Characterization of an Activation Protein-1-binding Site in the
Murine Interleukin-12 p40 Promoter
DEMONSTRATION OF NOVEL FUNCTIONAL ELEMENTS BY A REDUCTIONIST
APPROACH*
Chen
Zhu
,
Khatuna
Gagnidze
,
James H. M.
Gemberling§, and
Scott E.
Plevy
¶
From the
Immunobiology Center, The Mount Sinai School
of Medicine, New York, New York 10029-6574 and the
§ Howard Hughes Medical Institute, UCLA School of Medicine,
Los Angeles, California 90095-1662
Received for publication, January 17, 2001, and in revised form, March 9, 2001
 |
ABSTRACT |
Interleukin (IL)-12 is a heterodimeric cytokine
produced by macrophages in response to intracellular pathogens and
provides an obligatory signal for the differentiation of T-helper-1
cells. We previously reported an analysis of the IL-12 p40
promoter in RAW264.7 macrophages. Multiple control elements were
involved in activation of transcription by bacterial products. A
critical control element, located between
96 and
88, interacts with
C/EBP family members. In this study, using a strategy to demonstrate functional activity in a minimal promoter context, three novel cis-acting elements are found to have an important role in IL-12 p40
promoter activation by lipopolysaccharide. One of these elements is
characterized in detail. Mutations from
79 to
74 in the murine IL-12 p40 promoter significantly reduce lipopolysaccharide-induced promoter activity. Electrophoretic mobility shift assays demonstrate binding of AP-1 family members to this region. Spacing between the
C/EBP and AP-1 site is important for promoter activation, suggesting
cooperativity between these elements. c-Jun and a mutant c-Jun molecule
activate the IL-12 p40 promoter and synergistically activate the
promoter when co-expressed with C/EBP
. Finally, this region of the
promoter is demonstrated to be a target for mitogen-activated protein
kinase and toll-like receptor signaling pathways.
 |
INTRODUCTION |
IL-121 is a
heterodimeric cytokine produced by macrophages and dendritic cells.
Among its biologic activities, IL-12 production is necessary for the
differentiation of T-helper-1 (Th1) cells and for the secretion of the
Th1 cytokine, interferon-
(IFN-
) (1). Accordingly, IL-12 and Th1
cells are required for cell-mediated immunity against intracellular
microbes (2-4). The protective role of IL-12 in human infectious
diseases including leprosy (5), tuberculosis (6), and leishmaniasis (7)
has been well characterized. Conversely, overexpression of Th1
cytokines and IL-12 may contribute to the development of chronic
inflammatory disorders (8), including Crohn's disease and rheumatoid
arthritis. Thus, the regulated expression of IL-12 in
antigen-presenting cells is a critical event in the pathogenesis of
infectious and inflammatory diseases.
IL-12 is composed of two covalently linked glycosylated chains, p40 and
p35, that are encoded by separate genes and together form the
biologically active p70 heterodimer (9-11). The p35 gene is
constitutively expressed in many tissue types (9). p40 mRNA is
detected in macrophages and other cells that produce bioactive IL-12
(9) and is strongly induced by intracellular bacteria and bacterial
products (12). Therefore, studies of IL-12 transcriptional regulation
have focused on the p40 gene. Murphy and colleagues (13) identified an
NF-
B site between
122 and
132 relative to the transcription
start site in the murine p40 gene that was necessary for induction of
promoter activity by LPS. In another series of investigations, Ma and
colleagues (14-16) have identified an Ets protein DNA-binding sequence
between
212 and
207 in the human p40 gene that was implicated in
promoter activation by LPS. We have reported a comprehensive functional
analysis of the murine and human IL-12 p40 promoters in RAW264.7 cells
(17). The p40 promoter was strongly induced by heat-killed
Listeria monocytogenes and LPS. An important control element
defined by this analysis, located between
96 and
88 relative to the
murine transcription start site, interacts with C/EBP proteins. This
element was functionally synergistic with the NF-
B site. However,
mutations in several other elements had functional effects on promoter
activation. Thus, induction of the IL-12 p40 promoter by bacterial
products will be defined by a complex series of events.
In this study, a functional role for three novel cis-acting elements in
the p40 promoter is demonstrated in reporter assays by utilizing a
combination of multiple substitution and deletion mutants. One of these
elements is characterized in detail. Mutations from
79 to
74 in the
murine IL-12 p40 promoter significantly reduce LPS-induced promoter
activation and correlate with binding of AP-1 family members by
electrophoretic mobility shift assays. Spacing between the adjacent
C/EBP and AP-1 sites and orientation of the AP-1 site are important for
promoter activation, suggesting cooperativity between these two sites.
Expression of a dominant negative AP-1 protein, A-Fos, in RAW264.7
cells inhibits p40 promoter activation by LPS, demonstrating a
functional role for AP-1 family members. Furthermore, c-Jun and a
mutant c-Jun molecule TAM67 activate the IL-12 p40 promoter and
synergistically activate the promoter when co-expressed with C/EBP
.
Likewise, c-Jun and TAM67 activate the tumor necrosis factor-
(TNF)
promoter in RAW264.7 cells. Finally, experiments using inhibitors and
activators of signal transduction pathways identify the region of the
promoter from
101 to +55 as a target for the p38 kinase, the
N-terminal Jun kinase (JNK), and the toll-like receptor (TLR) signal
transduction pathways.
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EXPERIMENTAL PROCEDURES |
Plasmid Constructs--
The murine wild type IL-12 p40 promoter
350 to +55 region was inserted into the chloramphenicol
acetyltransferase (CAT) reporter vector pCAT basic (Promega) modified
by addition of KpnI and BglII sites in the
polylinker as described previously (17). All site-directed mutant
plasmids described in this paper were generated by two-step polymerase
chain reaction using overlapping internal primers containing a mutant
site (18). Polymerase chain reaction-generated mutant promoters were
cloned into pCAT basic vector (Promega) with a modified polylinker or
pGL2B vector (Promega) and sequenced before use in transfection
experiments. Mammalian expression plasmids for human wild type c-Jun,
c-Fos, JunB (Tom Curran), TAM67 (Michael Birrer), and C/EBP
(from
Steven McKnight) were described previously (17, 19). An expression
plasmid for the AP-1 dominant negative protein A-Fos was obtained from
Charles Vinson (see Ref. 20). The following plasmids were utilized for
signal transduction experiments: constitutively active MKK3, MKK3glu
(Roger Davis) (21); constitutively active MKK6, MKK6E (Jiahuai Han)
(22); JIP-1 (23); constitutively active MEKK1 (367
MEKK1)(Dennis
Templeton) (24); dominant negative TLR2 (Paul Godowski) (25);
constitutively active CD4-TLR4 fusion (26); and dominant negative
MyD88, dominant negative TRAF6 (Ruslan Medzhitov) (27). As MAP kinase
pathway controls, MEF2c-GAL4 was obtained from Jiahuai Han. pFA-Jun,
and pFR-luciferase were from Stratagene. A multimerized NF-
B element
luciferase reporter plasmid and a human TNF-
promoter (
283 to
+113) luciferase reporter plasmid were obtained from Adrian Ting. A
multimerized consensus AP-1 DNA-binding element CAT reporter gene
(TRE-CAT) was provided by Andre Nel.
Cell Lines and Reagents--
The RAW264.7 murine macrophage line
(American Type Culture Collection) was maintained in DMEM supplemented
with 10% fetal bovine serum and penicillin/streptomycin. LPS was
purchased from Sigma. L. monocytogenes was obtained from Hao
Shen, and Mycobacterium tuberculosis whole cell extract was
from John Belisle. Antibodies against Pu.1, C/EBP, AP1, ATF, and Rel
family members were purchased from Santa Cruz Biotechnology, Inc.
Transfections--
RAW267.4 cells were transiently transfected
using SuperFect Transfection Reagent (Qiagen) by the described protocol
with modifications. For each transfection, 2.5 µg of plasmid were
mixed with 100 µl of DMEM (without serum and antibiotics) and 10 µl
of SuperFect reagent. The mixture was incubated at room temperature for
10 min, and then 600 µl of DMEM complete medium was added and
immediately placed onto the cells in 6-well plates. After incubation
for 3-4 h at 37 °C, the cells were washed with PBS and split
equally into two wells. One well was activated with LPS (to final
concentration 5 µg/ml) 24 h later. Sixteen to eighteen hours
after activation, the cells were harvested by using 1× Reporter Lysis
Buffer (Promega). CAT assays were performed with 75 µg of total
protein from cell extracts. The conversion of
[14C]chloramphenicol to its acetylated forms was assessed
by thin layer chromatography and quantified by a PhosphorImager
(Molecular Dynamics). Luciferase activity was determined from 20 µl
of cell extract, as described previously (17). Cells were
co-transfected with a constitutively active HSP promoter that expresses
-galactosidase (from Bradley Cobb) to monitor for transfection
efficiency.
