From the Wistar Institute of Anatomy and Biology, Philadelphia, Pennsylvania 19104
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Monocytes/macrophages produce interleukin-12
(IL-12) in response to pathogenic stimulation, whereas most
Epstein-Barr virus-transformed (EBV+) B cells
constitutively secrete IL-12. The molecular mechanism regulating the
constitutive IL-12 gene expression in EBV+ B cells has not
been addressed. In this study, using the EBV+ B cell line
RPMI-8866, we localized to the human IL-12 p40 promoter two essential
cis elements, the NFB site and the Ets site. The NF
B
site was shown to interact with members of the NF
B family: p50 and
c-Rel. The Ets site constitutively bound a multi-component Ets-2-containing complex. While the NF
B and Ets sites appear equally
critical for inducible p40 promoter activity in macrophage cell lines,
NF
B plays a more dominant role in the constitutive p40 promoter
activity in EBV+ B cells. Transient expression of Ets-2 and
c-Rel in B, T, and monocytic cell lines synergistically activated the
IL-12 p40 promoter, apparently overcoming the requirement for cell
type- or stimulant-specific transcription factors. These data provide
new evidence that full activation of the human IL-12 p40 promoter may
result primarily from the interplay between NF
B and Ets family
members.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Interleukin 12 (IL-12),1
a heterodimeric cytokine composed of two disulfide-linked subunits of
35 (p35) and 40 (p40) kDa, was originally identified in the supernatant
fluid of Epstein-Barr virus-transformed (EBV+) human B-cell
lines (BCL) (1, 2). IL-12 exerts multiple effects, including:
(a) induction of cytokine production, particularly interferon- (IFN-
) by T and NK cells, (b) induction of
proliferation in T and NK cells and enhancement of their cytotoxic
activity, and (c) induction of T helper-type 1 (Th1)
responses and inhibition of Th2 responses (3). The finding that IL-12
p40 knockout mice have a severely depressed Th1 response (4) supports
the role of IL-12 in Th1 cell differentiation.
IL-12 is produced by phagocytic cells and other antigen-presenting cells in response to bacteria, bacterial products, intracellular pathogens, and viruses (1, 2, 5-11). Normal B cells appear to be relatively poor producers of IL-12 (6). Most EBV+ BCL and EBV+ lymphomas produce IL-12, with the highest levels observed in EBV+ BCL derived from AIDS-associated lymphomas (9, 12). SCID mice injected with human lymphocytes from EBV-seropositive donors developed EBV+ human B cell lymphoma secreting in vivo high levels of human IL-12,2 suggesting that a similar production of IL-12 during initial proliferation of EBV+ B cell in patients may affect the reactivity of their immune cells against the infected cells. Although any extrapolation to the in vivo situation from data observed with established cell lines should be taken with caution, it is tempting to speculate that, in healthy individuals, IL-12-producing EBV+ cells would be easily rejected by immune response and thus only cells that have lost the ability to produce IL-12 would be able to give rise to Burkitt's lymphomas; in immunodeficient AIDS patients, these protective mechanisms would be inefficient and their IL-12-producing EBV-transformed cells could give rise to lymphomas in a relatively high proportion of patients.
