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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.

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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.
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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.

Mentions: Having determined that DPK cell differentiation was dependent upon ras activation, we sought to determine whether the expression of the Egr gene family was also downstream of this signaling pathway. As shown in Fig. 4 A, induction of Egr-1 mRNA in DPK cells was blocked by expression of the dominant negative mutant of p21ras. Similarly, there was no detectable induction of Egr-2 (Krox-20) or Egr-3 mRNA in these cells after stimulation by anti-CD3ε mAb (Fig. 4 B). This is in contrast to CsA, which differentially affects the expression of the Egr family.


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 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.
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Related In: Results  -  Collection

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

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.
Mentions: Having determined that DPK cell differentiation was dependent upon ras activation, we sought to determine whether the expression of the Egr gene family was also downstream of this signaling pathway. As shown in Fig. 4 A, induction of Egr-1 mRNA in DPK cells was blocked by expression of the dominant negative mutant of p21ras. Similarly, there was no detectable induction of Egr-2 (Krox-20) or Egr-3 mRNA in these cells after stimulation by anti-CD3ε mAb (Fig. 4 B). This is in contrast to CsA, which differentially affects the expression of the Egr family.

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.

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