-Galactosidase assays were performed with 25 µg of
total protein (for CAT assays) or 20 µl (luciferase assays) from cell
extracts, as described previously (17).
Nuclear Extracts and DNA Binding Assays--
RAW267.4 cell
nuclear extracts were prepared as described previously (28, 29). EMSA
probes were made by annealing equal amount of single-stranded
oligonucleotides with 5'-GATC overhangs (Genosys Biotechnologies,
Inc.). 200 ng of annealed probe was labeled by filling in with
[
-32P]dGTP and[
-32P]dCTP by Klenow
enzyme. Labeled probes were purified with NucTrap purification columns
(Stratagene). Sequences of the IL-12 p40 EMSA probes are demonstrated
in the text (see Fig. 4A). The sequence of the canonical
AP-1 EMSA probe is 5' gatcCGCTTGATGACTCAGCCGGAA, and the canonical
C/EBP probe is 5' gatcAAGCTGCAGATTGCGCAATCTGCAGCTT 3'. Electrophoretic
mobility shift assays were performed as described previously (17),
using 105 cpm probe and 5 µg of nuclear extracts per
reaction. In supershift experiments, 1-2 µl of antibody was added to
each reaction, and the nuclear extract/antibody mixture was incubated
at room temperature for 30 min before adding probes. DNA-binding
complexes were separated by 5% polyacrylamide/Tris glycine/EDTA gel
run at 4 °C for 4 h at 150 V. Gels were dried and exposed to
Kodak X-AR 5 film at
80 °C in the presence of an intensifying screen.
 |
RESULTS |
Demonstration of Novel Functional Cis-acting Control Elements in
the IL-12 p40 Promoter--
In our previous analysis of the murine and
human IL-12 p40 promoters, the C/EBP element spanning
93 to
88, the
NF-
B element at
131 to
120, and the TATA box were the only sites
convincingly shown to be important for p40 promoter function (17).
However, two results suggested that other functionally important
elements exist within the promoter. First, deletion or substitution
mutations at other locations appeared to have less significant effects
on promoter activity. Second, 25% of the wild type promoter activity was retained following disruption of the NF-
B element. This activity appeared to be too strong to be directed solely by a C/EBP-binding site
and a TATA box. In particular, of the 21 clustered substitution mutants
analyzed in this previous study, four (
114/
109,
107/
102,
80/
75, and
62/
57) were identified that demonstrated a 2-3-fold decrease in promoter activation (17). These elements, which have
relatively minor effects individually, may have additive or synergistic
effects when combined with mutations in other sites.
As mutations in the C/EBP site abrogate promoter activity, the
functional significance of other potentially important sites (
114/
109,
107/
102,
80/
75,
62/
57) needs to be evaluated in the presence of an intact C/EBP site. The
114/
109,
107/
102, and
62/
57 substitution mutants were described previously (17). However, the
80/
75 mutant from the initial study was re-derived by
changing a 13-base pair sequence from
85 to
73. This more extensive
mutant may demonstrate more significant functional effects. Furthermore, as random mutants may also have artifactual effects, a
different random sequence was inserted to rule out this possibility.
Site-directed mutants in two elements other than the C/EBP site were
constructed in the
355 to +55 wild type p40 promoter-CAT reporter
(Fig. 1A). The murine
macrophage cell line RAW264.7 was transiently transfected with double
mutant, single mutant, and wild type plasmids. Consistent with our
previously reported results, a mutation in the NF-
B element (Fig.
1B,
132/
127) reduces LPS-induced promoter activation by
around 80%, and a mutation in the C/EBP site (Fig. 1B,
93/
88,) virtually eliminates promoter activity. Simultaneous disruption of the NF-
B element and the sequence from
85 to
73 markedly reduces promoter activation by LPS compared with
the respective single mutations (Fig. 1B; compare the double mutant [
85/
73 and
132/
127 to the single mutant
85/
73
and the single mutant
132/
127). Three other mutations
(
114/
109,
107/
102, and
62/
57), when combined with mutations
in the NF-
B site, also demonstrate significantly diminished p40
promoter activity (Fig. 1B). A double mutant in the
114/
109 and
107/
102 elements does not attenuate promoter
activation to a greater extent than the respective single mutants (Fig.
1B). Therefore, one control element likely resides within
these two adjacent sequences. In summary, although each of these single
mutations had a relatively small effect on promoter activation, their
combination with a mutation in the NF-
B site demonstrates dramatic
decreases in LPS-induced promoter activity. These results identify
three novel elements, from
114 to
102,
82 to
73, and
62 to
57, important for activation of IL-12 p40 transcription by LPS, and
provide clear evidence that an intact C/EBP site is not sufficient for promoter activation.

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Fig. 1.
Functionally redundant cis-acting control
elements in the IL-12 p40 promoter are demonstrated by mutation of two
elements other than the C/EBP site. A, diagram of the
IL-12 p40 promoter showing the two most important regulatory domains,
the NF- B ( 132/ 127) and C/EBP ( 93/ 88) elements, and other
potentially important regions detected in a previous analysis (17).
B, site-directed mutations in potentially important elements
( 114/ 109, 107/ 102, 85/ 73, 62/ 57) when combined with a
mutation in the NF- B site ( 132/ 127) demonstrated significant
diminution of LPS-induced promoter activity, compared with the
respective single mutant promoters (compare mutant 85/ 73 and
132/ 127 with mutant 85/ 73; mutant 114/ 109 and
132/ 127 with 114/ 109; mutant 107/ 102 and
132/ 127 with 107/ 102; mutant 62/ 57 and
132/ 127 with 62/ 57). The single mutant in the NF- B
site ( 132/ 127), the single mutant in the C/EBP site
( 93/ 88), and wild type promoter ( 350 to +55
p40-CAT) activities are represented at the top of the
figure. Two µg of each single and double mutant promoter CAT reporter
plasmid and 0.5 µg of HSP- -galactosidase reporter plasmid were
transfected into RAW264.7 macrophage cells. Transfected cells were
incubated for 24 h and then activated with LPS (5 µg/ml) for
16-18 h. CAT activity was quantitated following thin layer
chromatography by PhosphorImager analysis, and results were expressed
as percent conversion of [14C]chloramphenicol to
acetylated substrates, normalized to -galactosidase activity
obtained from a co-transfected control reporter plasmid. Each result
represents the mean ± the S.D. of data from three to five
experiments.
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As a second approach, a minimal inducible IL-12 p40 promoter was
defined. From our previous analysis, a promoter deletion of all
elements upstream of the C/EBP site mediated strong LPS-inducible promoter activity of greater than 10% of the wild type
355 to +55
promoter (17). Therefore, functional control elements likely exist
downstream of the C/EBP element. To test this hypothesis, site-directed
mutants were created in elements downstream of the C/EBP site in a
promoter from
101 to +55, which deletes all elements upstream of the
C/EBP element. As expected, a mutation in the C/EBP site within this
minimal promoter eliminates LPS-induced activation (Fig.
2,
101 to +55 and
93/
88)
in RAW264.7 cells. This experiment confirms the functional importance
of the element from
85 to
73, as a mutation in this site markedly
reduces promoter activity (Fig. 2,
101 to +55 and
82/
74). Furthermore, the
62 to
57 mutant sequence reduces
promoter activity by ~70% (Fig. 2,
101 to +55 and
62/
57).

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Fig. 2.
