By
From the * Lineberger Comprehensive Cancer Center, and Department of Biology, University of
North Carolina, Chapel Hill, North Carolina 27599-7295
NF-B is an important transcription factor required for T cell proliferation and other immunological functions. The NF-
B1 gene encodes a 105-kD protein that is the precursor of the p50 component of NF-
B. Previously, we and others have demonstrated that NF-
B regulates the
NF-
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-
B1 encoding transcripts than cells stimulated with tumor
necrosis factor-
, despite the fact that both stimuli activate NF-
B. Characterization of the NF-
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-
B and Egr-1. Furthermore, Egr-1 induction was required for endogenous NF-
B1 gene expression, since PMA/PHA-stimulated T cell lines expressing antisense
Egr-1 RNA were inhibited in their ability to upregulate NF-
B1 transcription. Our studies indicate that transcriptional synergy mediated by activation of both Egr-1 and NF-
B may have
important ramifications in T cell development by upregulating NF-
B1 gene expression.
Tcell activation results in a rapid activation of NF- Although little is known about the regulation of NF Analysis of the NF- Cell Culture and Transient Transfection Analysis.
CEM cells were
cultured in RPMI 1640 medium containing 10% FCS and antibiotics. Cells were stimulated by supplementing their growth medium with either PMA and PHA (Sigma Chemical Co., St. Louis,
MO) to a final concentration of 50 ng/ml and 5 µg/ml, respectively, or TNF- Plasmid Constructs and Site-directed Mutagenesis.
Both the NF Northern Blot and Electrophoresis Mobility Shift Analysis.
Total cellular RNA was isolated from CEM cells using the RNeasy total
RNA kit (Qiagen, Inc., Chatsworth, CA). Northern blot analysis
was performed as previously described (11). Nuclear extracts were
isolated and electrophoresis mobility shift analyses (EMSAs) were
performed as previously described (11, 18). Oligonucleotides corresponding to the NF- Generation of Stable Cell Lines.
The full-length human Egr-1
cDNA was cloned into the EcoR1 site of the CMV-5 expression
vector in the antisense orientation. CEM cells were cotransfected
with either CMV-AsEgr-1 or with the CMV-5 control vector in the
presence of pcDNA-3.1 Neo (Invitrogen, San Diego, CA). Transfected cells were grown in complete RPMI-1640 medium supplemented with 1.5 mg/ml of Geneticin (GIBCO BRL, Gaithersburg, MD) and resistant clones were isolated. RNase protection
assays were performed using the RPA II kit (Ambion, Inc., Austin, TX) to ensure that the CEM/AsEgr-1 cells were expressing
the CMV-driven antisense human Egr-1 transcript.
To determine whether differential expression of NF-
To determine whether
the increase in NF- To localize the PMA/PHA-responsive region within the
NF- Computer analysis of the SphI/ApoI
fragment of the NF-
To elucidate whether differential Egr-1 binding was observed in T cells after the addition of either PMA/PHA or
TNF- To determine
whether Egr-1 is required for PMA/PHA responsive activation of the NF-
To further explore the ability of each of these transcription factors to activate the NF To determine whether the difference between PMA/
PHA- and TNF- To determine the importance of the Egr-1 transcription factor on endogenous
NF-
In this paper we have demonstrated that to achieve full
activation of the NF-B
(1, 2) which appears to play an important role in T cell
proliferation, partly because of the involvement of this transcription factor in the upregulation of the IL-2 and cognate
receptor (IL-2R) genes (3). NF-
B was originally described to
be composed of heterodimeric subunits of p50 and p65
proteins (1, 2) and is typically found in the cytoplasm of
cells as an inactive precursor complex with an inhibitor
protein, I
B (4). The NF-
B1 gene product p105 is processed into the DNA-binding subunit of p50 through a
mechanism that requires serine phosphorylation (5) and the induction of an ATP-dependent ubiquitin-proteasome
pathway (6, 7). Cellular activation leads to an increase in
NF-
B1 transcripts, p105 processing, and subsequent p50
accumulation in both fibroblasts and in T cells (5, 8).