The key role of IL-12 in inflammation and in the immune response, and
the importance of this cytokine in anti-tumor resistance, have raised
considerable interest in the mechanisms of IL-12 gene transcription. We
(13) have shown previously that, in lipopolysaccharide (LPS)- and
IFN--treated human monocytes, the expression of IL-12 p40 and p35 is
primarily regulated at the transcriptional level. In a luciferase
reporter gene construct, a 3300-bp genomic DNA fragment including the
upstream sequences of the human p40 gene largely recapitulates its cell
type specificity and transcriptional regulation (13). The
cis elements in the p40 promoter region responsible for the
inducible activation of the genes have been studied extensively in
phagocytic cells, but no information is available for the elements
controlling the constitutive expression of the gene in EBV+
BCL. We have previously localized a DNA sequence spanning nucleotides
292 to
196 (relative to the transcription start site) responsible for induced promoter activity (13). The core element at position
212
binds a series of IFN-
- and LPS-induced nuclear proteins (termed the
F1 complex) including Ets-2, IFN-regulatory factor-1 (IRF-1), c-Rel,
and Ets-related factors (14). A downstream NF
B site between bp
117
and
107 was also characterized as an LPS response element in the
murine macrophage J774 cell line (15). Recently, Plevy et
al. (16) identified a third cis element located at
96
and
88 (downstream of the NF
B site) of the murine IL-12 p40
promoter, also conserved in humans, that binds members of the C/EBP
family of transcription factors in activated murine macrophage cell
line. The C/EBP element exhibits functional synergy with the upstream
NF
B site, although no physical interactions between C/EBP and Rel
proteins were observed (16). Together, these results suggest that in
myeloid cells a complex interplay exists between multiple inducible
transcription factors contributing to the regulation of IL-12 p40 gene
activation.
In this study, we focused upon the role of cis-acting
regulatory elements in the transcriptional activation of the human
IL-12 p40 gene in EBV+ BCL. In these cells, we showed
constitutive binding of multiple transcription factors to the NFB
and Ets sites of the p40 promoter. We found that cotransfection of
NF
B and Ets-2 transcription factors strongly and specifically
synergized in the induction of p40 promoter activity in
EBV+ BCL as well as in both inducible IL-12-producing
macrophage cell lines and IL-12-nonproducing EBV
and T
cell lines.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell Lines and Reagents--
Human EBV-transformed B
lymphoblastoid cell line (EBV+), RPMI-8866, PA682BM-2, and
AS283A (9), EBV Burkitt's lymphoma cell line BJAB (a
gift from E. Kieff, Harvard Medical School, Boston, MA), T cell line
Jurkat, and murine macrophage cell line RAW264.7 were all grown in RPMI
1640 medium (Mediatech, Herndon, VA) supplemented with 10% fetal calf
serum (Irvine Scientific, Santa Ana, CA). All tissue culture media and
supplements were endotoxin-free. All polyclonal antibodies used in
supershift experiments were purchased from Santa Cruz Biotechnology,
Inc. (Santa Cruz, CA). Recombinant murine (rmIFN-
) was a gift from
Dr. G. Garotta (Human Genome Sciences, Inc., Rockville, MD). Anti-CD3
monoclonal antibody (OKT3, ascites) was produced from cells obtained
from America Type Culture Collection (ATCC, Rockville, MD). LPS from Escherichia coli and
12-O-tetradecanoylphorbol-13-acetate (TPA) were purchased
from Sigma.
Nuclear Run-on Assay-- Untreated RPMI-8866 cells, TPA-treated BJAB cells (50 ng/ml, 4 h), and TPA- and anti-CD3-treated Jurkat cells (OKT3, ascites 1:1000, 4 h) were harvested and washed with phosphate-buffered saline (BioWhittaker, Walkersville, MD). Isolation of nuclei and in vitro transcription in the presence of [32P]UTP (3000 Ci/nmol, DuPont) were performed as described (17, 18).
Plasmids--
A 3300-bp fragment of the human IL-12 p40 promoter
was cloned into the luciferase reporter construct pXP2 (13) at the
PstI site. All deletion mutant constructs were generated by
PCR and fully sequenced for verification as described previously (13). NFB expression vectors were a gift from Dr. K. M. Murphy
(Washington University School of Medicine, St. Louis, MO). The
CMV-based murine Ets-2 expression vector was a gift from Dr. R. Maki.
The TNF-
promoter was obtained from a TNF cosmid library (ATCC,
catalog no. 57590) by PCR amplification of a 1294-bp fragment. The
sequence of the 5' primer used is 5'-TCTCTGAAATGCTGTCTGCTTGT-3' and the 3' primer used is 5'-CCTCTTAGCTGGTCCTCTGC-3'. The PCR product was
cloned into pXP2 expression vector. All plasmids were banded twice in
cesium chloride and verified by restriction mapping.