Functionally redundant cis-acting control
elements are identified downstream of the C/EBP site in the IL-12 p40
promoter using a minimal LPS-inducible promoter. The p40
promoter-CAT reporter plasmid 101 to +55 contains 5'-elements
beginning at the C/EBP site at position 101 relative to the
transcription start site. Within this minimal promoter, 6-10-base pair
substitution mutants were created between 93 and 51. The plasmids
were transfected into RAW264.7 cells, incubated for 24 h, and then
activated with LPS (5 µg/ml) for 16-18 h. Extracts from
LPS-activated cells were analyzed by CAT assay. Results (normalized for
-galactosidase activity) are expressed as the percentage of the CAT
activity obtained with the 101 to +55 plasmid. Plasmid 101 to
+55 and 93/ 88 represents a mutation in the C/EBP element. Two
mutations in plasmid 101 to +55 ( 82/ 74 and 62/ 57) had
significant effects on LPS-induced promoter activation. Each result
represents the mean ± S.D. of data from four to five
experiments.
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Detailed Characterization of a Functional Element within the
82
to
73 Region--
By two different approaches, the sequence between
82 and
73 is demonstrated to have an important functional role in
IL-12 p40 promoter activation by LPS. This control element may contain a binding site for one transcription factor or may represent a composite binding site for two or more proteins. To define better critical nucleotides within this element, 3-base pair site-directed mutants were constructed within this sequence (Fig.
3A). Two mutants,
79/
77
and
76/
74, demonstrate significantly decreased LPS-induced promoter
activity, whereas mutants in the flanking sequences retain wild type
activity (Fig. 3B). Thus, an important regulatory element is
localized to the region spanning
79 to
74.

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Fig. 3.
Detailed characterization of a functional
element within the 82 to 73 region of the IL-12 p40 promoter.
A, within the murine IL-12 p40 promoter from 350 to +55,
3-base pair substitution mutants were created between 85 and 71.
Specific substitution mutants are indicated below the
sequence. B, an important control element resides within the
79 to 74 region. Plasmids were transfected into RAW264.7 cells,
incubated for 24 h, then activated with LPS (5 µg/ml) for 16-18
h. Extracts from LPS-activated cells were analyzed by CAT assay.
Results (normalized for -galactosidase activity) are expressed as
the percentage of the CAT activity obtained with the 350 to +55 wild
type promoter. Each result represents the mean ± S.D. of data
from four to five experiments.
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Determination of DNA-Protein Interactions in the
79 to
74
Region: Identification of an AP-1-binding Element--
To identify
DNA-protein interactions in the
79 to
74 region, EMSA probes were
constructed that spanned 1) the C/EBP-binding site and
79/
74
(118/69), 2) the C/EBP site alone (112/78), and 3)
79/
74 excluding
the C/EBP site (88/63) (Fig.
4A). With the 88/63 probe, a
slow mobility DNA-protein complex is detected that is strongly
induced in nuclear extracts from RAW264.7 cells activated with LPS
(compare Fig. 4B, lanes 9 and 10, upper
arrow). This complex is present in EMSAs with probe 118/69 (Fig.
4B, lanes 1-4), containing the C/EBP site and downstream
sequences, but not with probe 112/78 (Fig. 4B, lanes 5-8)
that spans only the C/EBP site.

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Fig. 4.
Determination of DNA-protein interactions in
the 85/ 73 region of the murine IL-12 p40 promoter.
A, electrophoretic mobility shift assay probes. EMSA probe
88/63 spans the promoter sequence between 88 to 63 that
contains the newly described AP1-binding site. Probe 112/78
contains only the C/EBP-binding site sequence. Probe 118/69
contains both C/EBP-binding site and AP1-binding site. B,
EMSAs were performed with nuclear extracts from unactivated and
LPS-activated RAW264.7 cell. Cells were either untreated or activated
with LPS (5 µg/ml) for 4 h. Five µg of extracts from
unactivated (lanes 1, 5, and 9) and LPS-treated
(lanes 2-4, 6-8, and 10-12) cells were added
to 32P-labeled probes 118/69 (lanes 1-4),
112/78 (lanes 5-8), and 88/63 (lanes 9-12).
Extract and probe were incubated with 0.5 µg of poly(dI-dC) at room
temperature for 30 min. For competition experiments, 100-fold molar
excess of unlabeled consensus C/EBP or AP-1 oligonucleotide was then
added for 30 min, as indicated above each lane, prior to
electrophoresis. The upper arrow on the left side
of the figure indicates AP-1 DNA binding activity, which is visible
with probes 118/69 and 88/63 but not with probe 112/78. The two
lower arrows indicate C/EBP DNA binding activity.
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The DNA sequence from
81 to
73, CTAGTCAGT, has homology to a
canonical AP-1 DNA-binding element (30). The fos/jun
proto-oncogenes encode proteins that are major components of the AP-1
transcription factor complex. Several Fos-related (c-Fos, FosB, Fra-1,
and Fra-2) and Jun-related (c-Jun, JunB, and JunD) proteins have been
described. These leucine zipper proteins form the transcriptionally
active AP-1 complex as Fos/Jun heterodimers or Jun/Jun homodimers (31, 32). Competition with an unlabeled oligonucleotide containing a
consensus AP-1-binding site in 100-fold molar excess completely inhibits formation of this complex in EMSAs, strongly suggesting that
AP-1 family members interact with this sequence in the IL-12 p40
promoter (Fig. 4B, lanes 4 and 12). A consensus
C/EBP-binding site partially competes AP-1 DNA binding activity in gel
shift experiments, suggesting that either C/EBP family members are
present in this complex or that AP-1 dimers may to some extent bind to a consensus C/EBP element (Fig. 4B, lanes 3 and
11). Competition with a consensus NF-
B DNA-binding site
oligonucleotide does not inhibit complex formation on all three
radiolabeled probes (data not shown). Mutants within the 88/63 EMSA
probe, 79/77m (mutated from
79 to
77) and 76/74m (mutated from
76
to
74) eliminate binding of the AP-1 complex (Fig.
5, lanes 3-6). These specific mutants correlate with decreased promoter activity in functional assays
(Fig. 3B).

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Fig. 5.
AP-1 DNA binding activity is eliminated by
mutations that decrease IL-12 p40 promoter activation by LPS in
reporter assays. Two mutant EMSA probes (79/77m and
76/74m) were created within the wild type probe 88/63.
32P-Labeled wild type probe 88/63 (lanes 1 and
2), mutant probe 79/77m (lanes 3 and
4), and mutant probe 76/74m (lanes 5 and
6) were incubated with 0.5 µg of poly(dI-dC) and 5 µg of
nuclear extracts from untreated (lanes 1, 3, and
5) and LPS-activated (lanes 2, 4, and
6) RAW264.7 cells for 30 min prior to electrophoresis. Both
mutant probes, which correlate with the decreased promoter activity in
CAT assays (see Fig. 3B), do not demonstrate AP-1 DNA
binding activity (arrow on left).
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The presence of AP-1 family members in this DNA-binding complex was
confirmed by supershift experiments. A polyclonal antibody to the AP-1
family Fos (Fig. 6, lanes 5 and 15, K-25) strongly inhibits and supershifts the
DNA-protein complex. By using antibodies that recognize specific Fos
family members, it appears that the most abundant Fos DNA binding
activity is attributable to c-Fos (Fig. 6, lanes 6 and
16). Antibodies to Fra-1 and Fra-2 cause minor inhibition of
complex formation (Fig. 6, lane 8, 9, 18, and
19). An antibody that recognizes the Jun family member,
JunB, strongly inhibits and supershifts the DNA-binding complex (Fig. 6, lanes 3 and 13). Additionally, an antibody to
c-Jun causes inhibition and supershift of the complex (Fig. 6,
lanes 2 and 12). Antibodies directed against the
leucine zipper proteins ATF-1, ATF-2, and C/EBP
demonstrate little,
if any, inhibition of complex formation in nuclear extracts from
LPS-treated cells (data not shown). Likewise, antibodies against
unrelated transcription factors Pu.1 (Fig. 6, lanes 10 and
20), NF-
B p50, NF-
B p65, and the glucocorticoid
receptor (data not shown) do not supershift or inhibit the DNA-binding
complex. Thus, the AP-1 DNA-binding complex on the IL-12 p40 promoter
is composed of JunB and c-Jun homodimers and/or heterodimers with
c-Fos. However, other AP-1 family members and leucine zipper proteins
may be present as well.

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Fig. 6.
Identification of specific AP-1 family
members in the IL-12 p40 DNA-binding complex by supershift assay.