B1 in T lymphocytes, its expression has been demonstrated to be essential for proliferation after costimulation
through CD2 or CD28 receptors (9). Recently, it has been
demonstrated that lymphocytes derived from p50
/
-deficient mice display defects in immune responses (10). In addition Sha et al. (10) noted that T cells derived from these animals were unable to effectively proliferate in response to TCR and CD28 costimulation. Although disruption of the
NF-
B1 gene did not result in lethality, possibly because
NF-
B2/p52 could substitute for p50, these studies strongly
suggest that the p50 DNA-binding component of NF-
B
is critical for T cell proliferation.
B1 promoter revealed several NF-
B
DNA-binding sites within the upstream regulatory region
which are capable of binding p50 homodimers, p50/p65,
or p50/c-Rel heterodimers and, in part, account for the
ability of NF-
B to regulate NF-
B1 gene expression (11,
12). Although the NF-
B1 promoter has been shown to
require NF-
B for activity, it is presently unclear whether
NF-
B needs other transcription factors to mediate a transcriptional response. This is especially important because on
many responsive promoters NF-
B alone is insufficient at
activating transcription (2). T cell stimulation mediated by
the addition of either PMA and PHA or TNF-
results in
an increase in NF-
B1 expression (13). Both stimuli activate
NF-
B nuclear translocation and DNA-binding within 15 to 30 min (14); however, phorbol esters in combination
with mitogens result in a greater accumulation of NF-
B1
transcripts (11, 13). Our research demonstrates that full
PMA/PHA-mediated activation of the NF-
B1 promoter
not only required the NF-
B transcription factor, but also
the activation of the early growth response gene product
(Egr-1)1. Egr-1, also known as NGFI-A, Zif268, Krox-24,
and Tis 8, is transiently induced during the activation of T
cells from the G0 to G1 phase in response to various cellular
stimuli (15), has been recently demonstrated to be required
for T cell proliferation, and is known to regulate transcription of several genes (15). Our data establishes Egr-1
and RelA as important transcription factors capable of synergistically transactivating the NF-
B1 promoter.
(10 ng/ml; Promega Corp., Madison, WI). Transient transfections by electroporation (11) and luciferase or chloramphenicol acetyl-transferase assays were performed as previously
described (11, 18, 19).
B1 promoter constructs, p50HS- and p50SS-CAT, were previously
described (11). The SpS-, HiS-, and AS-CAT constructs were generated by digesting the p50HS-CAT reporter with HindIII, and
SphI, HinfI, or ApoI, respectively. The SSLUC reporter plasmid was
generated by cloning the StuI/SmaI fragment from p50HS-CAT into the pGL2 basic reporter containing the luciferase gene (Promega Corp.). The StuI/SmaI fragment of the NF-
B1 promoter was subjected to site-directed mutagenesis using a two-staged PCR (20).
The mNLUC construct, containing the disrupted 289-bp NF-
B
site, was made using a 5
primer (5
-ATGTAACTGAGACACGCTTAAATGGAATA-3
) and a 3
primer (5
-AGGCCATCAGCGCCGCGCCATGGCCGCA-3
) in the first round of
amplification and two internal primers (5
-AGGCGCTTCCTGGAAGCTTGAATACCGGCTCCAG-3
and 5
-CTGGAGCCGGTATTCAAGCTTCCAGGAAGCGCCT-3
) which mutated the
B element to a HindIII site. The NF-
B1 promoter
sequences containing a site-directed mutation within the Egr-1
DNA binding site (mELUC) was created using the same 5
and 3
primers described above plus two internal primers (5
-GCGCACGCAGCGAATTCGGGAGGTAGGGTC-3
) and (5
-GACCCTACCTCCCGAATTCGCTGCGTGCGC-3
) which destroyed
both the SP1/Egr-1 motif and created a unique EcoRI site. The
mNELUC construct was created by mixing the mutant Egr-1
oligos described above and using the mNF-
B fragment as a template. The PCR amplified NF-
B1 promoter fragments were cloned
into the SSLUC construct by replacing the wild-type PvuII/SmaI
fragment with the corresponding site-directed mutant promoter
sequences. Nucleotide sequences of mN, mE, and mNELUC
constructs were confirmed by sequence analysis.
