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T cell receptor-independent basal signaling via Erk and Abl kinases suppresses RAG gene expression.

Roose JP, Diehn M, Tomlinson MG, Lin J, Alizadeh AA, Botstein D, Brown PO, Weiss A - PLoS Biol. (2003)

Bottom Line: This TCR-like pathway results in constitutive low-level activity of Erk and Abl kinases.Inhibition of Abl by the drug STI-571 or inhibition of signaling events upstream of Erk increases RAG-1 expression.Our data suggest that physiologic gene expression programs depend upon tonic activity of signaling pathways independent of receptor ligation.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of California, San Francisco, USA.

ABSTRACT
Signal transduction pathways guided by cellular receptors commonly exhibit low-level constitutive signaling in a continuous, ligand-independent manner. The dynamic equilibrium of positive and negative regulators establishes such a tonic signal. Ligand-independent signaling by the precursors of mature antigen receptors regulates development of B and T lymphocytes. Here we describe a basal signal that controls gene expression profiles in the Jurkat T cell line and mouse thymocytes. Using DNA microarrays and Northern blots to analyze unstimulated cells, we demonstrate that expression of a cluster of genes, including RAG-1 and RAG-2, is repressed by constitutive signals requiring the adapter molecules LAT and SLP-76. This TCR-like pathway results in constitutive low-level activity of Erk and Abl kinases. Inhibition of Abl by the drug STI-571 or inhibition of signaling events upstream of Erk increases RAG-1 expression. Our data suggest that physiologic gene expression programs depend upon tonic activity of signaling pathways independent of receptor ligation.

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Erk and Abl Kinases Transduce Repressive Signals That Control RAG Gene Expression(A) Northern blot analysis for RAG-1 gene expression in STI-571-treated Jurkat T cells and comparison to PP2- or Ro-318220-treated samples.(B) Wild-type thymocytes were treated with the indicated inhibitors for 20 h and analyzed for Rag-1 expression.(C) Northern blot analysis of Rag-1 expression in inhibitor-treated TCR transgenic OT-I thymocytes or wild-type thymocytes of littermate controls. The relative expression level of Rag-1 was calculated from two independent experiments, and the mean expression and standard deviation (SD) are indicated.(D) Western blot analysis using RIPA lysates of the indicated cell lines. Protein levels of phosphorylated Erk-1 and Erk-2, Erk-1 and Erk-2, and c-Abl were determined in 4 × 106 resting cells per sample.(E) Analysis of phospho-Erk-1 and Erk-2 levels in Jurkat T cells treated for 24 h with the indicated inhibitors prior to RIPA lysis. Equal loading is indicated by Erk-1 and Erk-2 levels determined by stripping and reprobing the same blot.
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pbio.0000053-g005: Erk and Abl Kinases Transduce Repressive Signals That Control RAG Gene Expression(A) Northern blot analysis for RAG-1 gene expression in STI-571-treated Jurkat T cells and comparison to PP2- or Ro-318220-treated samples.(B) Wild-type thymocytes were treated with the indicated inhibitors for 20 h and analyzed for Rag-1 expression.(C) Northern blot analysis of Rag-1 expression in inhibitor-treated TCR transgenic OT-I thymocytes or wild-type thymocytes of littermate controls. The relative expression level of Rag-1 was calculated from two independent experiments, and the mean expression and standard deviation (SD) are indicated.(D) Western blot analysis using RIPA lysates of the indicated cell lines. Protein levels of phosphorylated Erk-1 and Erk-2, Erk-1 and Erk-2, and c-Abl were determined in 4 × 106 resting cells per sample.(E) Analysis of phospho-Erk-1 and Erk-2 levels in Jurkat T cells treated for 24 h with the indicated inhibitors prior to RIPA lysis. Equal loading is indicated by Erk-1 and Erk-2 levels determined by stripping and reprobing the same blot.

Mentions: We noticed that primary murine thymocytes were more sensitive to apoptosis induced by prolonged exposure to chemical inhibitors. Nevertheless, we were able to determine the effects of the signaling inhibitors on Rag-1 expression in wild-type thymocytes by adding a general caspase inhibitor (Z-DEVD-FMK) to our incubations in order to block apoptosis. Addition of Z-DEVD-FMK by itself had no effect on Rag-1 gene expression, but did improve survival without affecting the relative proportions of the different thymocyte subsets (determined by CD4, CD8, CD25, and CD44 staining; data not shown). As observed in cell lines, inhibition of components of the downstream TCR signaling pathway in total thymocytes significantly increased Rag-1 gene expression (Figure 4C). The inhibitors delineated the same pathway as in the Jurkat T cell and the thymocyte cell lines, including Src kinases, PLCγ1, PKC, PI3K, MEK-1, and calcineurin. The magnitude of induced Rag-1 expression was more moderate in thymocytes than the level observed in our model cell line. The fact that thymocytes in the process of receptor rearrangement already express Rag-1 may account for the smaller induction over the existing level. We therefore treated thymocytes from TCR transgenic OT-I mice with inhibitors in the same manner. OT-I transgenic mice have a thymus containing substantial numbers of DP thymocytes. In absence of the peptide from chicken ovalbumin, these DP thymocytes are positively selected on self-antigens and effectively downregulate Rag expression (McGargill et al. 2000). As a result, the level of Rag-1 expression in thymocytes of these animals is lower than that in wild-type littermate thymi (Figure 5C, lanes 1 and 3). Treatment of OT-1 thymocytes with PP2 or U-0126 led to a strong induction in Rag-1 expression, comparable to the effects observed in Jurkat T cells (Figure 5C). These data argue that detection of constitutive Rag repression in wild-type thymocytes is hindered by normal Rag expression during rearrangement.