Transient Transfection--
DNA transfection experiments were
performed by electroporation as described (13). Briefly, 0.4 ml of the
cell suspension was mixed with 60 µg of DNA (10 µg of p40-luc/3.3
kb plus 3 µg of pCMV--galactosidase (transfection efficiency
control) and 47 µg of BS/KSII+) and electroporated in 0.45-cm
electroporation cuvettes (Gene Pulser, Bio-Rad) at 960 microfarads and
250 V for BJAB and Jurkat cells, 300 V for RPMI-8866 cells, and 350 V
for RAW264.7 cells. Cells were harvested 24 h post-transfection,
pelleted by centrifugation, and resuspended in 100 µl of lysis buffer
(Promega, Madison, WI). Lysates were used for both luciferase and
-galactosidase assay (13).
Gel Electrophoretic Mobility Shift Assay (EMSA)-- Nuclear extracts were isolated from RPMI-8866 as described (19) and from RAW264.7 also described (20). EMSA and supershift were performed as described previously (14).
Oligonucleotides Used for EMSAs--
Oligonucleotides
292/
196,
243/
196, and
222/
196 have been described (14).
Oligonucleotide
135/
99 used as a probe has the following sequence
(plus strand):
5'-AAACAAAAAAGGAACTTCTTGAAATTCCCCCAGAAGG-3' and
encompasses the potential PU.1 and NF
B sites (underlined) of the
human IL-12 p40 promoter found, respectively, in positions
143 and
132 of the murine IL-12 p40 promoter (15). Oligonucleotide
135/
99
m differs from oligonucleotide
135/
99 by a CC
GG transversion
at position
108 and
107. Oligonucleotide
123/
99 has the
following sequence: 5'-AACTTCTTGAAATTCCCCCAGAAGG-3'. An IRF-1 repeated consensus binding sequence was used as nonspecific oligonucleotide in competition assays (21). NF
B oligonucleotide is a
high affinity binding site for NF
B/c-Rel homodimeric and heterodimeric complexes (22).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Constitutive Transcription of the Human IL-12 p40 Gene in
EBV-transformed B Cells--
Based on our previous observation that
EBV+ BCL constitutively produce IL-12 protein (1, 6, 9), we
tested whether the p40 and p35 genes were also constitutively
transcribed in nuclear run-on assays performed with unstimulated
EBV+ BCL (Fig.
1A). Nascent p40 transcripts
were constitutively present in RPMI-8866 (Fig. 1A) and in
all other EBV+ BCL tested (PA682BM-2 and AS283A), and
expression was not further enhanced by TPA treatment (data not shown).
Jurkat T cells and BJAB EBV BCL, which do not express p40
mRNA or protein, were used as negative controls (Fig.
1A). Transcription of the IL-12 p40 gene, unlike that of the
TNF-
gene, was not detectable in unstimulated cells or in the
EBV
BCL or T cell lines stimulated with TPA alone or TPA
in combination with
-CD3, respectively. However, TNF-
transcription was shown to be further up-regulated by TPA treatment of
all cells examined. Nascent p35 transcripts were present in all cell
lines and under all conditions tested. Thus, the IL-12 p40 gene appears
to be constitutively transcribed in EBV+ but not in
EBV
BCL or T cells. We then analyzed the IL-12 p40
promoter activity in the same EBV+, EBV
and T
cell lines using a full-length promoter construct linked to a
luciferase reporter gene (
3300/+108) in a transient transfection assay. The promoter construct was active in EBV+ BCL, but
poorly or not active at all in EBV
and T cell lines (Fig.