B1 promoter Egr-1 DNA binding site
(5
-TCGACTCCCGCCCCCGCTGCG-3
and 5
-TCGACGCAGCGGGGGCGGGAG-3
) were radiolabeled using
-[32P]dCTP
and the Klenow fragment of DNA polymerase I. For antibody super shift assays and competition experiments, nuclear extracts were preincubated (10 min, room temperature) with either 1 µg of
antiserum or with a nonradioactive double stranded oligonucleotide before the addition of the radiolabeled gel shift probe. The
mutant Egr-1 probe (annealed 5
-TCGACTCCCGAATTCGCTGCG-3
and 5
-TCGACGCAGCGAATTCGGGAG-3
) was
used in competition assays. The Egr-1-specific antibody (SC No. 189) was purchased from Santa Cruz Biotechnology (Santa
Cruz, CA).
Differential Expression of NF-B1 mRNA After PMA/
PHA- or TNF-
-Stimulation.
B1 mRNA was observed after
stimulation by PMA and PHA or TNF-
, Northern blot
analysis was performed on total cellular RNA isolated from the human T cell line, CEM. As shown in Fig. 1 A, NF
B1 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-
-stimulated cells also displayed increases in NF-
B1 transcripts
with similar kinetics, a substantially lower accumulation of
mRNA was observed over the 4-h time course.
Fig. 1.
NF-B1 gene expression and
promoter analysis in CEM cells after PMA/
PHA or TNF-
stimulation. (A) RNAs
were isolated from CEM cells after addition
of either PMA/PHA (50 ng and 5 µg/ml) or TNF-
(10 ng/ml) over the time course
indicated. RNAs were detected using a 32Plabeled NF-
B1-specific probe. Transcripts
encoding NF-
B1 were quantitated by densitometric scanning of autoradiograms and
the fold accumulation of mRNAs were calculated after normalization to
-actin. (B)
Schematic of the NF-
B1 promoter depicting the Egr-1 and NF-
B 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-
(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.
[View Larger Versions of these Images (14 + 16K GIF file)]
B1 Promoter Region Required for
PMA/PHA-Responsive Upregulation.
B1 mRNA, seen in Fig. 1 A, was due
to transcriptional upregulation of the promoter and not due
to PMA/PHA-induced message stabilization, CEM cells were transiently transfected with the p50HS-CAT NF-
B1
promoter construct and stimulated with either PMA/PHA
or TNF-
for 24 h. As shown in Fig. 1 B, the NF-
B1
promoter (p50HS-CAT) was strongly activated in CEM
cells after PMA/PHA stimulation, but only weakly after
TNF-
addition, suggesting that PMA/PHA-mediated activation of the NF-
B1 promoter was through a transcription-dependent mechanism.
B1 promoter, a series of deletion promoter constructs
were made. CEM cells were transiently transfected with
each reporter construct and then either stimulated with
PMA/PHA or with TNF-
. As shown in Fig. 1 B, no significant difference in stimulation was observed between the
HS-, the SS-, or the SpS-CAT constructs. However, the
HiS-CAT reporter, which lacked the
289 NF-
B site,
displayed a significant decrease in NF-
B1 promoter activity after stimulation by either PMA/PHA or TNF-
. The
AS-CAT reporter, lacking both the
103 and
11
B sites,
was greatly diminished in response to PMA/PHA- and
TNF-
-mediated activation (Fig. 1 B). Collectively, this
data indicates that the region between the SphI site (
425) and the ApoI site (
9) was critical for both inducers, but
that PMA/PHA stimulation resulted in a higher fold activation than TNF-
. Based on the deletion analysis of the
NF-
B1 promoter, these results suggest that both stimuli may
use NF-
B, but that the PMA/PHA stimulation must activate additional transcription factors not induced by TNF-
.