32P-Labeled EMSA probe 88/63 was incubated with 0.5 µg
poly(dI-dC), and 5 µg of nuclear extracts from untreated (lanes
1-10) or LPS-treated (lanes 11-20) RAW264.7 cells at
room temperature for 30 min. Then 1.5 µl of polyclonal antibodies
against c-Jun (lanes 2 and 12), JunB (lanes
3 and 13), JunD (lanes 4 and 14),
c-Fos (K-25, lanes 5 and 15), c-Fos
(H-125, lanes 6 and 16), FosB
(lanes 7 and 17), Fra1 (lanes 8 and
18), Fra2 (lanes 9 and 19), and Pu.1
(lanes 10 and 20) were added. Results demonstrate
that the IL-12 p40 DNA-binding complex contains Fos family members,
JunB and c-Jun, as demonstrated by inhibition and supershift of DNA
binding activity. However, other basic ZIP proteins are likely present
in smaller amounts, as demonstrated by inhibition of the DNA-binding
complex by other antibodies but not by antibodies against non-basic ZIP
proteins (lanes 10 and 20, and data not shown).
The arrow to the left of the figure denotes the
location of the AP-1 DNA-binding complex.
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Determination of Spacing Requirements between Control Elements in
the IL-12 p40 Promoter--
Proper spacing of cis-acting elements may
be necessary for the interaction of transcriptional activators with the
basal transcription machinery, or it may be required for cooperative
binding of transcription factors (33, 34). Spacing restrictions may
involve distance or alignment of sites on the DNA double helix. The
functional effects of spacing between the TATA box and regulatory
elements as well as between control elements within the IL-12 p40
promoter were next determined. Insertion mutants were constructed
within the
355 to +55 p40 promoter that increase the distance between the AP-1 and the C/EBP site by 5, 10, and 15 bp, approximating one-half, one, and one and a half helical turns of DNA, respectively (Fig. 7A). In addition,
insertion mutants adding 5 and 10 bp between the C/EBP site and NF-
B
element and 5 and 10 bp between the C/EBP site and the TATA box were
created to determine spacing requirements between C/EBP, NF-
B, and
the basal transcription machinery. All mutations were inserted within
sequences that were not critical for promoter activation (
45/
40 for
mutants between TATA and C/EBP site,
121/
117 for mutants between
NF-
B site and C/EBP site, and
84/
85 for C/EBP and AP-1 elements)
(17). These mutants were tested by transient transfection in
unactivated and LPS-activated RAW264.7 cells. Spacing mutations between
the TATA box and C/EBP site have little effect on promoter activity
(>80% of wild type activity; data not shown). However, mutations
between the NF-
B and C/EBP sites, and the C/EBP and AP-1 elements
reveal significant effects on promoter induction. A 10-bp insertion
between the NF-
B and C/EBP elements decreases LPS-induced promoter
activation by 65% (Fig. 7B). A 10-bp insertion between the
C/EBP and AP-1 sites diminishes LPS-induced promoter activation by
one-third, whereas a 5- and 15-bp insertion decreases activation by
50% (Fig. 7B). As random sequences inserted within the
promoter may fortuitously create a protein-binding site, the 5- and
10-bp mutants between the C/EBP and AP-1 elements were re-derived with
different sequences, and similar results were demonstrated in reporter
assays (data not shown).

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Fig. 7.
Determination of spacing requirements between
control elements in the IL-12 p40 promoter. A, 5-, 10-, and 15-base pair insertion mutants were created at 119 and 84
within the IL-12 p40 350 to +55 promoter-CAT reporter plasmid.
B, insertion mutant plasmids were transfected into RAW264.7
cells, incubated for 24 h, and then activated with LPS (5 µg/ml)
for 16-18 h. Extracts from LPS-activated cells were analyzed by CAT
assay. Results (normalized for -galactosidase activity) are
expressed as the percentage of the CAT activity obtained with the 350
to +55 wild type promoter. These results suggest that spacing between
the C/EBP and NF- B elements, as well as between the C/EBP and AP-1
sites, are important for
promoter activation. Each result represents the mean ± S.D.
of data from four to five experiments. C, spacing mutants
between the C/EBP and AP-1 elements have more dramatic effects on
promoter activation by LPS in the context of a minimal deleted
promoter, 101 to +55. Results (normalized for -galactosidase
activity) were expressed as the percentage of the CAT activity obtained
with the 101 to +55 plasmid. Each result represents the mean ± S.D. of data from four to five experiments. D, the specific
sequence and orientation of the AP-1 site in the IL-12 p40 promoter is
important for activation by LPS. Within the 350 to +55 p40
promoter-CAT reporter plasmid, the AP-1-binding site from 81 to 75
was mutated to a canonical AP-1 site ( 81 to 75 from CTAGTCA to
TGAGTCA). This mutation strikingly increases LPS-induced promoter
activation to almost 6 times wild type activity ( 350 to +55) and
demonstrates a strong basal signal (20% of LPS-induced wild type
activity) in unactivated cells. Also, the IL-12 p40 AP-1 site at 81
to 75 was reversed in orientation from CTAGTCA to ACTGATC. This
mutation decreases LPS-induced promoter activity to 20% of wild type
( 350 to +55). Plasmids were transfected into RAW264.7 cells,
incubated for 24 h, and then activated with LPS (5 µg/ml) for
16-18 h. Extracts from LPS-activated cells were analyzed by CAT assay.
Results (normalized for -galactosidase activity) are expressed as
the percentage of the CAT activity obtained with the 350 to +55 wild
type promoter. Each result represents the mean ± S.D. of data
from three experiments.
|
|
As the effect of spacing between the C/EBP and AP-1 sites may have been
minimized by redundant control elements, insertion mutants were then
constructed within the
101 to +55 minimal promoter. In this context,
the effect of 5-, 10-, and 15-bp insertions are more dramatic, reducing
promoter activity more than 60% (Fig. 7C).
Based on comparison to consensus sequences, there are two potential
AP-1-binding sites within the
79 to
74 region (Fig. 3). To localize
better the AP-1 DNA-binding element, and to determine whether the
sequence and orientation of this site in the IL-12 p40 promoter
influences activity, additional mutants were created. Within each of
these two potential AP-1 sites, a canonical AP-1-binding site was
created, and the orientation of the AP-1 site was reversed. From
79
to
73, a canonical AP-1-binding site was created by changing the
sequence AGTCAGT to ACTGAGT. This mutation decreases LPS-induced
promoter activation by almost 75% (data not shown). A second mutant
reversed the orientation from
79 to
73, by changing AGTCAGT to
TGACTGA. This mutation also diminishes LPS-induced promoter activity by
75% (data not shown). From
81 to
75, a canonical AP-1 site was
constructed by changing the sequence CTAGTCA to TGAGTCA. When
transfected into RAW264.7 cells, this mutation strikingly increases
LPS-induced promoter activation to almost 6 times wild type activity
(Fig. 7D). Significantly, this mutant demonstrates a strong
basal signal (20% of LPS-induced wild type activity) in unactivated
cells (Fig. 7D). Next, the IL-12 p40 AP-1 site at
81 to
75 was reversed in orientation from CTAGTCA to ACTGATC. This mutation
reduces LPS-induced promoter activity by 5-fold (Fig. 7D).
An EMSA probe was created containing this consensus AP-1 sequence from
81 to
75 in the p40 promoter. Compared with the wild type probe
88/63 (see Fig. 4), AP-1 DNA binding activity is markedly increased in
unactivated and LPS-treated nuclear extracts from RAW264.7 cells (data
not shown). Supershift experiments suggest that DNA binding activities
on this mutant probe are attributable to JunB, c-Fos, and c-Jun (data
not shown), as demonstrated for the wild type probe. The most plausible
interpretation of these results is that the IL-12 p40 AP-1 site lies
within the
81 to
75 region. Hence, the consensus AP-1 site created
from
79 to
73 would actually weaken the natural AP-1 element at
81 to
75. Also, orientation of this AP-1 site is important for
optimal promoter activation, as reversal of the sequence of this
element significantly diminishes promoter activity.