1B), confirming that the construct contains adequate
sequence information for appropriate cell type-specific expression
(13).
|
Important cis-Regulatory Elements Located in the 222/+108 bp
Region of the IL-12 p40 Promoter--
To delineate the
cis-acting elements regulating the constitutive IL-12 p40
expression in EBV+ B cells, we first examined the activity
of the full-length promoter (
3300/+108) and a series of nested 5'
deletion constructs (Fig. 2) in
EBV+ BCL. Luciferase activity of the
3300/+108 construct
was 65-fold that of the parental, promoterless pXP2 control vector
(Fig. 3). Promoter activity was
progressively decreased when the full-length promoter was deleted from
the 5' end at position
471 (32-fold over pXP2 control vector),
292
(38-fold),
265 (28-fold), and
243 (29-fold). Surprisingly, the
activity of
222 bp plasmid was only slightly reduced compared with
the full-length promoter. An additional 100-bp deletion to
position
122 abolished half of the full-length promoter activity. Truncation of
the promoter to
28 bp (containing the TATA box) markedly diminished
the constitutive promoter expression to a level only 10-fold greater
than the promoterless control plasmid pXP2. These data suggest that the
region between nucleotide positions
222 and
28 is most critical for
constitutive promoter activity in the EBV+ BCL.
|
|
Functional Role of the NFB and Ets Sites--
Since the
222/+108 promoter region was shown to exhibit significant promoter
activity, we analyzed the activity of this construct in further detail.
To evaluate the functional role of the putative NF
B half-site at
117 and the Ets site at
212, we prepared a series of additional
promoters with defined mutations. The
222NF
Bm construct has a
substitution of the core
109CC
108 with
109GG
108. The
222ets-2
construct has a
5-bp deletion that eliminates the
211TTTCC
207 sequence (13). The same
mutations were made in the context of the
3300/+108 full-length
promoter and were designated respectively,
3300NF
Bm and
3300ets-2
. Analysis of the activity of these constructs in
transiently transfected EBV+ BCL (RPMI-8866) (Fig.
4A) revealed a dramatic
decrease in the luciferase activity of promoter constructs mutated at
the NF
B site (
3300NF
Bm and
222NF
Bm) (80-88% less than
their corresponding wild-type constructs), but only a 50% decrease for
the Ets-2 deletion mutants. In contrast, analysis of these constructs
in the macrophage cell line RAW264.7 (Fig. 4B) demonstrated
that the mutations of both the NF
B and Ets sites almost completely
abolished the inducibility of the p40 promoter by IFN-
and LPS
(reduced to 12-20% of the
3300/+108 wild-type promoter activity),
the latter result being consistent with our previous finding that the
Ets site plays an important role in this system (13). Together, these
results suggest that the B cells may use the same cis
elements and transcription factors as do macrophages, but the relative
contribution of each factor may vary.
|
Characterization of the DNA-Protein Complexes Associated with the
NFB Site--
To establish the relationship between promoter
activity and specific DNA-protein interactions at the NF
B site
(
117/
107) in EBV+ BCL, EMSA were performed using a
37-mer oligonucleotide probe (
135/
99) encompassing the putative
NF
B and PU.1 sites. Three DNA-protein complexes were observed via
EMSA (Fig. 5A); two of them
indicate specific binding to nuclear proteins since they were competed
by an excess of the unlabeled oligonucleotide corresponding to the
probe
135/
99, but not by an unrelated (NS) oligonucleotide competitor. Competition of the slower mobility complex was observed using a 25-mer oligo (
123/
99) containing the NF
B site, but not
the putative upstream PU.1 site located at
127 of the promoter. Use
of an oligonucleotide with recognized binding activity for members of
the NF
B/Rel family (22) as competitor resulted in complete
disappearance of the top band, indicating that this DNA-protein complex
likely involves members of the NF
B/Rel family. Further characterization of the NF
B protein members bound to the
135 to
99 promoter region (Fig. 5B) revealed a slowly migrating
supershifted complex in the presence of NF
B anti-p50 and anti-c-Rel
antibodies. The appearance of this supershifted band was accompanied by
a partial disappearance of the upper complex. Thus, p50 and
c-Rel-containing complexes constitutively bind to a transcriptionally
active 6B site in the IL-12 p40 gene in EBV+ BCL. Anti-PU.1
antibody decreased the intensity of the faster-migrating band,
suggesting the presence of PU.1 in this complex. No supershifted bands
were detected with anti-c-Fos used as a control.
