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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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NF-κB1 gene expression and  promoter analysis in CEM cells after PMA/ PHA or TNF-α stimulation. (A) RNAs  were isolated from CEM cells after addition  of either PMA/PHA (50 ng and 5 μg/ml)  or TNF-α (10 ng/ml) over the time course  indicated. RNAs were detected using a 32Plabeled NF-κB1-specific probe. Transcripts  encoding NF-κB1 were quantitated by densitometric scanning of autoradiograms and  the fold accumulation of mRNAs were calculated after normalization to β-actin. (B)  Schematic of the NF-κB1 promoter depicting the Egr-1 and NF-κB binding sites.  CEM cells were transfected with either the  HS-CAT reporter or with various deletion  constructs (SS-, SpS-, HiS-, and AS-CAT).  18 h after transfection, cells were stimulated  with either PMA/PHA (50 ng and 5 μg/ ml) or TNF-α (10 ng/ml) and CAT activity  was analyzed. Transfection experiments  were performed in triplicate and the mean  fold induction and the standard deviations  are shown.
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Figure 1: NF-κB1 gene expression and promoter analysis in CEM cells after PMA/ PHA or TNF-α stimulation. (A) RNAs were isolated from CEM cells after addition of either PMA/PHA (50 ng and 5 μg/ml) or TNF-α (10 ng/ml) over the time course indicated. RNAs were detected using a 32Plabeled NF-κB1-specific probe. Transcripts encoding NF-κB1 were quantitated by densitometric scanning of autoradiograms and the fold accumulation of mRNAs were calculated after normalization to β-actin. (B) Schematic of the NF-κB1 promoter depicting the Egr-1 and NF-κB binding sites. CEM cells were transfected with either the HS-CAT reporter or with various deletion constructs (SS-, SpS-, HiS-, and AS-CAT). 18 h after transfection, cells were stimulated with either PMA/PHA (50 ng and 5 μg/ ml) or TNF-α (10 ng/ml) and CAT activity was analyzed. Transfection experiments were performed in triplicate and the mean fold induction and the standard deviations are shown.

Mentions: To determine whether differential expression of NF-κB1 mRNA was observed after stimulation by PMA and PHA or TNF-α, Northern blot analysis was performed on total cellular RNA isolated from the human T cell line, CEM. As shown in Fig. 1 A, NFκB1 mRNA began to accumulate within 1 h of stimulation with PMA and PHA, and by 4 h a significant induction (10-fold) was observed in CEM cells. Although TNF-α–stimulated cells also displayed increases in NF-κB1 transcripts with similar kinetics, a substantially lower accumulation of mRNA was observed over the 4-h time course.


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)

NF-κB1 gene expression and  promoter analysis in CEM cells after PMA/ PHA or TNF-α stimulation. (A) RNAs  were isolated from CEM cells after addition  of either PMA/PHA (50 ng and 5 μg/ml)  or TNF-α (10 ng/ml) over the time course  indicated. RNAs were detected using a 32Plabeled NF-κB1-specific probe. Transcripts  encoding NF-κB1 were quantitated by densitometric scanning of autoradiograms and  the fold accumulation of mRNAs were calculated after normalization to β-actin. (B)  Schematic of the NF-κB1 promoter depicting the Egr-1 and NF-κB binding sites.  CEM cells were transfected with either the  HS-CAT reporter or with various deletion  constructs (SS-, SpS-, HiS-, and AS-CAT).  18 h after transfection, cells were stimulated  with either PMA/PHA (50 ng and 5 μg/ ml) or TNF-α (10 ng/ml) and CAT activity  was analyzed. Transfection experiments  were performed in triplicate and the mean  fold induction and the standard deviations  are shown.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196044&req=5

Figure 1: NF-κB1 gene expression and promoter analysis in CEM cells after PMA/ PHA or TNF-α stimulation. (A) RNAs were isolated from CEM cells after addition of either PMA/PHA (50 ng and 5 μg/ml) or TNF-α (10 ng/ml) over the time course indicated. RNAs were detected using a 32Plabeled NF-κB1-specific probe. Transcripts encoding NF-κB1 were quantitated by densitometric scanning of autoradiograms and the fold accumulation of mRNAs were calculated after normalization to β-actin. (B) Schematic of the NF-κB1 promoter depicting the Egr-1 and NF-κB binding sites. CEM cells were transfected with either the HS-CAT reporter or with various deletion constructs (SS-, SpS-, HiS-, and AS-CAT). 18 h after transfection, cells were stimulated with either PMA/PHA (50 ng and 5 μg/ ml) or TNF-α (10 ng/ml) and CAT activity was analyzed. Transfection experiments were performed in triplicate and the mean fold induction and the standard deviations are shown.
Mentions: To determine whether differential expression of NF-κB1 mRNA was observed after stimulation by PMA and PHA or TNF-α, Northern blot analysis was performed on total cellular RNA isolated from the human T cell line, CEM. As shown in Fig. 1 A, NFκB1 mRNA began to accumulate within 1 h of stimulation with PMA and PHA, and by 4 h a significant induction (10-fold) was observed in CEM cells. Although TNF-α–stimulated cells also displayed increases in NF-κB1 transcripts with similar kinetics, a substantially lower accumulation of mRNA was observed over the 4-h time course.

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