B1 Promoter Contains an Egr-1 Consensus Site and
T Cells Stimulated with PMA and PHA, but not TNF-
, Display
Egr-1 DNA Binding.
B1 promoter identified potential
DNA-binding motifs for five different transcription factors
including AP-1, E2F, Egr-1, NF-
B, and SP1. Of these,
NF-
B, Egr-1, and AP-1 are known to display increased transactivation potential after PMA stimulation (2, 15, 21). Since PMA/PHA and TNF-
both activated NF-
B, we excluded this transcription factor as the nuclear factor responsible for differentially regulating the NF-
B1 promoter.
In addition, the putative AP-1 site located in the NF-
B1
promoter was not found to affect PMA-induced transactivation of the NF-
B1 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-
B1 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-
(data not shown), we chose to focus on
the inducible binding of the Egr-1 transcription factor.
Fig. 2.
Identification of an Egr-1 DNA binding site within the NFB1 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-
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.
[View Larger Versions of these Images (94 + 31K GIF file)]
, nuclear proteins were isolated from stimulated
CEM cells over a 4 h time course and EMSAs were performed using the NF-
B1 Egr-1 consensus site. After stimulation with PMA/PHA, Egr-1 binding begins to appear
within 30 min and continues to increase at both 1 and 2 h
with a slight decrease seen at 4 h (Fig. 2 B). Interestingly, no Egr-1 binding was observed in CEM cell extracts after
TNF-
stimulation (Fig. 2 B). However, both PMA/
PHA- and TNF-
-stimulated CEM cells demonstrated
NF-
B-specific binding after the addition of each inducer,
confirming that CEM cells were capable of responding to
TNF-
(data not shown). Identical results to those obtained in the CEM cell line were demonstrated in Jurkat
T cells (data not shown). Collectively, these results indicate
that the
68 site located in the NF-
B1 promoter is capable of binding the Egr-1 transcription factor and that, unlike PMA/PHA stimulation, TNF-
fails to stimulate Egr-1
binding in T cell lines.
B1
Promoter Requires the Egr-1 DNA Binding Site.
B1 promoter, site-directed mutagenesis was performed. Since the NF-
B1 promoter is regulated
directly by NF-
B (11, 12), we chose to mutate the
289
B motif alone or in combination with the
68 Egr-1
binding site. The four luciferase NF-
B1 promoter constructs included (a) SSLUC, the wild-type construct, (b)
mNLUC, which contained a mutated NF-
B site at position
289, (c) mELUC, which contained the disrupted 68-bp
Egr-1 site, and (d ) mNELUC, which contained mutated
NF-
B and Egr-1 sites. As shown in Fig. 3 A, site-directed
mutagenesis of the Egr-1 binding element (mELUC) significantly diminished PMA/PHA-responsive activation of
the NF-
B1 promoter, but had very little or no effect on
TNF-
-mediated stimulation. In contrast, mutating the
NF-
B
289 site alone had very little effect on either
PMA/PHA- or TNF-
-induced activation (data not shown).
These results in conjunction with the promoter deletion
studies (shown in Fig. 1 B) would strongly suggest that other
B binding sites (namely
103 and
11) are important for transcriptional activation. Recently, McElhinny et al.
(22) demonstrated that the
11 NF-
B site was required
for HIV-mediated induction of NF-
B1 promoter in monocytes. Although the
289 motif is probably not the only
functional NF-
B motif, site-directed mutagenesis of both
the Egr-1 and NF-
B elements (mNELUC), resulted in a
further decrease in PMA/PHA-responsive stimulation compared to the mELUC construct (Fig. 3 A). Taking into account that mutagenesis of the Egr-1 site in the NF-
B1
promoter significantly diminished PMA/PHA-responsive
stimulation, these results indicate that the
68 Egr-1 site
plays an important role in the activation of this gene in T
lymphocytes.
