti-CD4 and anti-CD8 mAbs. Shown are the percentages of CD4⁺8^lo/- DPK cells in the designated regions.

Figure 3

Expression of a dominant negative mutant of p21ras blocks DPK cell differentiation. (A) RT-PCR assay of N-ras expression in DPK cells or thymocytes derived from wild-type or MHC-deficient mice. (B) Cell lysates from DPK cells and four independent lines that express Ha-ras N17 were analyzed by Western blot and probed with anti-ras antibody. Endogenous p21ras is not visible in this exposure. (C) DPK or 17N4 cells were cultured with DCEK-ICAM fibroblast antigen presenting cells in the presence (bold lines) or absence (thin lines) of 2 μM pigeon cytochrome c peptide. After 3 d of culture, cells were collected, stained with mAb to CD69 and analyzed by flow cytometry. (D) DPK or 17N4 cells were cultured as in (C) except that cells were harvested on day 1 or 3 as indicated and stained with anti-CD4 and anti-CD8 mAbs. Shown are the percentages of CD4⁺8^lo/- DPK cells in the designated regions.

Figure 4

Expression of Egr gene family is dependent upon ras signaling pathways. (A) Competitive RT-PCR was used to compare expression of Egr-1 and CD4 genes in DPK or 17N4 cells activated for 6 h with immobilized anti-CD3ε mAb. (B) RT-PCR analysis of total RNA derived from DPK or 17N4 cells activated for 6 h with immobilized anti-CD3ε mAb. Independent PCR reactions using Egr-2 (Krox-20), CD4, or Egr-3 primers were performed.

Figure 5

Egr-1 mRNA and DNA binding activity in the thymus is MHC dependent. (A) Thymocytes derived from wild-type or MHCdeficient mice were two color-stained for CD4 and CD8. (B) Competitive RT-PCR was used to determine the level of expression of CD4 and Egr-1 genes in thymocytes derived from wild-type or MHC-deficient mice. (C) Electrophoretic mobility shift assay using nuclear lysates prepared from freshly isolated thymocytes derived from wild-type or MHCdeficient mice using a probe containing an Egr-1 binding site. Nuclear extracts derived from 5 × 10⁵ cells containing equivalent amounts of protein were used in binding reactions. In some instances as indicated, binding reactions contained anti-Egr-1 antibody or normal rabbit serum (NRS). For comparison, a binding reaction containing recombinant Egr-1 is shown.

Figure 6

Expression of Egr-1 mRNA in double positive thymocytes. (A) Total thymocytes and CD4⁺8⁺ thymocytes (isolated by cell sorting, 95% DP) derived from the same animal, were assayed for expression of Egr and CD4 mRNA by competitive RT-PCR. Shown is the relative level of Egr-1 cDNA in the sample, normalized to the level of CD4 cDNA. (B) Total thymocytes derived from an MHC-deficient mouse were cultured with hamster immunoglobulin-coated or anti-CD3ε mAbcoated beads for 90 min before determination of Egr-1 and CD4 gene expression as in Fig. 1 C.

Figure 7

Expression of Egr-2 and Egr-3 mRNA in thymocytes is MHC dependent. Total RNA prepared from freshly isolated wild-type or MHC knockout thymocytes was subjected to RT-PCR analysis using CD4, Egr-2 (Krox-20), or Egr-3 primers as indicated.

Figure 8

Expression of Egr-1 protein in the thymus. Thin sections of normal thymus (A–C) or MHC knockout thymus (D) were fixed in formaldehyde and stained with a specific rabbit anti-Egr-1 peptide antiserum (A, C, D) or the same antibody preincubated with specific peptide (B). Regions of cortex (C) and medulla (M) are indicated. Sections were counterstained with hematoxylin and photographed at ×20 (A, B) or ×40 (C, D). C shows a magnification of the same section photographed in A.

Figure 9

Expression of Egr-1 protein in thymocyte subsets. Thymocytes from a young adult mouse were 4-color stained for expression of CD4, CD8, Egr-1, and CD69 or CD3, and analyzed by FACS^® as described in Materials and Methods. Indicated in the dot plots are the percentage of thymocytes within each quadrant, or in parenthesis (upper right dot plot), the percentage of Egr-1⁺ thymocytes within each thymocyte subset. Where indicated, staining is shown for gated populations of thymocytes (either Egr-1⁺thymocytes as shown in histogram, or CD4⁺8⁺ thymocytes). Similar results were obtained from three other individual animals.