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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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Egr-1 is required for endogenous NF-κB1 gene expression. (A) Total  RNAs were isolated from CEM/CMV  control cells or from CEM/AsEgr-1 cells  stimulated with either PMA/PHA or  TNF-α over a 4 h period. RNAs were detected using a 32P-labeled NF-κB1 specific  probe. (B) Total proteins were isolated from  CEM/CMV or CEM/AsEgr-1 cells after  the addition of either PMA/PHA or TNF-α.  Proteins were subjected to PAGE and analyzed using an Egr-1–specific antibody and  enhanced chemiluminescent assay. NS, shows  the presence of a nonspecific band and  demonstrates that equal amounts of protein  were loaded in each lane.
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Figure 4: Egr-1 is required for endogenous NF-κB1 gene expression. (A) Total RNAs were isolated from CEM/CMV control cells or from CEM/AsEgr-1 cells stimulated with either PMA/PHA or TNF-α over a 4 h period. RNAs were detected using a 32P-labeled NF-κB1 specific probe. (B) Total proteins were isolated from CEM/CMV or CEM/AsEgr-1 cells after the addition of either PMA/PHA or TNF-α. Proteins were subjected to PAGE and analyzed using an Egr-1–specific antibody and enhanced chemiluminescent assay. NS, shows the presence of a nonspecific band and demonstrates that equal amounts of protein were loaded in each lane.

Mentions: To determine the importance of the Egr-1 transcription factor on endogenous NF-κB1 gene expression, we developed a CEM cell line which constitutively expressed antisense human Egr-1 RNA. Total RNAs were isolated from either CEM/AsEgr-1 cells (which express the human Egr-1 antisense RNA) or from CEM/CMV control cells (which harbor the empty expression vector) after the addition of either PMA/PHA or TNF-α over the time course indicated. As shown in Fig. 4 A, CEM/AsEgr-1 cells did not display significant increases in NF-κB1 transcripts until 4 h after PMA/PHA addition, while the control cells (CEM/CMV) responded within 1 to 2 h with similar kinetics as the parental CEM cell line (Fig. 1 A). Interestingly, TNF-α–stimulated CEM/ AsEgr-1 cells demonstrated a reduction in NF-κB1 transcripts 30 min after activation. Although TNF-α does not activate Egr-1, it would appear that under conditions in which endogenous Egr-1 levels are reduced (as with CEM/ AsEgr-1 cells), TNF-α stimulation represses NF-κB1 transcription (Fig. 4 A). Although the level of Egr-1 protein in the CEM/AsEgr-1 cells after PMA/PHA stimulation was significantly lower (fivefold) than protein levels observed in the control line, the antisense Egr-1 transcripts were not able to completely block PMA/PHA-mediated increases in Egr-1 protein (Fig. 4 B). Perhaps for this reason CEM/ AsEgr-1 cells are still able to display PMA/PHA-responsive upregulation of NF-κB1 transcripts but with slower kinetics.


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)

Egr-1 is required for endogenous NF-κB1 gene expression. (A) Total  RNAs were isolated from CEM/CMV  control cells or from CEM/AsEgr-1 cells  stimulated with either PMA/PHA or  TNF-α over a 4 h period. RNAs were detected using a 32P-labeled NF-κB1 specific  probe. (B) Total proteins were isolated from  CEM/CMV or CEM/AsEgr-1 cells after  the addition of either PMA/PHA or TNF-α.  Proteins were subjected to PAGE and analyzed using an Egr-1–specific antibody and  enhanced chemiluminescent assay. NS, shows  the presence of a nonspecific band and  demonstrates that equal amounts of protein  were loaded in each lane.
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Related In: Results  -  Collection

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Figure 4: Egr-1 is required for endogenous NF-κB1 gene expression. (A) Total RNAs were isolated from CEM/CMV control cells or from CEM/AsEgr-1 cells stimulated with either PMA/PHA or TNF-α over a 4 h period. RNAs were detected using a 32P-labeled NF-κB1 specific probe. (B) Total proteins were isolated from CEM/CMV or CEM/AsEgr-1 cells after the addition of either PMA/PHA or TNF-α. Proteins were subjected to PAGE and analyzed using an Egr-1–specific antibody and enhanced chemiluminescent assay. NS, shows the presence of a nonspecific band and demonstrates that equal amounts of protein were loaded in each lane.
Mentions: To determine the importance of the Egr-1 transcription factor on endogenous NF-κB1 gene expression, we developed a CEM cell line which constitutively expressed antisense human Egr-1 RNA. Total RNAs were isolated from either CEM/AsEgr-1 cells (which express the human Egr-1 antisense RNA) or from CEM/CMV control cells (which harbor the empty expression vector) after the addition of either PMA/PHA or TNF-α over the time course indicated. As shown in Fig. 4 A, CEM/AsEgr-1 cells did not display significant increases in NF-κB1 transcripts until 4 h after PMA/PHA addition, while the control cells (CEM/CMV) responded within 1 to 2 h with similar kinetics as the parental CEM cell line (Fig. 1 A). Interestingly, TNF-α–stimulated CEM/ AsEgr-1 cells demonstrated a reduction in NF-κB1 transcripts 30 min after activation. Although TNF-α does not activate Egr-1, it would appear that under conditions in which endogenous Egr-1 levels are reduced (as with CEM/ AsEgr-1 cells), TNF-α stimulation represses NF-κB1 transcription (Fig. 4 A). Although the level of Egr-1 protein in the CEM/AsEgr-1 cells after PMA/PHA stimulation was significantly lower (fivefold) than protein levels observed in the control line, the antisense Egr-1 transcripts were not able to completely block PMA/PHA-mediated increases in Egr-1 protein (Fig. 4 B). Perhaps for this reason CEM/ AsEgr-1 cells are still able to display PMA/PHA-responsive upregulation of NF-κB1 transcripts but with slower kinetics.

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