T cell receptor-independent basal signaling via Erk and Abl kinases suppresses RAG gene expression.

Roose JP, Diehn M, Tomlinson MG, Lin J, Alizadeh AA, Botstein D, Brown PO, Weiss A - PLoS Biol. (2003)

Erk and Abl Kinases Transduce Repressive Signals That Control RAG Gene Expression(A) Northern blot analysis for RAG-1 gene expression in STI-571-treated Jurkat T cells and comparison to PP2- or Ro-318220-treated samples.(B) Wild-type thymocytes were treated with the indicated inhibitors for 20 h and analyzed for Rag-1 expression.(C) Northern blot analysis of Rag-1 expression in inhibitor-treated TCR transgenic OT-I thymocytes or wild-type thymocytes of littermate controls. The relative expression level of Rag-1 was calculated from two independent experiments, and the mean expression and standard deviation (SD) are indicated.(D) Western blot analysis using RIPA lysates of the indicated cell lines. Protein levels of phosphorylated Erk-1 and Erk-2, Erk-1 and Erk-2, and c-Abl were determined in 4 × 106 resting cells per sample.(E) Analysis of phospho-Erk-1 and Erk-2 levels in Jurkat T cells treated for 24 h with the indicated inhibitors prior to RIPA lysis. Equal loading is indicated by Erk-1 and Erk-2 levels determined by stripping and reprobing the same blot.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC261890&req=5

pbio.0000053-g005: Erk and Abl Kinases Transduce Repressive Signals That Control RAG Gene Expression(A) Northern blot analysis for RAG-1 gene expression in STI-571-treated Jurkat T cells and comparison to PP2- or Ro-318220-treated samples.(B) Wild-type thymocytes were treated with the indicated inhibitors for 20 h and analyzed for Rag-1 expression.(C) Northern blot analysis of Rag-1 expression in inhibitor-treated TCR transgenic OT-I thymocytes or wild-type thymocytes of littermate controls. The relative expression level of Rag-1 was calculated from two independent experiments, and the mean expression and standard deviation (SD) are indicated.(D) Western blot analysis using RIPA lysates of the indicated cell lines. Protein levels of phosphorylated Erk-1 and Erk-2, Erk-1 and Erk-2, and c-Abl were determined in 4 × 106 resting cells per sample.(E) Analysis of phospho-Erk-1 and Erk-2 levels in Jurkat T cells treated for 24 h with the indicated inhibitors prior to RIPA lysis. Equal loading is indicated by Erk-1 and Erk-2 levels determined by stripping and reprobing the same blot.
Mentions: We noticed that primary murine thymocytes were more sensitive to apoptosis induced by prolonged exposure to chemical inhibitors. Nevertheless, we were able to determine the effects of the signaling inhibitors on Rag-1 expression in wild-type thymocytes by adding a general caspase inhibitor (Z-DEVD-FMK) to our incubations in order to block apoptosis. Addition of Z-DEVD-FMK by itself had no effect on Rag-1 gene expression, but did improve survival without affecting the relative proportions of the different thymocyte subsets (determined by CD4, CD8, CD25, and CD44 staining; data not shown). As observed in cell lines, inhibition of components of the downstream TCR signaling pathway in total thymocytes significantly increased Rag-1 gene expression (Figure 4C). The inhibitors delineated the same pathway as in the Jurkat T cell and the thymocyte cell lines, including Src kinases, PLCγ1, PKC, PI3K, MEK-1, and calcineurin. The magnitude of induced Rag-1 expression was more moderate in thymocytes than the level observed in our model cell line. The fact that thymocytes in the process of receptor rearrangement already express Rag-1 may account for the smaller induction over the existing level. We therefore treated thymocytes from TCR transgenic OT-I mice with inhibitors in the same manner. OT-I transgenic mice have a thymus containing substantial numbers of DP thymocytes. In absence of the peptide from chicken ovalbumin, these DP thymocytes are positively selected on self-antigens and effectively downregulate Rag expression (McGargill et al. 2000). As a result, the level of Rag-1 expression in thymocytes of these animals is lower than that in wild-type littermate thymi (Figure 5C, lanes 1 and 3). Treatment of OT-1 thymocytes with PP2 or U-0126 led to a strong induction in Rag-1 expression, comparable to the effects observed in Jurkat T cells (Figure 5C). These data argue that detection of constitutive Rag repression in wild-type thymocytes is hindered by normal Rag expression during rearrangement.

Bottom Line: This TCR-like pathway results in constitutive low-level activity of Erk and Abl kinases.Inhibition of Abl by the drug STI-571 or inhibition of signaling events upstream of Erk increases RAG-1 expression.Our data suggest that physiologic gene expression programs depend upon tonic activity of signaling pathways independent of receptor ligation.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of California, San Francisco, USA.

ABSTRACT
Signal transduction pathways guided by cellular receptors commonly exhibit low-level constitutive signaling in a continuous, ligand-independent manner. The dynamic equilibrium of positive and negative regulators establishes such a tonic signal. Ligand-independent signaling by the precursors of mature antigen receptors regulates development of B and T lymphocytes. Here we describe a basal signal that controls gene expression profiles in the Jurkat T cell line and mouse thymocytes. Using DNA microarrays and Northern blots to analyze unstimulated cells, we demonstrate that expression of a cluster of genes, including RAG-1 and RAG-2, is repressed by constitutive signals requiring the adapter molecules LAT and SLP-76. This TCR-like pathway results in constitutive low-level activity of Erk and Abl kinases. Inhibition of Abl by the drug STI-571 or inhibition of signaling events upstream of Erk increases RAG-1 expression. Our data suggest that physiologic gene expression programs depend upon tonic activity of signaling pathways independent of receptor ligation.

Show MeSH
Related in: MedlinePlus