Fig. 3.
The Egr-1 DNA binding site is required for PMA/PHA-responsive activation and for transcriptional synergy mediated by RelA and Egr-1. (A)
CEM cells were transfected with either the wild-type NF-B1 promoter (SSLUC) or with each of the site-directed mutants including (a) mNLUC,
which contained a mutated NF-
B site at position
289, (b) mELUC, which contained a disrupted 68-bp Egr-1 site, and (c) mNELUC, which contained mutated NF-
B and Egr-1 sites, and 18 h later cells were stimulated with either PMA/PHA or TNF-
. 24 h after stimulation, cell extracts were
harvested and assayed for luciferase activity. (B) CEM cells were co-transfected with either the SSLUC construct or with mutant reporters (mN, ME,
mNE; 5 µg each) and with various expression vectors (5 µg) including an empty vector control, RelA, Egr-1, or RelA plus Egr-1. Cells were harvested
48 h after transfection and luciferase activities were determined. (C) The SSLUC construct was transfected into CEM cells along with either the vector
control or with an Egr-1 expression construct. 18 h after transfection, cells were either left untreated or stimulated with PMA/PHA or TNF-
. Cells
were harvested 24 h after stimulation and extracts were analyzed for luciferase activity. All transfection assays were performed in triplicate in three independent experiments.
[View Larger Versions of these Images (29 + 35 + 26K GIF file)]
B and Egr-1 Transcription Factors Synergistically Transactivate the NF-
B1 Promoter.
B1 promoter, the wild-type as well as the site-directed
mutant reporters were cotransfected with the empty expression plasmid or with constructs encoding either RelA,
the p65 subunit of NF-
B, Egr-1, or a combination of
both. As shown in Fig. 3 B, the SSLUC reporter was
strongly transactivated after cotransfection with the RelA
encoding expression construct, but only weakly activated
by the overexpression of Egr-1. Interestingly, transfection
with both RelA and Egr-1 resulted in a potent synergistic transactivation of the NF-
B1 promoter. Although the
289
B site was important for RelA-mediated transactivation, mutagenesis of this element did not affect activation
by Egr-1, nor did it affect RelA/Egr-1 synergy (Fig. 3 B). This
result would suggest that, in the presence of Egr-1 binding,
other
B sites located within the NF-
B1 promoter are
used for the synergistic response. Unlike the mNLUC reporter, mutagenesis of the Egr-1 site (mELUC) resulted in
a significant reduction in RelA-, Egr1-, and RelA/Egr1mediated activation of the NF-
B1 promoter (Fig. 3 B).
However, disruption of both the
289 and
68 sites
(mNELUC) did not further diminish RelA/Egr-1-mediated synergy. This would suggest that overexpression of
both of these transcription factors may indirectly lead to the
activation of the NF-
B1 promoter by upregulating other
trans-acting elements, or that Egr-1 (like NF-
B) recognizes other DNA-binding sites within the NF-
B1 promoter. Collectively, co-transfection studies strongly suggest
that the Egr-1 DNA binding site is critical for both Egr1-mediated transactivation and for RelA/Egr-1-dependent synergy. Although the Rel homology domain of RelA has
been shown to be responsible for protein-protein interactions with transcription factors outside of the NF-
B/Rel
family (23), we were unable to demonstrate a physical
interaction between RelA and Egr-1, nor were we able to
generate synergistic transactivation mediated by these factors using either a 3×-NF-
B or 3×-Egr-1 CAT reporter (data not shown).
to Stimulate the NF-
B1 Promoter.
-stimulated NF-
B1 gene expression was
due to the inability of TNF-
to activate Egr-1, CEM cells
were transfected with the SSLUC reporter along with the
empty vector control or with the Egr-1 expression construct and 24 h later cells were stimulated with PMA/PHA
or TNF-
. As shown in Fig. 3 C, the fold increase between cells transfected with the vector control or with the Egr-1 expression construct was comparable to previous experiments, while PMA/PHA-stimulated cells (coexpressing
Egr-1) displayed the highest level of NF-
B1 activation.
