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

Related in: MedlinePlus

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.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2196139&req=5

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.

Mentions: Egr-1 is one of a family of transcription factors that share a highly conserved DNA binding domain consisting of three zinc finger motifs. To determine whether gene expression of other family members was also upregulated upon activation of immature T cells, we examined gene expression of Krox-20, the murine homologue of human Egr-2 (16, 18), and Egr-3 (20) in DPK cells. Neither Krox20 (Egr-2) nor Egr-3 mRNA was detected by RT-PCR in unactivated DPK cells, while both genes were induced after anti-CD3ε mAb stimulation (Fig. 2 C). Both Krox-20 (Egr-2) and Egr-3 RT-PCR products were sequenced to confirm identity of the expressed genes.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

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.
Mentions: Egr-1 is one of a family of transcription factors that share a highly conserved DNA binding domain consisting of three zinc finger motifs. To determine whether gene expression of other family members was also upregulated upon activation of immature T cells, we examined gene expression of Krox-20, the murine homologue of human Egr-2 (16, 18), and Egr-3 (20) in DPK cells. Neither Krox20 (Egr-2) nor Egr-3 mRNA was detected by RT-PCR in unactivated DPK cells, while both genes were induced after anti-CD3ε mAb stimulation (Fig. 2 C). Both Krox-20 (Egr-2) and Egr-3 RT-PCR products were sequenced to confirm identity of the expressed genes.

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
Related in: MedlinePlus