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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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Stimuli Bypassing LAT Deficiency in J.CaM2 Reduce Elevated RAG-1 Expression(A) LAT-deficient J.CaM2 cells were stimulated with PMA or PDBu and analyzed for RAG-1 mRNA expression by Northern blot analysis. In (A), (B), and (C), the same blot was hybridized with a fragment encoding the constant region of TCRα and with β-actin to control for stimulation and loading.(B) Analysis of PMA-induced reduction of RAG-1 expression levels in absence of protein synthesis blocked by cycloheximide treatment of J.CaM2 cells. Control hybridizations were carried out by hybridization with the constant region of TCRα and with β-actin.(C) Analysis of RAG-1 transcripts in J.CaM2-HM1-1.1 cells stimulated for various time intervals with carbachol to trigger the human muscarinic (HM) receptor subtype I on the surface of these cells.
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pbio.0000053-g002: Stimuli Bypassing LAT Deficiency in J.CaM2 Reduce Elevated RAG-1 Expression(A) LAT-deficient J.CaM2 cells were stimulated with PMA or PDBu and analyzed for RAG-1 mRNA expression by Northern blot analysis. In (A), (B), and (C), the same blot was hybridized with a fragment encoding the constant region of TCRα and with β-actin to control for stimulation and loading.(B) Analysis of PMA-induced reduction of RAG-1 expression levels in absence of protein synthesis blocked by cycloheximide treatment of J.CaM2 cells. Control hybridizations were carried out by hybridization with the constant region of TCRα and with β-actin.(C) Analysis of RAG-1 transcripts in J.CaM2-HM1-1.1 cells stimulated for various time intervals with carbachol to trigger the human muscarinic (HM) receptor subtype I on the surface of these cells.

Mentions: Rag-1 and Rag-2 are expressed in DP thymocytes and turned off during subsequent thymic maturation. This process can be mimicked in thymocytes in vitro by cross-linking the TCR, which leads to activation of PLCγ and subsequent generation of the second messengers diacylglycerol (DAG) and inositol trisphosphate (IP3) (Turka et al. 1991). The same effect is obtained when DAG and IP3 generation are replaced by stimulation with synthetic analogues phorbol myristate acetate (PMA) and ionomycin (Turka et al. 1991). PMA and ionomycin stimulation also bypasses LAT and SLP-76 deficiencies in transcriptional responses in the Jurkat line (Finco et al. 1998; Yablonski et al. 1998). Stimulation of J.CaM2 by PMA or another phorbol ester, phorbol-12,13-dibutyrate (PDBu), resulted in complete downregulation of the elevated RAG-1 transcripts within 12 h (Figure 2A). Induction of a calcium flux by ionomycin, however, did not substantially diminish RAG-1 gene expression in J.CaM2 cells (data not shown). PMA or PDBu stimulation induced a similar decrease in J14 cells (data not shown). The same kinetics of decreased RAG-1 mRNA levels were observed when transcription was blocked in J.CaM2 cells by actinomycin D, demonstrating the instability of RAG-1 mRNA (data not shown). In contrast, transcripts of TCRα were induced following PMA stimulation of J.CaM2 (Figure 2A), as has been described for wild-type Jurkat T cells (Lindsten et al. 1988). The reduction of RAG-1 levels by PMA stimulation was still observed when protein synthesis was blocked with cycloheximide (Figure 2B). The cycloheximide and actinomycin D experiments suggest that signals through the PKC/MAPK pathways are able to repress RAG-1 gene transcription in a relatively direct fashion, leading to loss of detectable RAG-1 mRNA. The fact that RAG-1 expression is elevated in these cells, but can be reduced by a PMA signal, suggests that the LAT/SLP-76 module is essential for a constitutive, suppressive signal that operates, in part, through the downstream signaling pathways. The MAPK pathway can also be activated through seven-transmembrane receptor signaling (Crespo et al. 1994). In J.CaM2 cells, this can be achieved by carbachol treatment of cells that stably express the human muscarinic receptor (J.CaM2-HM1-1.1) (Goldsmith et al. 1989). Carbachol stimulation decreased RAG-1 and increased TCRα expression to the same extent as observed with PMA (Figure 2C). Thus, pharmacologic and physiologic activation of the PKC and MAPK pathways can lead to inhibition of RAG-1 gene expression.


