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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 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+8lo/- DPK cells in the designated regions.
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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+8lo/- DPK cells in the designated regions.

Mentions: As shown in Fig. 3 A, N-ras mRNA can be detected in normal thymocytes, thymocytes derived from MHC-deficient mice (>90% double positive, Fig. 2 A), and DPK cells. This is consistent with previous reports of expression of both N-ras and K-ras in double positive thymocytes (53). To study the role of p21ras in Egr gene expression in immature T cells, retroviral mediated gene transfer was used to express a dominant negative mutant of p21ras, Haras N17 (with an asparagine substituted for serine at position 17) in DPK cells. This mutant blocks endogenous ras function by competing for guanine nucleotide exchange proteins, thereby preventing formation of ras-GTP complexes (42). Fig. 3 B shows expression of Ha-ras N17 in four independent DPK cell transformants that express high levels of this dominant negative mutant. The results obtained with line 17N4 are described here, although all four cell lines had similar responses.


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 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+8lo/- DPK cells in the designated regions.
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Related In: Results  -  Collection

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

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+8lo/- DPK cells in the designated regions.
Mentions: As shown in Fig. 3 A, N-ras mRNA can be detected in normal thymocytes, thymocytes derived from MHC-deficient mice (>90% double positive, Fig. 2 A), and DPK cells. This is consistent with previous reports of expression of both N-ras and K-ras in double positive thymocytes (53). To study the role of p21ras in Egr gene expression in immature T cells, retroviral mediated gene transfer was used to express a dominant negative mutant of p21ras, Haras N17 (with an asparagine substituted for serine at position 17) in DPK cells. This mutant blocks endogenous ras function by competing for guanine nucleotide exchange proteins, thereby preventing formation of ras-GTP complexes (42). Fig. 3 B shows expression of Ha-ras N17 in four independent DPK cell transformants that express high levels of this dominant negative mutant. The results obtained with line 17N4 are described here, although all four cell lines had similar responses.

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