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

Shao H, Kono DH, Chen LY, Rubin EM, Kaye J - J. Exp. Med. (1997)

Bottom Line: A similar pattern of expression is found for family members Egr-2 and Egr-3.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, The Scripps Research Institute, La Jolla, California 92037, USA.

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.

Show MeSH
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.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2196139&req=5

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.

Mentions: These results suggested that activation of the Egr-1 gene occurred during MHC-dependent selection of double positive thymocytes. However, the data were also consistent with the possibility that Egr-1 was upregulated only in mature single positive thymocytes, subsequent to positive selection. To distinguish these possibilities we analyzed the expression of Egr-1 in purified double positive thymocytes derived from normal mice. Given the rapid induction of Egr-1 mRNA in DPK cells (Fig. 1) one would predict that if Egr-1 was induced during thymic selection, it would be expressed before loss of the double positive phenotype. This was found to be the case, and a significant amount of Egr-1 mRNA was detected in double positive thymocytes isolated by cell sorting (Fig. 6 A). Isolated double positive thymocytes also expressed Egr-1 DNA binding activity, as did CD4 single positive thymocytes (not shown). To demonstrate that the Egr-1 gene could be induced by TCR activation in double positive thymocytes, thymocytes derived from MHC-deficient mice were stimulated with immobilized anti-CD3ε mAb. As shown in Fig. 6 B, TCR activation results in rapid upregulation of Egr-1 mRNA in these cells. In contrast, Egr-1 is not upregulated in thymocytes derived from MHC-deficient mice upon treatment in culture with doses of dexamethasone or ionizing radiation that induce cell death (not shown).


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

Shao H, Kono DH, Chen LY, Rubin EM, Kaye J - J. Exp. Med. (1997)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196139&req=5

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.
Mentions: These results suggested that activation of the Egr-1 gene occurred during MHC-dependent selection of double positive thymocytes. However, the data were also consistent with the possibility that Egr-1 was upregulated only in mature single positive thymocytes, subsequent to positive selection. To distinguish these possibilities we analyzed the expression of Egr-1 in purified double positive thymocytes derived from normal mice. Given the rapid induction of Egr-1 mRNA in DPK cells (Fig. 1) one would predict that if Egr-1 was induced during thymic selection, it would be expressed before loss of the double positive phenotype. This was found to be the case, and a significant amount of Egr-1 mRNA was detected in double positive thymocytes isolated by cell sorting (Fig. 6 A). Isolated double positive thymocytes also expressed Egr-1 DNA binding activity, as did CD4 single positive thymocytes (not shown). To demonstrate that the Egr-1 gene could be induced by TCR activation in double positive thymocytes, thymocytes derived from MHC-deficient mice were stimulated with immobilized anti-CD3ε mAb. As shown in Fig. 6 B, TCR activation results in rapid upregulation of Egr-1 mRNA in these cells. In contrast, Egr-1 is not upregulated in thymocytes derived from MHC-deficient mice upon treatment in culture with doses of dexamethasone or ionizing radiation that induce cell death (not shown).

Bottom Line: A similar pattern of expression is found for family members Egr-2 and Egr-3.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, The Scripps Research Institute, La Jolla, California 92037, USA.

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.

Show MeSH