Interestingly, cells transfected with Egr-1 and stimulated
with TNF-
displayed a significant increase in promoter
activity, compared to TNF-
-stimulated cells transfected with the vector control. The ability of Egr-1 to augment
TNF-
-mediated activation of the NF-
B1 promoter was
a direct effect since the transcriptional synergy was not observed with the mELUC construct containing the disrupted Egr-1 binding site (data not shown). These results
demonstrate that the Egr-1 transcription factor can complement TNF-
-mediated stimulation of the NF-
B1 promoter. Furthermore, our data suggests that the inability of
TNF-
to activate Egr-1 may account for the difference
between PMA/PHA- and TNF-
-mediated NF-
B1 gene
expression in T cells.
B1 Gene Expression Is Affected in T Cells
Expressing Antisense Egr-1 RNA.
B1 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-
over the time course indicated. As shown in Fig.
4 A, CEM/AsEgr-1 cells did not display significant increases in NF-
B1 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-
-stimulated CEM/
AsEgr-1 cells demonstrated a reduction in NF-
B1 transcripts 30 min after activation. Although TNF-
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-
stimulation represses NF-
B1 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-
B1 transcripts but with slower kinetics.
Fig. 4.
Egr-1 is required for endogenous NF-B1 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-
over a 4 h period. RNAs were detected using a 32P-labeled NF-
B1 specific
probe. (B) Total proteins were isolated from
CEM/CMV or CEM/AsEgr-1 cells after
the addition of either PMA/PHA or TNF-
.
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.
[View Larger Versions of these Images (15 + 17K GIF file)]
B1 promoter, T cell signals require not
only the activation of NF-
B, but also the Egr-1 transcription factor. Interestingly, it appears that the potent activation of NF-
B DNA binding activity by TNF-
is insufficient at inducing high levels of NF-
B1 transcription. This
is consistent with the recent report that activation of NF-
B
binding by TNF-
is insufficient at activating VCAM-1
transcription in some cells, but the co-activation of NF-
B
and IRF-1 in endothelial cells functions to stimulate transcription (27, 28). Moreover, like NF-
B, Egr-1 is a relatively poor activator of NF-
B1 transcription. Thus, it is
the combined action of NF-
B and Egr-1, both of which
alone result in weak activation, that functions to synergistically stimulate the NF-
B1 promoter. A number of NF
Bresponsive genes have been characterized and found to require
Egr-1 DNA binding motifs for maximum transactivation. Such genes include TNF-
, IL-2, and ICAM-1 (17, 29, 30). In T cells, TNF-
does not fully upregulate its own promoter, presumably because of the inability of cytokines to activate
Egr-1 (17). However, primary human fibroblasts and macrophage cell lines display dramatic increases in Egr-1 after
TNF-
stimulation (15). Collectively, these studies would
suggest that the synergistic potential mediated by the NF
B and Egr-1 transcription factors are not only restricted
by the type of stimuli, but also may depend on the particular cell type. Thus, it appears that the functional interaction
of NF-
B and Egr-1 in the context of a promoter is likely
to mediate critical aspects of T cell signaling and potentially other important aspects of cellular growth control. The potent synergy between NF-
B and Egr-1 may explain, at
least partially, the requirements for these transcription factors in T cell proliferation with NF-
B1 being a critical
downstream target for Egr-1.
Address correspondence to Albert S. Baldwin, Lineberger Comprehensive Cancer Center, Campus Box No. 7295, University of North Carolina, Chapel Hill, NC 27599.
Received for publication 20 September 1996
This work was funded by the National Institute of Health (NIH) grant AI35098 awarded to A.S Baldwin and by the NIH postdoctoral fellowship grant 1F32-CA69790-01 awarded to M.W. Mayo.We would like to thank Dr. Vikas P. Sukhatme for providing the human Egr-1 cDNA. We would also like to thank S. Scott Drouin for technical assistance in the preparation of this manuscript.
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