Expression of a Dominant Negative c-Fos Molecule Inhibits IL-12 p40
Promoter Activation by LPS--
To demonstrate a functional role for
AP-1 in IL-12 p40 promoter activity, a dominant negative AP-1 molecule,
A-Fos (20), was transfected into RAW264.7 macrophages. A-Fos contains
an acidic amphipathic protein sequence appended to the N terminus of
the Fos leucine zipper and lacks a DNA binding domain (20). A-Fos inhibits LPS-induced
350 to +55 IL-12 p40 promoter activation in a
dose-dependent manner, with maximal inhibition of ~50%
(data not shown). A-Fos also inhibits activation of the p40 promoter by
C/EBP
by 50% (data not shown). In the context of the minimal
101
to +55 p40 promoter, the effects of A-Fos are more significant, with
inhibition of LPS-induced promoter activation by 70% and inhibition of
C/EBP
-induced activity by greater than 50% (Fig. 8). These inhibitory effects are
dependent on the AP-1 DNA-binding element, as LPS- and C/EBP
-induced
activity of the
350 to +55 p40 promoter with a mutated AP-1 site
(
85/
73) is not inhibited by A-Fos (data not shown). This result
strongly suggests that A-Fos inhibits IL-12 p40 promoter activation by
heterodimerizing with endogenous Jun family members but will not
inhibit C/EBP-mediated transcriptional activation in the absence of an
adjacent AP-1 element. Furthermore, the inhibitory effect of A-Fos does
not represent a nonspecific phenomenon such as transcriptional
squelching. A-Fos expression does not decrease the activity of two
constitutively active promoters, a cytomegalovirus viral enhancer and
an SP-1 multimer upstream of a minimal core promoter (data not
shown).

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Fig. 8.
Expression of a dominant negative c-Fos
molecule inhibits IL-12 p40 promoter activation by LPS and
C/EBP . The 101 to +55 p40 minimal
promoter-CAT reporter plasmid was co-transfected with an expression
plasmid for A-Fos at different concentrations (0, 0.1, 0.25, 0.5, and
0.75 µg). Plasmids were transfected into RAW264.7 cells, incubated
for 24 h, and then activated with LPS (5 µg/ml) for 16-18 h.
Extracts from LPS-activated cells were analyzed by CAT assay
(black bars). In a parallel series of experiments, a
constant amount of expression plasmid for the C/EBP (0.1 µg) was
co-transfected with A-Fos (white bars). In these
experiments, promoter activity was determined in untreated cells.
Results (normalized for -galactosidase activity) are expressed as
the percentage of the CAT activity obtained with the 101 to +55 wild
type promoter co-transfected with 0.1 µg of C/EBP
expression plasmid (white bars). Results represent the
mean ± S.D. of four to five experiments.
|
|
c-Jun and a Mutant c-Jun Molecule Activate the
IL-12 p40 Promoter and Synergize with C/EBP
--
To investigate a
role for individual AP-1 proteins, mammalian expression plasmids for
AP-1 family members were co-transfected with a
101 to +55 p40
promoter-luciferase reporter into RAW264.7 cells. Neither JunB nor
c-Fos expression activate the promoter (Fig.
9A). However, expression of
c-Jun strongly activates the promoter (Fig. 9A) in a
dose-dependent manner (data not shown). Co-transfection of
Jun family members with c-Fos does not augment promoter activity (data
not shown). Within the TNF promoter, a functional interaction between a
C/EBP element immediately adjacent to an AP-1 site from
106 to
74
has been described (35). Expression of c-Jun and a mutant c-Jun
molecule that lacks the C-terminal 67 amino acid transactivation
domain, TAM67, were both capable of activating the TNF promoter (35).
As TNF and IL-12 are critical mediators of inflammatory responses, we
tested whether TAM67 activates the TNF and IL-12 p40 promoters in
macrophages. Expression of TAM67 in RAW264.7 cells activates the
101
to +55 IL-12 p40 promoter to the same extent as c-Jun (Fig.
9A). Furthermore, TAM67 activates a
283 to +113 TNF
promoter luciferase reporter in RAW264.7 cells (Fig. 9B).
The effect of TAM67 on the TNF and IL-12 p40 promoters appears to be
specific; c-Jun, but not TAM67, activates a multimerized consensus AP-1
DNA-binding element CAT reporter plasmid (TRE-CAT) in
untreated RAW264.7 cells (data not shown). Furthermore, TAM67, originally described as a dominant negative inhibitor of AP-1-mediated transcription (19), strongly inhibits LPS-induced TRE-CAT
activity (85%), whereas it augments LPS-induced IL-12 p40 promoter
activity by 2-fold in RAW264.7 cells (data not shown).

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Fig. 9.
c-Jun and a deleted c-Jun mutant, TAM67,
activate the IL-12 p40 and the TNF- promoters
in RAW264.7 cells. A, 101 to +55 IL-12 p40
promoter-luciferase reporter plasmid was co-transfected with 0.5 µg
of expression plasmids for c-Jun, TAM67, JunB, and c-Fos. Plasmid
amounts were normalized to 2.5 µg by the addition of empty expression
plasmid. Following transfection, RAW264.7 cells were incubated for
36 h. Experiments with AP-1 expression plasmids were conducted in
unactivated cells. As controls, cells were transfected with 2.5 µg of
empty expression plasmid and remained unactivated or were activated
with LPS (5 µg/ml) for 18 h prior to termination. B,
to TNF- promoter-luciferase reporter was co-transfected with 0.5 µg of expression plasmids for c-Jun, TAM67, JunB, and c-Fos, as in
A. C, c-Jun and TAM67 synergistically activate
the IL-12 p40 promoter when co-expressed with C/EBP . A 101 to +55
IL-12 p40 promoter-luciferase reporter plasmid was co-transfected with
expression plasmids for C/EBP (0.1 µg) and AP-1 proteins (0.5 µg), as in A. Results are expressed as fold activation
compared with cells transfected with C/EBP alone. A-C,
results (relative light units normalized for -galactosidase
activity) are expressed as the percentage of luciferase activity
obtained with the 101 to +55 wild type promoter activated with LPS.
Each result represents the mean ± S.D. of three to five
experiments.
|
|
To assess functionally additive or synergistic effects between C/EBP
and AP-1 in IL-12 p40 promoter activation, C/EBP
was co-expressed
with AP-1 family members in RAW264.7 cells. Expression of C/EBP
with
c-Jun results in 8-fold greater promoter activation than with C/EBP
alone and 4-fold greater promoter activity than with c-Jun alone (Fig.
9C). Similar results are demonstrated with TAM67 (Fig.
9C). However, synergistic activation is not apparent when
JunB and C/EBP
are co-expressed (Fig. 9C).
MAP Kinase and TLR Signaling Pathways Activate the IL-12 p40
Promoter through Control Elements in the
101 to +55
Region--
Recently, the p38 MAP kinase and the toll-like receptor
(TLR) signal transduction pathways have been linked to IL-12 p40 gene expression (25, 36). However, downstream targets of these pathways that
mediate IL-12 p40 promoter activation have not been identified.
Therefore, a series of dominant negative and constitutively active
molecules from the p38 kinase, JNK, and TLR signal transduction pathways were tested for effects on IL-12 p40 promoter activity. All
signal transduction molecules and soluble inhibitors display dose-dependent effects on promoter activity (data not
shown). To demonstrate activation and inhibition of the p38 pathway in RAW264.7 cells, activity of a GAL4-luciferase reporter by a MEF2c-GAL4 DNA-binding domain fusion protein (37) was assessed. In RAW264.7 cells,
LPS strongly induces p38 activity. The soluble p38 inhibitor SB202190
(1 µM) inhibits LPS-induced p38-mediated transcription by
60%, and constitutively active MKK3 and MKK6 strongly induced this
reporter in unactivated cells (data not shown). For the IL-12 p40
promoter, SB202190 inhibits LPS-induced activity of the
101 to +55
promoter, whereas the structurally similar control SB202474 does not
demonstrate inhibition (Fig.
10A). Furthermore,
expression of constitutively active MKK3 or MKK6 induces
101 to +55
p40 promoter activity in untreated cells, although not to the same extent as LPS (Fig. 10A).

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Fig. 10.
MAP kinase and TLR signal transduction
pathways modulate IL-12 p40 promoter activity through elements within
the 101 to +55 region. For each experiment, 0.5 µg of the
respective expression plasmid was transfected into RAW264.7 cells.
Cells were incubated for 24 h and then activated with LPS (5 µg/ml) for 16-18 h or left unactivated over this time. Results
(relative light units normalized for -galactosidase activity) are
expressed as the percentage of luciferase activity obtained with the
101 to +55 IL-12 p40 promoter activated with LPS. Each result
represents the mean ± S.D. of four to six experiments.