|
|
Differences in Ets Site Binding Activities between B and Macrophage
Cell Lines--
We previously demonstrated the functional predominance
of the Ets site at 212/
207 in macrophage cells and the
characteristics of the nuclear complexes formed around this site in
response to stimulation (13, 14). To extend this analysis to
EBV+ BCL with respect to differences in the relative
importance of the Ets site between EBV+ and macrophage
cells, we performed EMSAs using the same probe (spanning
292 to
196) as in our previous studies (14). Nuclear extract from IFN-
-
and LPS-stimulated macrophage (RAW264.7) cells formed the previously
characterized complex F1, whereas extract from EBV+ BCL
(RPMI-8866) formed a slightly faster migrating complex, designated F1a.
F1a was efficiently competed by a 50-fold molar excess of the
292/
196 DNA (Fig. 7A).
Furthermore, F1a was supershifted by an anti-Ets-2 and an anti-c-Rel
affinity-purified polyclonal antibody (Fig. 7B), as observed
previously for the F1 complex (14). However, formation of the F1a
complex was blocked by an anti-IRF-2 antibody and not, as in the case
of the F1 complex, by an anti-IRF-1. These data suggest that Ets-2,
c-Rel, and IRF-2, but not IRF-1, may be constituents of the F1a complex
in EBV+ BCL.
|
Transactivation of the IL-12 p40 Promoter by NFB and
Ets-2--
To assess the ability of several NF
B proteins and the
Ets-2 protein to activate the p40 promoter, we transfected
EBV+ BCL with the luciferase-based p40 reporter construct
(
3300/+108) together with an expression vector for either NF
B RelA
(p65), NF
B c-Rel, NF
B p50, or Ets-2. No transactivation of the
3300/+108 construct was detected by c-Rel, RelA, and p50, alone or
any combination. Ets-2 alone was able to activate the promoter 4-fold,
similarly to its actions in macrophages (13). Interestingly,
co-expression of Ets-2 and c-Rel, but not RelA or p50, synergistically
enhanced the p40 promoter activity (Fig.
8A). The decreased activity
seen in Ets-2 and p50 cotransfection may have been due to the formation of the p50/p50 homodimer, which acts as a repressor, and may be a
default method of regulation, as we have observed high levels of
endogenous p50 produced by EBV+ BCL (data not shown).
Transactivation by Ets-2 and c-Rel was dependent upon the integrity of
their respective sites, since a 5-base deletion of the Ets site
(
3300ets2
) or a CC
GG substitution in the NF
B core sequence
(
3300NF
Bm) in the context of the 3.3-kb promoter resulted in the
failure of the promoter to respond to Ets-2 and c-Rel (Fig.
8B). In addition, mutation at the NF
B site abolished the
transactivating effect of exogenous Ets-2 (Fig. 8B). This
effect, seen with the wild type construct, may have resulted from a
moderate level of synergy between exogenous Ets-2 and constitutively
present endogenous NF
B in EBV+ BCL.
|
Cell Type and Promoter Specificity of the Synergistic Activation by
Ets-2 and c-Rel--
To determine whether the same combination of
expression vectors for the NFB and Ets family could transactivate
the p40 promoter in uninduced macrophage cells and in Jurkat T cells
(which do not produce IL-12), c-Rel and Ets-2 expression vectors were
transiently cotransfected in these cells. In both systems, the
expression vectors strongly induced the activity of the full-length p40
promoter reporter construct (
3300/+108) (Fig.
9). Mutations of the NF
B or the Ets
site dramatically decreased the level of transactivation by the
effector constructs (Fig. 9, A and C). The
remaining activity observed in macrophage cells using the
3300NF
Bm
reporter construct, and in T cells using the
3300ets2
, might
reflect the presence of additional noncanonical NF
B or Ets-2 sites,
or a cooperativity with other endogenous transcription factors. Thus,
the synergy between co-transfected NF
B and Ets-2 transcription
factors in IL-12 p40 gene regulation is observed regardless of
whether the endogenous gene is expressed in different cell types. This
synergy is not observed in macrophage cells transfected with a
luciferase construct under the control of the TNF-
promoter (Fig.
