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Involvement of Egr-1/RelA synergy in distinguishing T cell activation from tumor necrosis factor-alpha-induced NF-kappa B1 transcription.

Cogswell PC, Mayo MW, Baldwin AS - J. Exp. Med. (1997)

Bottom Line: Characterization of the NF-kappa B1 promoter identified an Egr-1 site which was found to be essential for both the PMA/PHA-mediated induction as well as the synergistic activation observed after the expression of the RelA subunit of NF-kappa B and Egr-1.Furthermore, Egr-1 induction was required for endogenous NF-kappa B1 gene expression, since PMA/PHA-stimulated T cell lines expressing antisense Egr-1 RNA were inhibited in their ability to upregulate NF-kappa B1 transcription.Our studies indicate that transcriptional synergy mediated by activation of both Egr-1 and NF-kappa B may have important ramifications in T cell development by upregulating NF-kappa B1 gene expression.

View Article: PubMed Central - PubMed

Affiliation: Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill 27599-7295, USA.

ABSTRACT
NF-kappa B is an important transcription factor required for T cell proliferation and other immunological functions. The NF-kappa B1 gene encodes a 105-kD protein that is the precursor of the p50 component of NF-kappa B. Previously, we and others have demonstrated that NF-kappa B regulates the NF-kappa B1 gene. In this manuscript we have investigated the molecular mechanisms by which T cell lines stimulated with phorbol 12-myristate 13-acetate (PMA) and phytohemagglutin (PHA) display significantly higher levels of NF-kappa B1 encoding transcripts than cells stimulated with tumor necrosis factor-alpha, despite the fact that both stimuli activate NF-kappa B. Characterization of the NF-kappa B1 promoter identified an Egr-1 site which was found to be essential for both the PMA/PHA-mediated induction as well as the synergistic activation observed after the expression of the RelA subunit of NF-kappa B and Egr-1. Furthermore, Egr-1 induction was required for endogenous NF-kappa B1 gene expression, since PMA/PHA-stimulated T cell lines expressing antisense Egr-1 RNA were inhibited in their ability to upregulate NF-kappa B1 transcription. Our studies indicate that transcriptional synergy mediated by activation of both Egr-1 and NF-kappa B may have important ramifications in T cell development by upregulating NF-kappa B1 gene expression.

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Identification of an Egr-1 DNA binding site within the NFκB1 promoter. (A) Nuclear extracts were isolated from CEM cells after a  2 h PMA/PHA stimulation in either the absence or presence of cycloheximide (50 μg/ml, 30 min pretreatment). Nuclear extracts (5 μg) were incubated with a 32P-labeled probe corresponding to the 68-bp Egr-1 site.  Competition assays and antibody supershift experiments were performed  by preincubating nuclear extracts (15 min) with either unlabeled mutant  Egr-1 (mEgr-1) or wild-type Egr-1 oligonucleotide (50 ng total) before  the addition of probe. Lane 1, unstimulated cells; lane 2, PMA/PHA for  2 h; lane 3, PMA/PHA + wild-type Egr-1 oligo; lane 4, PMA/PHA +  mEgr-1 oligo; lane 5, PMA/PHA + Egr-1–specific antibody; lane 6, pretreatment with cycloheximide (30 min) + PMA/PHA for 2 h. SS indicates the position of the Egr-1 + antibody supershift complex; NS identifies nonspecific bands. (B) Nuclear proteins from CEM cells were isolated  after the addition of either PMA/PHA or TNF-α over the indicated time  course and EMSAs were performed using the 32P-labeled Egr-1 site. Note  that the unbound 32P-labeled probe is not shown in both A and B.
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Figure 2: Identification of an Egr-1 DNA binding site within the NFκB1 promoter. (A) Nuclear extracts were isolated from CEM cells after a 2 h PMA/PHA stimulation in either the absence or presence of cycloheximide (50 μg/ml, 30 min pretreatment). Nuclear extracts (5 μg) were incubated with a 32P-labeled probe corresponding to the 68-bp Egr-1 site. Competition assays and antibody supershift experiments were performed by preincubating nuclear extracts (15 min) with either unlabeled mutant Egr-1 (mEgr-1) or wild-type Egr-1 oligonucleotide (50 ng total) before the addition of probe. Lane 1, unstimulated cells; lane 2, PMA/PHA for 2 h; lane 3, PMA/PHA + wild-type Egr-1 oligo; lane 4, PMA/PHA + mEgr-1 oligo; lane 5, PMA/PHA + Egr-1–specific antibody; lane 6, pretreatment with cycloheximide (30 min) + PMA/PHA for 2 h. SS indicates the position of the Egr-1 + antibody supershift complex; NS identifies nonspecific bands. (B) Nuclear proteins from CEM cells were isolated after the addition of either PMA/PHA or TNF-α over the indicated time course and EMSAs were performed using the 32P-labeled Egr-1 site. Note that the unbound 32P-labeled probe is not shown in both A and B.