A, p38 MAP kinase pathway: LPS-induced activity of the IL-12
p40 101 to +55 promoter in RAW264.7 cells is inhibited by the soluble
p38 inhibitor, SB202190 (1 µM), but not a structurally
similar control, SB202474 (1 µM). Basal promoter activity
is induced by expression of constitutively active MKK3 (0.5 µg of
expression plasmid) and MKK6 (0.5 µg of expression plasmid).
B, JNK pathway: LPS-induced activity of the IL-12 p40 101
to +55 promoter in RAW264.7 cells is inhibited by expression of JIP-1
(0.5 µg of expression plasmid), and basal activity is strongly
induced by expression of constitutively active MEKK1 (0.5 µg of
expression plasmid). C, TLR signaling pathway: LPS-induced
activity of the IL-12 p40 101 to +55 promoter in RAW264.7 cells is
inhibited by expression of DN MyD88, DN TRAF6, or DN TLR2, and basal
activity is induced by expression of constitutively active CD4-TLR4
(0.5 µg of expression plasmid in each experiment). DN,
dominant negative.
|
|
The role of the JNK pathway in IL-12 p40 promoter activation has not
been determined, although the description of an AP-1 site suggests that
this pathway may be involved. LPS activates the JNK pathway in RAW264.7
cells, as assessed by activation of a GAL4-luciferase reporter by a
c-Jun-GAL4 DNA-binding domain fusion protein (data not shown). A
dominant negative inhibitor of JNK, JIP-1, inhibits LPS-induced
JNK-mediated transcription, while constitutively active MEKK1 strongly
up-regulated activity in untreated cells (data not shown). Consistent
with involvement of the JNK pathway in IL-12 p40 promoter activation,
JIP-1 inhibits LPS-induced
101 to +55 promoter activity, and active
MEKK1 strongly activates this minimal p40 promoter (Fig.
10B). As MEKK1 also induces NF-
B (38), it is important to
note that this effect occurs with a p40 promoter that lacks an NF-
B element.
Dominant negative signal transduction molecules in the Toll/IL-1
receptor pathway inhibit IL-12 p40 promoter activation by bacterial
products (25). As NF-
B is a well described target of TLR signaling,
we asked whether this signal transduction pathway affects IL-12 p40
promoter activation through elements other than NF-
B. Expression of
a dominant negative TLR2, TRAF6, and MyD88 inhibits LPS-induced
activity of the IL-12 p40
101 to +55 promoter (Fig. 10C)
to the same magnitude as they inhibit activation of a multimerized
NF-
B element luciferase reporter in RAW264.7 cells (data not shown).
Similar results were obtained when RAW264.7 cells were activated with
heat-killed L. monocytogenes and M. tuberculosis
whole cell extract (data not shown). Furthermore, a constitutively
active CD4-TLR4 fusion protein activates the
101 to +55 promoter in
untreated cells, although not to the same degree as LPS (Fig.
10C). Once again, similar results are obtained for IL-12 p40
promoter constructs containing and lacking the NF-
B site (data not
shown), suggesting that the TLR pathway may influence promoter
activation through elements other than NF-
B.
 |
DISCUSSION |
In summary, functional and DNA binding assays reveal complex
interactions within a small region of the IL-12 p40 promoter. A C/EBP
DNA-binding element was previously characterized at
96 to
88. In
the current study, mutations from
79 to
74 in the murine IL-12 p40
promoter significantly reduce LPS-induced promoter activation and
correlate with DNA binding of AP-1 by EMSA. AP-1 family members
detected in the IL-12 p40 promoter DNA-binding complex include JunB,
c-Jun, and c-Fos. Spacing between the C/EBP and AP-1 site and
orientation of the AP-1 site are important for promoter activation,
suggesting cooperativity between these two sites. Expression of a
dominant negative AP-1 protein, A-Fos, in RAW264.7 cells inhibits p40
promoter activation by LPS and C/EBP
. c-Jun and a c-Jun mutant TAM67
activate the IL-12 p40 promoter and synergize with C/EBP
in
transient expression studies. c-Jun and TAM67 also activate the TNF
promoter in RAW264.7 cells. Finally, this small region of the promoter
downstream of
101 is identified as a target for MAP kinase and TLR
signal transduction pathways.
This group had reported a comprehensive functional analysis of the
murine and human IL-12 p40 promoters in RAW264.7 cells (17). A critical
control element for promoter activation by bacterial products, located
between
96 and
88, interacts with C/EBP family members. This
element was functionally synergistic with the NF-
B site between
131 and
120 (13). In this analysis, when the C/EBP site was
mutated, virtually all LPS-induced promoter activity was lost. Thus, it
was possible to conclude that the C/EBP site was both necessary and
sufficient for promoter activation. To characterize effects of other
cis-acting elements, a strategy was developed to assess the functional
role of other elements in the presence of an intact C/EBP site. First,
mutations were created in two cis-acting elements other than the C/EBP
site. A second approach utilized a minimal promoter where all elements upstream of the C/EBP site were deleted. These experiments demonstrate the existence of three important cis-acting elements at
114 to
102,
82 to
73, and
62 to
57. Of note, the functional regions described in this study correspond to constitutive and bacterial product-inducible DNase I footprints in the previous analysis (17).
Furthermore, these regions likely correspond to DNA-protein interactions detected on the endogenous IL-12 p40 promoter in primary
macrophages by in vivo genomic DNase I footprinting (39). Taken together, these results provide clear evidence that the C/EBP
site may be necessary but is not sufficient for LPS-induced promoter activation.
A cis-acting control element at
207 to
211 in the human IL-12 p40
promoter that binds a multiprotein complex consisting of Ets-2,
interferon regulatory factor-1, interferon consensus sequence binding
protein, and c-Rel has been extensively characterized (14-16, 40).
Although we could not demonstrate a functional effect for a single
site-directed substitution mutant in a previous analysis (17), a
functional effect may be demonstrable by methodologies employed in the
current study. Thus, this analysis and others suggest that at least six
control elements within a small region of the IL-12 p40 promoter may be
necessary for activated gene expression.
One novel site defined by this analysis from
84 to
73 was
characterized in detail. A control element was identified from
79 to
74 that binds AP-1 family members. Supershift experiments detect
predominantly c-Fos, JunB, and to a lesser extent, c-Jun in the IL-12
p40 EMSA complex. However, relative amounts of AP-1 family members
cannot be accurately deduced from these results, as the degree of
inhibition or supershift is a function of the antibody as well as the
relative amount of protein in the DNA-binding complex. Furthermore, a
recent study demonstrated that c-Rel, a relatively minor component of
the IL-12 p40 NF-
B DNA-binding complex, is the relevant activator of
p40 transcription in cell line models and in vivo (41).
Therefore, from our functional results, c-Jun may be the relevant
activator of the IL-12 p40 promoter despite the apparent abundance of
JunB and c-Fos in the EMSA complex.
To demonstrate a role for AP-1 in IL-12 p40 promoter activation, a
dominant negative AP-1 molecule, A-Fos, was expressed in RAW264.7
macrophages. A-Fos contains an acidic amphipathic protein sequence
appended onto the N terminus of the c-Fos leucine zipper. This
extension physically interacts with the Jun basic region and prevents
the basic region from binding DNA. This interaction was demonstrated to
be specific. A-Fos inhibited transcriptional activation of
Jun-dependent reporters but not C/EBP-dependent reporters (20). Expression of A-Fos significantly inhibits promoter activation by LPS or C/EBP
(Fig. 8), demonstrating that AP-1 DNA
binding is critical for IL-12 p40 promoter activation. Then, the
potential role of individual AP-1 family members in IL-12 p40 promoter
activation was determined. Interestingly, c-Jun and a deleted c-Jun
mutant, TAM67, activate the p40 promoter in untreated RAW264.7 to a
similar magnitude as LPS and synergistically activate the promoter when
co-expressed with C/EBP
. Furthermore, JunB and c-Fos, although
abundantly detected in EMSA complexes, are unable to activate the
promoter or augment C/EBP
-induced activation.
Important information about functional synergy between the AP-1 and
C/EBP element was obtained from experiments that altered spacing
between these sites. A 5-, 10-, and 15-bp insertion between AP-1 and
C/EBP markedly decreases promoter activation by LPS. In other
promoter/enhancers, spatial arrangement of transcription factor binding
sites on the double helix is critical for promoter activation (33, 34).