9B), also known to contain functional NF
B and Ets sites
(23-25). In addition, overexpression of the two transcription
factors failed to transactivate the thymidine kinase minimal promoter,
lacking NF
B and Ets sites (data not shown). Taken together, these
results demonstrate that the functional synergy between NF
B and
Ets-2 is promoter-specific.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The present study was performed to delineate the molecular
mechanisms by which EBV transformation of B lymphocytes results in the
activation of IL-12 gene expression. We demonstrate that two regulatory
elements, Ets at 212/
207 and NF
B at
117/
107, play a role in
the high level expression of the human IL-12 p40 promoter in
EBV+ BCL and in its inducible expression in macrophages. In
EBV+ BCL, activity of a 3300-bp p40 promoter parallels
transcription of the endogenous p40 gene, whereas no promoter activity
was detectable in EBV
BCL or in Jurkat T cells in which
the endogenous p40 gene is inactive. In EBV+ BCL, a segment
of the p40 promoter containing 222 bp upstream and 108 bp downstream of
the transcription start site (
222/+108) retains most of the activity
of the full-length promoter, indicating that constitutive transcription
from the p40 promoter is regulated by transcription factors acting on
this region. This basal promoter activity is dependent mostly on the
integrity of the NF
B site (
117/
107), as demonstrated by mutation
analysis. A DNA probe encompassing this site binds the p50 and c-Rel
NF
B family members, consistent with previous reports that NF
B
proteins in mature B cell lines are constitutively present in the
nucleus (26-28). Similar experiments on activated macrophages revealed
that the NF
B site, bound by p50/RelA, p50/c-Rel, and RelA/c-Rel
heterodimers, is also critical for p40 promoter activity. When this
site is mutated, macrophage cells completely lose their ability to
activate the p40 promoter after IFN-
and LPS stimulation. The data
provided here are corroborated by the studies of Murphy et
al. (15) on the murine p40 promoter in activated macrophages. Our
studies indicate that NF
B transcription factors are essential for
constitutive IL-12 p40 gene expression in EBV+ BCL and for
p40 gene inducibility in macrophage cells. However, in both B and
macrophage cells, p50, c-Rel, and RelA individually are unable to
transactivate the IL-12 p40 promoter, suggesting the requirement for
additional signals. Ets-2 appeared to be a likely candidate as the
IL-12 p40 promoter contains a functional Ets motif, TTTCCT or AGGAAA
(
212 to
207) essential for promoter activity in stimulated
macrophage cells (13). Complete activation of the IL-12 p40 promoter in
Ets-2 transiently transfected macrophages has been shown to require
IFN-
or LPS stimulation (13), suggesting that, although vital, Ets-2
alone is not sufficient to achieve optimal p40 promoter activation.
Here, we clearly demonstrate, in both B and unstimulated macrophage
cells, that Ets-2 exerts a strong enhancing effect on p40 promoter
activity only in the presence of the NF
B c-Rel. Ets-2 and c-Rel are
sufficient for potent transactivation of the p40 promoter even in T and
EBV
BCL (data not shown), where the p40 promoter is
normally inactive. The specificity of this response is demonstrated by
mutation or deletion of the NF
B or Ets binding sites resulting in
greatly diminished promoter activation. Cooperativity between these
transcription factors seems to be specific for the IL-12 p40 promoter,
since no synergistic activation was observed for the TNF-
promoter, also known to contain NF
B and Ets sites (23-25).