Mentions: Computer analysis of the SphI/ApoI fragment of the NF-κB1 promoter identified potential DNA-binding motifs for five different transcription factors including AP-1, E2F, Egr-1, NF-κB, and SP1. Of these, NF-κB, Egr-1, and AP-1 are known to display increased transactivation potential after PMA stimulation (2, 15, 21). Since PMA/PHA and TNF-α both activated NF-κB, we excluded this transcription factor as the nuclear factor responsible for differentially regulating the NF-κB1 promoter. In addition, the putative AP-1 site located in the NF-κB1 promoter was not found to affect PMA-induced transactivation of the NF-κB1 promoter (12). To test whether extracts from PMA/PHA-stimulated cells would display Egr-1 binding, EMSAs were performed. As shown in Fig. 2 A, a 32P double stranded oligonucleotide probe corresponding to the 68-bp Egr-1 site within the NF-κB1 promoter bound a nuclear protein which was present in PMA/PHA-stimulated cell extracts (lane 2), but not in unstimulated cell extracts (lane 1). This DNA–protein complex could also be competed with an excess of wild-type unlabeled oligonucleotide (lane 3), but not by equal concentrations of a corresponding mutant Egr-1 site (lane 4). Moreover, this complex contained Egr-1 protein since incubation with an Egr-1–specific antibody completely supershifted the nuclear protein bound to the 32P-probe (lane 5). Pretreatment with cycloheximide, which has been previously demonstrated to block PMA-mediated increases in Egr-1 protein (15), prevented the detection of Egr-1–specific binding (lane 6). Although we do not effectively detect SP1 binding under our EMSA conditions (Fig. 2 A), we observed constitutive SP1 binding in nuclear extracts isolated from CEM cells using a modified binding protocol (data not shown). Since SP1 binding to the −68 site was constitutive and completely independent of cellular stimulation mediated by either PMA/ PHA or TNF-α (data not shown), we chose to focus on the inducible binding of the Egr-1 transcription factor.


Involvement of Egr-1/RelA synergy in distinguishing T cell activation from tumor necrosis factor-alpha-induced NF-kappa B1 transcription.

Cogswell PC, Mayo MW, Baldwin AS - J. Exp. Med. (1997)

Identification of an Egr-1 DNA binding site within the NFκB1 promoter. (A) Nuclear extracts were isolated from CEM cells after a  2 h PMA/PHA stimulation in either the absence or presence of cycloheximide (50 μg/ml, 30 min pretreatment). Nuclear extracts (5 μg) were incubated with a 32P-labeled probe corresponding to the 68-bp Egr-1 site.  Competition assays and antibody supershift experiments were performed  by preincubating nuclear extracts (15 min) with either unlabeled mutant  Egr-1 (mEgr-1) or wild-type Egr-1 oligonucleotide (50 ng total) before  the addition of probe. Lane 1, unstimulated cells; lane 2, PMA/PHA for  2 h; lane 3, PMA/PHA + wild-type Egr-1 oligo; lane 4, PMA/PHA +  mEgr-1 oligo; lane 5, PMA/PHA + Egr-1–specific antibody; lane 6, pretreatment with cycloheximide (30 min) + PMA/PHA for 2 h. SS indicates the position of the Egr-1 + antibody supershift complex; NS identifies nonspecific bands. (B) Nuclear proteins from CEM cells were isolated  after the addition of either PMA/PHA or TNF-α over the indicated time  course and EMSAs were performed using the 32P-labeled Egr-1 site. Note  that the unbound 32P-labeled probe is not shown in both A and B.
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Related In: Results  -  Collection