For the p40 promoter, it appears that distance rather than arrangement
of these sites on the double helix is critical for optimal activation,
as the addition of any length decreased reporter activity. A promoter
mutant that created a canonical AP-1 site from
81 to
75
demonstrates markedly increased LPS-induced promoter activity and
strong basal activity (Fig. 7D), suggesting that this region
contains the AP-1 site. However, precise mapping of the IL-12 p40 AP-1
element will require a careful analysis of DNA-protein contacts using
methylation interference assays and further mutagenesis of the region.
When the AP-1 site is reversed in orientation, a marked decrease in
LPS-induced activity is found. This result may suggest a
protein-protein interaction with C/EBP or another factor or may be
related to the ability of AP-1 to bend DNA, thus facilitating
protein-protein interactions (42).
AP-1 family members are prototypic targets for the c-Jun N-terminal
kinase (JNK) MAP kinase pathway. Although p38 and extracellular signal-regulated kinases have been implicated in IL-12 p40 gene expression (36, 43), the role of the JNK pathway is unclear. In this
study, the JNK pathway is implicated in IL-12 p40 promoter activation
using a dominant negative inhibitor of JNK, JIP-1, and a constitutively
active MEKK1 molecule. Interaction of conserved bacterial ligands with
TLRs is important for activation of IL-12 p40 gene expression in
macrophages (25). However, specific promoter region and transcription
factor targets that modulate IL-12 p40 gene expression have not been
identified. For TLR signaling, NF-
B is a critical downstream target
(26), although MAP kinases may be activated through TLRs (44). The p38,
JNK, and TLR pathways, in part, appear to influence IL-12 p40 gene
expression through elements downstream of
101. However, specific
transcription factors activated by these pathways remain elusive. For
example, in nuclear extracts from LPS-treated RAW264.7 cells and
primary bone marrow-derived macrophages, protein expression and DNA
binding for C/EBP or AP-1 family members is not affected by treatment
with a soluble p38 inhibitor (data not shown).
The IFN-
(45) and the T-cell receptor
(46) gene enhancers
provide important biochemical details of how transcriptional activators
are assembled into a nucleoprotein complex called the "enhanceosome" that promotes their interaction and cooperative binding to DNA. Extensive analysis of the IL-12 p40 promoter begins to
describe an IL-12 p40 enhanceosome. In particular, a small region from
96 to
74 of the promoter is a critical focus for protein-DNA and
possibly protein-protein interactions. This small sequence that
contains a C/EBP and AP-1 DNA-binding element is of particular interest
because it appears that the strategy used by macrophages to induce
IL-12 p40 expression may define a common mechanism for other important
inflammatory genes. As discussed, within the TNF-
promoter, there is
a C/EBP element immediately adjacent to an AP-1 site. Functional
synergy and cooperative DNA binding between c-Jun and C/EBP
has been
demonstrated in activation of the TNF-
promoter (35). Similarly,
AP-1 and C/EBP sites have been characterized at similar locations with
respect to a TATA box in the IL-8 gene (47, 48). Thus, transcriptional synergy and cooperativity between the C/EBP and AP-1 families of
transcription factors may be involved in the regulation of several
important inflammatory genes expressed in macrophages.
These studies, performed in cell lines, suggest mechanisms by which
IL-12 p40 may be transcriptionally activated in vivo. Confirming the findings of this analysis in primary cells and in
vivo will require a combination of approaches to determine transcription factor occupancy on the endogenous promoter and their
responses to biologic inducers and inhibitors of IL-12 gene expression.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grants K11 DK02358, the New York City Council Speaker's Fund for Biomedical Research, and the Irma T. Hirschl Trust Career Scientist Award.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: Immunobiology Center, Box 1630, The Mount Sinai School of
Medicine, 1 Gustave L. Levy Place, New York, NY 10029-6574. Tel.:
212-659-9408; Fax: 212-849-2525; E-mail: scott.plevy@mssm.edu.
Published, JBC Papers in Press, March 9, 2001, DOI 10.1074/jbc.M100440200
 |
ABBREVIATIONS |
The abbreviations used are:
IL, interleukin;
LPS, lipopolysaccharide;
AP-1, activation protein-1;
C/EBP, CCAAT
enhancer-binding protein;
MAP kinase, mitogen-activated protein kinase;
IFN, interferon;
Th1, T-helper-1;
JNK, N-terminal c-Jun kinase;
TLR, toll-like receptor;
CAT, chloramphenicol acetyltransferase;
MKK, mitogen-activated kinase kinase;
MEKK, MAP kinase/extracellular
signal-regulated kinase kinase;
TNF, tumor necrosis factor-
;
JIP-1, JNK interacting protein-1;
EMSA, electrophoretic mobility shift assay;
bp, base pair;
DMEM, Dulbecco's modified Eagle's medium.
 |
REFERENCES |
1.
|
Trinchieri, G.
(1995)
Annu. Rev. Immunol.
13,
251-276[CrossRef][Medline]
[Order article via Infotrieve]
|
2.
|
Abbas, A. K.,
Murphy, K. M.,
and Sher, A.
(1996)
Nature
383,
787-793[CrossRef][Medline]
[Order article via Infotrieve]
|
3.
|
Mosmann, T. R.,
and Coffman, R. L.
(1989)
Annu. Rev. Immunol.
7,
145-173[CrossRef][Medline]
[Order article via Infotrieve]
|
4.
|
Magram, J.,
Connaughton, S. E.,
Warrier, R. R.,
Carvajal, D. M.,
Wu, C. Y.,
Ferrante, J.,
Stewart, C.,
Sarmiento, U.,
Faherty, D. A.,
and Gately, M. K.
(1996)
Immunity
4,
471-481[Medline]
[Order article via Infotrieve]
|
5.
|
Sieling, P. A.,
and Modlin, R. L.
(1994)
Immunobiology
191,
378-387[Medline]
[Order article via Infotrieve]
|
6.
|
Zhang, M.,
Gately, M. K.,
Wang, E.,
Gong, J.,
Wolf, S. F.,
Lu, S.,
Modlin, R. L.,
and Barnes, P. F.
(1994)
J. Clin. Invest.
93,
1733-1739[Medline]
[Order article via Infotrieve]
|
7.
|
Pirmez, C.,
Yamamura, M.,
Uyemura, K.,
Paes-Oliveira, M.,
Conceicao-Silva, F.,
and Modlin, R. L.
(1993)
J. Clin. Invest.
91,
1390-1395[Medline]
[Order article via Infotrieve]
|
8.
|
Seder, R. A.,
Kelsall, B. L.,
and Jankovic, D.
(1996)
J. Immunol.
157,
2745-2748[Abstract]
|
9.
|
Gubler, U.,
Chua, A. O.,
Schoenhaut, D. S.,
Dwyer, C. M.,
McComas, W.,
Motyka, R.,
Nabavi, N.,
Wolitzky, A. G.,
Quinn, P. M.,
Familletti, P. C.,
and Gately, M. K.
(1991)
Proc. Natl. Acad. Sci. U. S. A.
88,
4143-4147[Abstract]
|
10.
|
Schoenhaut, D. S.,
Chua, A. O.,
Wolitzky, A. G.,
Quinn, P. M.,
Dwyer, C. M.,
McComas, W.,
Familletti, P. C.,
Gately, M. K.,
and Gubler, U.
(1992)
J. Immunol.
148,
3433-3440[Abstract/Free Full Text]
|
11.
|
Wolf, S. F.,
Temple, P. A.,
Kobayashi, M.,
Young, D.,
Dicig, M.,
Lowe, L.,
Dzialo, R.,
Fitz, L.,
Ferenz, C.,
Hewick, R. M.,
Kelleher, K.,
Herrmann, S. H,
Clark, S. C.,
Azzoni, L.,
Chan, S. H.,
Trinchieri, G.,
and Perussia, B.
(1991)
J. Immunol.
146,
3074-3081[Abstract/Free Full Text]
|
12.
|
D'Andrea, A.,
Rengaraju, M.,
Valiante, N. M.,
Chehimi, J.,
Kubin, M.,
Aste, M.,
Chan, S. H.,
Kobayashi, M.,
Young, D.,
Nickbarg, E.,
Chizzonita, R.,
Wolf, S. F.,
and Trinchieri, G.
(1992)
J. Exp. Med.
176,
1387-1398[Abstract]
|
13.
|
Murphy, T. L.,
Cleveland, M. G.,
Kulesza, P.,
Magram, J.,
and Murphy, K. M.