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)

Stimuli Bypassing LAT Deficiency in J.CaM2 Reduce Elevated RAG-1 Expression(A) LAT-deficient J.CaM2 cells were stimulated with PMA or PDBu and analyzed for RAG-1 mRNA expression by Northern blot analysis. In (A), (B), and (C), the same blot was hybridized with a fragment encoding the constant region of TCRα and with β-actin to control for stimulation and loading.(B) Analysis of PMA-induced reduction of RAG-1 expression levels in absence of protein synthesis blocked by cycloheximide treatment of J.CaM2 cells. Control hybridizations were carried out by hybridization with the constant region of TCRα and with β-actin.(C) Analysis of RAG-1 transcripts in J.CaM2-HM1-1.1 cells stimulated for various time intervals with carbachol to trigger the human muscarinic (HM) receptor subtype I on the surface of these cells.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC261890&req=5

pbio.0000053-g002: Stimuli Bypassing LAT Deficiency in J.CaM2 Reduce Elevated RAG-1 Expression(A) LAT-deficient J.CaM2 cells were stimulated with PMA or PDBu and analyzed for RAG-1 mRNA expression by Northern blot analysis. In (A), (B), and (C), the same blot was hybridized with a fragment encoding the constant region of TCRα and with β-actin to control for stimulation and loading.(B) Analysis of PMA-induced reduction of RAG-1 expression levels in absence of protein synthesis blocked by cycloheximide treatment of J.CaM2 cells. Control hybridizations were carried out by hybridization with the constant region of TCRα and with β-actin.(C) Analysis of RAG-1 transcripts in J.CaM2-HM1-1.1 cells stimulated for various time intervals with carbachol to trigger the human muscarinic (HM) receptor subtype I on the surface of these cells.
Mentions: Rag-1 and Rag-2 are expressed in DP thymocytes and turned off during subsequent thymic maturation. This process can be mimicked in thymocytes in vitro by cross-linking the TCR, which leads to activation of PLCγ and subsequent generation of the second messengers diacylglycerol (DAG) and inositol trisphosphate (IP3) (Turka et al. 1991). The same effect is obtained when DAG and IP3 generation are replaced by stimulation with synthetic analogues phorbol myristate acetate (PMA) and ionomycin (Turka et al. 1991). PMA and ionomycin stimulation also bypasses LAT and SLP-76 deficiencies in transcriptional responses in the Jurkat line (Finco et al. 1998; Yablonski et al. 1998). Stimulation of J.CaM2 by PMA or another phorbol ester, phorbol-12,13-dibutyrate (PDBu), resulted in complete downregulation of the elevated RAG-1 transcripts within 12 h (Figure 2A). Induction of a calcium flux by ionomycin, however, did not substantially diminish RAG-1 gene expression in J.CaM2 cells (data not shown). PMA or PDBu stimulation induced a similar decrease in J14 cells (data not shown). The same kinetics of decreased RAG-1 mRNA levels were observed when transcription was blocked in J.CaM2 cells by actinomycin D, demonstrating the instability of RAG-1 mRNA (data not shown). In contrast, transcripts of TCRα were induced following PMA stimulation of J.CaM2 (Figure 2A), as has been described for wild-type Jurkat T cells (Lindsten et al. 1988). The reduction of RAG-1 levels by PMA stimulation was still observed when protein synthesis was blocked with cycloheximide (Figure 2B). The cycloheximide and actinomycin D experiments suggest that signals through the PKC/MAPK pathways are able to repress RAG-1 gene transcription in a relatively direct fashion, leading to loss of detectable RAG-1 mRNA. The fact that RAG-1 expression is elevated in these cells, but can be reduced by a PMA signal, suggests that the LAT/SLP-76 module is essential for a constitutive, suppressive signal that operates, in part, through the downstream signaling pathways. The MAPK pathway can also be activated through seven-transmembrane receptor signaling (Crespo et al. 1994). In J.CaM2 cells, this can be achieved by carbachol treatment of cells that stably express the human muscarinic receptor (J.CaM2-HM1-1.1) (Goldsmith et al. 1989). Carbachol stimulation decreased RAG-1 and increased TCRα expression to the same extent as observed with PMA (Figure 2C). Thus, pharmacologic and physiologic activation of the PKC and MAPK pathways can lead to inhibition of RAG-1 gene expression.

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