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Figure 2: Identification of an Egr-1 DNA binding site within the NFκB1 promoter. (A) Nuclear extracts were isolated from CEM cells after a 2 h PMA/PHA stimulation in either the absence or presence of cycloheximide (50 μg/ml, 30 min pretreatment). Nuclear extracts (5 μg) were incubated with a 32P-labeled probe corresponding to the 68-bp Egr-1 site. Competition assays and antibody supershift experiments were performed by preincubating nuclear extracts (15 min) with either unlabeled mutant Egr-1 (mEgr-1) or wild-type Egr-1 oligonucleotide (50 ng total) before the addition of probe. Lane 1, unstimulated cells; lane 2, PMA/PHA for 2 h; lane 3, PMA/PHA + wild-type Egr-1 oligo; lane 4, PMA/PHA + mEgr-1 oligo; lane 5, PMA/PHA + Egr-1–specific antibody; lane 6, pretreatment with cycloheximide (30 min) + PMA/PHA for 2 h. SS indicates the position of the Egr-1 + antibody supershift complex; NS identifies nonspecific bands. (B) Nuclear proteins from CEM cells were isolated after the addition of either PMA/PHA or TNF-α over the indicated time course and EMSAs were performed using the 32P-labeled Egr-1 site. Note that the unbound 32P-labeled probe is not shown in both A and B.
Mentions: Computer analysis of the SphI/ApoI fragment of the NF-κB1 promoter identified potential DNA-binding motifs for five different transcription factors including AP-1, E2F, Egr-1, NF-κB, and SP1. Of these, NF-κB, Egr-1, and AP-1 are known to display increased transactivation potential after PMA stimulation (2, 15, 21). Since PMA/PHA and TNF-α both activated NF-κB, we excluded this transcription factor as the nuclear factor responsible for differentially regulating the NF-κB1 promoter. In addition, the putative AP-1 site located in the NF-κB1 promoter was not found to affect PMA-induced transactivation of the NF-κB1 promoter (12). To test whether extracts from PMA/PHA-stimulated cells would display Egr-1 binding, EMSAs were performed. As shown in Fig. 2 A, a 32P double stranded oligonucleotide probe corresponding to the 68-bp Egr-1 site within the NF-κB1 promoter bound a nuclear protein which was present in PMA/PHA-stimulated cell extracts (lane 2), but not in unstimulated cell extracts (lane 1). This DNA–protein complex could also be competed with an excess of wild-type unlabeled oligonucleotide (lane 3), but not by equal concentrations of a corresponding mutant Egr-1 site (lane 4). Moreover, this complex contained Egr-1 protein since incubation with an Egr-1–specific antibody completely supershifted the nuclear protein bound to the 32P-probe (lane 5). Pretreatment with cycloheximide, which has been previously demonstrated to block PMA-mediated increases in Egr-1 protein (15), prevented the detection of Egr-1–specific binding (lane 6). Although we do not effectively detect SP1 binding under our EMSA conditions (Fig. 2 A), we observed constitutive SP1 binding in nuclear extracts isolated from CEM cells using a modified binding protocol (data not shown). Since SP1 binding to the −68 site was constitutive and completely independent of cellular stimulation mediated by either PMA/ PHA or TNF-α (data not shown), we chose to focus on the inducible binding of the Egr-1 transcription factor.

Bottom Line: Characterization of the NF-kappa B1 promoter identified an Egr-1 site which was found to be essential for both the PMA/PHA-mediated induction as well as the synergistic activation observed after the expression of the RelA subunit of NF-kappa B and Egr-1.Furthermore, Egr-1 induction was required for endogenous NF-kappa B1 gene expression, since PMA/PHA-stimulated T cell lines expressing antisense Egr-1 RNA were inhibited in their ability to upregulate NF-kappa B1 transcription.Our studies indicate that transcriptional synergy mediated by activation of both Egr-1 and NF-kappa B may have important ramifications in T cell development by upregulating NF-kappa B1 gene expression.

View Article: PubMed Central - PubMed

Affiliation: Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill 27599-7295, USA.

ABSTRACT
NF-kappa B is an important transcription factor required for T cell proliferation and other immunological functions. The NF-kappa B1 gene encodes a 105-kD protein that is the precursor of the p50 component of NF-kappa B. Previously, we and others have demonstrated that NF-kappa B regulates the NF-kappa B1 gene. In this manuscript we have investigated the molecular mechanisms by which T cell lines stimulated with phorbol 12-myristate 13-acetate (PMA) and phytohemagglutin (PHA) display significantly higher levels of NF-kappa B1 encoding transcripts than cells stimulated with tumor necrosis factor-alpha, despite the fact that both stimuli activate NF-kappa B. Characterization of the NF-kappa B1 promoter identified an Egr-1 site which was found to be essential for both the PMA/PHA-mediated induction as well as the synergistic activation observed after the expression of the RelA subunit of NF-kappa B and Egr-1. Furthermore, Egr-1 induction was required for endogenous NF-kappa B1 gene expression, since PMA/PHA-stimulated T cell lines expressing antisense Egr-1 RNA were inhibited in their ability to upregulate NF-kappa B1 transcription. Our studies indicate that transcriptional synergy mediated by activation of both Egr-1 and NF-kappa B may have important ramifications in T cell development by upregulating NF-kappa B1 gene expression.

Show MeSH
Related in: MedlinePlus