(1995)
Mol. Cell. Biol.
15,
5258-5267[Abstract]
|
14.
|
Ma, X.,
Chow, J. M.,
Gri, G.,
Carra, G.,
Gerosa, F.,
Wolf, S. F.,
Dzialo, R.,
and Trinchieri, G.
(1996)
J. Exp. Med.
183,
147-157[Abstract]
|
15.
|
Ma, X.,
Gri, G.,
and Trinchieri, G.
(1996)
Ann. N. Y. Acad. Sci.
795,
357-360[Medline]
[Order article via Infotrieve]
|
16.
|
Ma, X.,
Neurath, M.,
Gri, G.,
and Trinchieri, G.
(1997)
J. Biol. Chem.
272,
10389-10395[Abstract/Free Full Text]
|
17.
|
Plevy, S. E.,
Gemberling, J. H.,
Hsu, S.,
Dorner, A. J.,
and Smale, S. T.
(1997)
Mol. Cell. Biol.
17,
4572-4588[Abstract]
|
18.
|
Ho, S. N.,
Hunt, H. D.,
Horton, R. M.,
Pullen, J. K.,
and Pease, L. R.
(1989)
Gene (Amst.)
77,
51-59[CrossRef][Medline]
[Order article via Infotrieve]
|
19.
|
Wise, S. C.,
Burmeister, L. A.,
Zhou, X. F.,
Bubulya, A.,
Oberfield, J. L.,
Birrer, M. J.,
and Shemshedini, L.
(1998)
Oncogene
16,
2001-2009[CrossRef][Medline]
[Order article via Infotrieve]
|
20.
|
Olive, M.,
Krylov, D.,
Echlin, D. R.,
Gardner, K.,
Taparowsky, E.,
and Vinson, C.
(1997)
J. Biol. Chem.
272,
18586-18594[Abstract/Free Full Text]
|
21.
|
Raingeaud, J.,
Whitmarsh, A. J.,
Barrett, T.,
Derijard, B.,
and Davis, R. J.
(1996)
Mol. Cell. Biol.
16,
1247-1255[Abstract]
|
22.
|
Han, J.,
Lee, J. D.,
Jiang, Y.,
Li, Z.,
Feng, L.,
and Ulevitch, R. J.
(1996)
J. Biol. Chem.
271,
2886-2891[Abstract/Free Full Text]
|
23.
|
Dickens, M.,
Rogers, J. S.,
Cavanagh, J.,
Raitano, A.,
Xia, Z.,
Halpern, J. R.,
Greenberg, M. E.,
Sawyers, C. L.,
and Davis, R. J.
(1997)
Science
277,
693-696[Abstract/Free Full Text]
|
24.
|
Yan, M.,
Dai, T.,
Deak, J. C.,
Kyriakis, J. M.,
Zon, L. I.,
Woodgett, J. R.,
and Templeton, D. J.
(1994)
Nature
372,
798-800[Medline]
[Order article via Infotrieve]
|
25.
|
Brightbill, H. D.,
Libraty, D. H.,
Krutzik, S. R.,
Yang, R. B.,
Belisle, J. T.,
Bleharski, J. R.,
Maitland, M.,
Norgard, M. V.,
Plevy, S. E.,
Smale, S. T.,
Brennan, P. J.,
Bloom, B. R.,
Godowski, P. J.,
and Modlin, R. L.
(1999)
Science
285,
732-736[Abstract/Free Full Text]
|
26.
|
Medzhitov, R.,
Preston-Hurlburt, P.,
and Janeway, C. A., Jr.
(1997)
Nature
388,
394-397[CrossRef][Medline]
[Order article via Infotrieve]
|
27.
|
Medzhitov, R.,
Preston-Hurlburt, P.,
Kopp, E.,
Stadlen, A.,
Chen, C.,
Ghosh, S.,
and Janeway, C. A., Jr.
(1998)
Mol. Cell
2,
253-258[Medline]
[Order article via Infotrieve]
|
28.
|
Dignam, J. D.,
Lebovitz, R. M.,
and Roeder, R. G.
(1983)
Nucleic Acids Res.
11,
1475-1489[Abstract]
|
29.
|
Lo, K.,
Landau, N. R.,
and Smale, S. T.
(1991)
Mol. Cell. Biol.
11,
5229-5243[Medline]
[Order article via Infotrieve]
|
30.
|
Wingender, E.,
Dietze, P.,
Karas, H.,
and Knuppel, R.
(1996)
Nucleic Acids Res.
24,
238-241[Abstract/Free Full Text]
|
31.
|
Brown, P. H.,
Kim, S. H.,
Wise, S. C.,
Sabichi, A. L.,
and Birrer, M. J.
(1996)
Cell Growth Differ.
7,
1013-1021[Abstract]
|
32.
|
Ransone, L. J.,
Visvader, J.,
Wamsley, P.,
and Verma, I. M.
(1990)
Proc. Natl. Acad. Sci. U. S. A.
87,
3806-3810[Abstract]
|
33.
|
Du, W.,
Thanos, D.,
and Maniatis, T.
(1993)
Cell
74,
887-898[Medline]
[Order article via Infotrieve]
|
34.
|
Nikolajczyk, B. S.,
Nelsen, B.,
and Sen, R.
(1996)
Mol. Cell. Biol.
16,
4544-4554[Abstract]
|
35.
|
Zagariya, A.,
Mungre, S.,
Lovis, R.,
Birrer, M.,
Ness, S.,
Thimmapaya, B.,
and Pope, R.
(1998)
Mol. Cell. Biol.
18,
2815-2824[Abstract/Free Full Text]
|
36.
|
Lu, H. T.,
Yang, D. D.,
Wysk, M.,
Gatti, E.,
Mellman, I.,
Davis, R. J.,
and Flavell, R. A.
(1999)
EMBO J.
18,
1845-1857[Abstract/Free Full Text]
|
37.
|
Han, J.,
Jiang, Y.,
Li, Z.,
Kravchenko, V. V.,
and Ulevitch, R. J.
(1997)
Nature
386,
296-299[CrossRef][Medline]
[Order article via Infotrieve]
|
38.
|
Lee, F. S.,
Hagler, J.,
Chen, Z. J.,
and Maniatis, T.
(1997)
Cell
88,
213-222[Medline]
[Order article via Infotrieve]
|
39.
|
Weinmann, A. S.,
Plevy, S. E.,
and Smale, S. T.
(1999)
Immunity
11,
665-675[Medline]
[Order article via Infotrieve]
|
40.
|
Wang, I. M.,
Contursi, C.,
Masumi, A.,
Ma, X.,
Trinchieri, G.,
and Ozato, K.
(2000)
J. Immunol.
165,
271-279[Abstract/Free Full Text]
|
41.
|
Sanjabi, S.,
Hoffmann, A.,
Liou, H. C.,
Baltimore, D.,
and Smale, S. T.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
12705-12710[Abstract/Free Full Text]
|
42.
|
Leonard, D. A.,
Rajaram, N.,
and Kerppola, T. K.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
4913-4918[Abstract/Free Full Text]
|
43.
|
Feng, G. J.,
Goodridge, H. S.,
Harnett, M. M.,
Wei, X. Q.,
Nikolaev, A. V.,
Higson, A. P.,
and Liew, F. Y.
(1999)
J. Immunol.
163,
6403-6412[Abstract/Free Full Text]
|
44.
|
Yang, H.,
Young, D. W.,
Gusovsky, F.,
and Chow, J. C.
(2000)
J. Biol. Chem.
275,
20861-20866[Abstract/Free Full Text]
|
45.
|
Thanos, D.,
and Maniatis, T.
(1995)
Cell
83,
1091-1100[Medline]
[Order article via Infotrieve]
|
46.
|
Giese, K.,
Kingsley, C.,
Kirshner, J. R.,
and Grosschedl, R.
(1995)
Genes Dev.
9,
995-1008[Abstract]
|
47.
|
Wu, G. D.,
Lai, E. J.,
Huang, N.,
and Wen, X.
(1997)
J. Biol. Chem.
272,
2396-2403[Abstract/Free Full Text]
|
48.
|
Matsusaka, T.,
Fujikawa, K.,
Nishio, Y.,
Mukaida, N.,
Matsushima, K.,
Kishimoto, T.,
and Akira, S.
(1993)
Proc. Natl. Acad. Sci. U. S. A.
90,
10193-10197[Abstract]
|
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