Physical interactions between NFB and Ets-like protein have been
reported to regulate expression of the IL-2 receptor
gene (29) and
the activation of the human immunodeficiency virus enhancer (30). The
Ets domain was shown to be necessary and sufficient to mediate this
interaction, suggesting that the highly conserved Ets domain may act
broadly to facilitate multiple protein-protein interactions. Indeed, it
is now well established that Ets family proteins can activate
transcription in conjunction with other proteins (31-35). Here, we
have shown an association between Ets-2 and c-Rel in the F1a complex in
EBV+ BCL similar to the F1 complex of activated macrophages
(14). We are currently investigating whether the F1a and NF
B
complexes physically interact at the p40 promoter.
The composition of the F1a complex differs from the previously
characterized F1 of activated macrophages (14), in that the former
contains the IRF-2 protein but not IRF-1. IRF-1-deficient mice fail to
produce IL-12, resulting in a severely compromised Th1 immune response
in vivo and in vitro (36, 37) strongly implicating a role for IRF-1 in IL-12 activation. IRF-1 and IRF-2 are
highly homologous and bind to common DNA sequence elements (38, 39)
with similar affinities. As a consequence of mutating the Ets site, we
demonstrated here, in EBV+ BCL, only a partial reduction in
the constitutive p40 promoter activity. In contrast, mutation of the
same site in macrophages resulted in total abrogation of inducible p40
promoter activity. In other promoters, such as that of IFN-, it has
been shown that IRF-2 antagonizes the activating function of IRF-1
(40). Therefore, the possibility that IRF-2 binding to the Ets site in
EBV+ BCL may function as a repressor of IRF-1 activity by
preventing its binding; thus, decreasing the relative participation of
this site in the constitutive activation of the p40 promoter in
EBV+ BCL should be considered. However, the ability of
IRF-2 to act as a suppressor is by no means absolute, as both IRF-1 and
IRF-2 have been shown to up-regulate expression of two genes, the
histone H4 gene FO108 and the EBNA 1 gene (41, 42).
We provided evidence of a pivotal role of the NFB site in
constitutive IL-12 p40 gene expression in EBV+ BCL and a
functional cooperativity between NF
B and Ets-2 transcription factors, critical for the constitutive and inducible activation of the
IL-12 p40 gene. Parallel to our findings, it was recently demonstrated
that a synergy exists between C/EBP and NF
B in human and mouse p40
promoter regulation (16), but whether C/EBP plays a role in
constitutive p40 gene expression in EBV+ BCL remains to be
determined. Together, this information suggests that the expression of
IL-12 p40 gene requires the coordinated interactions between multiple
regulatory proteins. The B cell lines used in our studies expressed the
EBV latent-infection membrane protein 1 (LMP1) capable of activating
the NF
B transcription factors (43-46). However, transient or stable
transfection of LMP1 in EBV
B cells was insufficient to
transactivate the p40
promoter.3 This suggests that
it is likely that EBV employs more elaborate mechanisms stimulating
multiple transcription factors critical for the activation of the IL-12
p40 gene.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Paolo Salomoni, Dr. Susan Robertson, and Dr. Frank Rauscher III for helpful suggestions during this work. We thank Sana Chehimi for excellent technical assistance and Marina Hoffman for editing the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by Public Health Service Grants CA10815, CA20833, CA32898, and AI34412.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Wistar Institute, 3601 Spruce St., Philadelphia, PA 19104. Tel.: 215-898-3992; Fax:
215-898-2357; E-mail: trinchieri{at}wista.wistar.upenn.edu.
1
The abbreviations used are: IL, interleukin;
EBV, Epstein-Barr virus; BCL, B-cell line; Th1, T helper-type 1; EMSA,
electrophoretic mobility shift assay; LPS, lipopolysaccharide; IFN-,
interferon-
; TPA, 12-O-tetradecanoylphorbol-13-acetate;
bp, base pair(s); TNF, tumor necrosis factor; CMV, cytomegalovirus;
PCR, polymerase chain reaction; IRF, IFN-regulatory factor; C/EBP,
CCAAT-enhancer-binding protein.
2 R. Baiocchi, G. Gri, and M. Caligiuri, unpublished results.
3 G. Gri, D. Savio, G. Trinchieri, and X. Ma, unpublished observation.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|