STAT-1 and c-Fos interaction in nitric oxide synthase-2 gene activation
Weiling Xu,1
Suzy A. A. Comhair,1
Shuo Zheng,1
Shan C. Chu,2
Joanna Marks-Konczalik,2
Joel Moss,2
S. Jaharul Haque,1 and
Serpil C. Erzurum1
1Pulmonary and Critical Care Medicine and Cancer
Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland,
Ohio 44195; and 2Pulmonary-Critical Care Medicine
Branch, National Heart, Lung, and Blood Institute, National Institutes of
Health, Bethesda, Maryland 20892
Submitted 30 December 2002
; accepted in final form 12 March 2003
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ABSTRACT
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Interferon-
(IFN-
) is required for induction of the human
nitric oxide synthase-2 (NOS2) gene in lung epithelium. Although the
human NOS2 promoter region contains many cytokine-responsive
elements, the molecular basis of induction is only partially understood. Here,
the major cis-regulatory elements that control IFN-
-inducible
NOS2 gene transcription in human lung epithelial cells are identified
as composite response elements that bind signal transducer and activator of
transcription 1 (STAT-1) and activator protein 1 (AP-1), which is comprised of
c-Fos, Fra-2, c-Jun, and JunD. Notably, IFN-
activation of the human
NOS2 promoter is shown to require functional AP-1 regulatory
region(s), suggesting a role for AP-1 activation/binding in the IFN-
induction of genes. We show that c-Fos interacts with STAT-1 after IFN-
activation and the c-Fos/STAT-1 complex binds to the
-activated site
(GAS) element in close proximity to AP-1 sites located at 4.9 kb upstream of
the transcription start site. Taken together, our findings support a model in
which a physical interaction between c-Fos and STAT-1 participates in
NOS2 gene transcriptional activation.
gene regulation; signal transduction
INTERFERON-
(IFN-
) induces gene expression through
activation of specific members of the Janus kinase (JAK) family, which in turn
phosphorylate signal transducer and activator of transcription 1 (STAT-1; see
Refs. 14 and
15). The phosphorylated STAT-1
molecules form homodimers, translocate to the nucleus, and bind to
-activated sites (GAS) in the 5'-flanking regions of genes, such
as the nitric oxide synthase-2 (NOS2) gene. The importance of STAT-1
for IFN signaling is clearly demonstrated by STAT-1-deficient mice, which fail
to respond to IFNs and are consequently highly sensitive to microbial
infection, which is the result of lack of induction of downstream target
genes, such as NOS2
(5,
29,
32).
NOS2, which is inducible in diverse cell types by cytokines,
converts L-arginine to L-citrulline, and nitric oxide
(NO; see Refs. 30 and
36). NO, a short-lived, free
radical gas, functions in essential biological processes, including regulation
of vascular tone and host defense
(30,
36). In addition to its
beneficial effects, increased production of NO is associated with inflammatory
tissue damage in human diseases, e.g., septic shock and asthma
(6,
11,
25,
27,
44). The molecular basis for
induction of the human NOS2 gene is only partially understood. The
8.3-kb human NOS2 promoter region contains clusters of
cytokine-responsive elements, perfectly or partially matched to consensus
sequences, including GAS and activator protein 1 (AP-1; see Refs.
3,
21,
35,
39). Previous studies have
demonstrated that IFN-
is necessary for induction of the murine and
human NOS2 promoter
(3,
4,
8,
9,
24,
45,
47) and the GAS element in the
NOS2 promoter is essential for IFN-
activation
(8,
9,
45). However, prior work has
also emphasized the importance of AP-1 binding sites in activation of the
NOS2 promoter in response to cytokine combinations
(3,
26). AP-1 is a complex
composed of proteins of the Fos (c-Fos, FosB, Fra-1, and Fra-2) and Jun
(c-Jun, JunB, and JunD) protooncogene families
(2,
35,
43). In general, Fos and Jun
family proteins function as dimeric transcription factors that bind to AP-1
regulatory elements in the promoter and enhancer regions of genes
(2,
43).
Induction of NOS2 gene expression in the lung epithelial cell line
A549 requires activation of AP-1, but the relevance of AP-1 to in vivo
activation of the human NOS2 gene is not clear, since IFN-
alone is sufficient for induction of NOS2 in primary human airway
epithelial cells (HAEC), the key cellular source of NO in the lung
(12,
42). IFN regulatory factor
(IRF)-1, another essential transcriptional activator of NOS2
gene expression (18,
41), is itself induced by
IFN-
activation of STAT-1, which binds to GAS in the IRF-1
promoter (20,
38). IFN-
also
activates c-fos gene expression through the sis-inducible
element within the c-fos promoter, which binds sis-inducible
factor complexes, comprised of homodimers of STAT-3, heterodimers of STAT-3
and STAT-1, and homodimers of STAT-1
(31,
46). STAT proteins demonstrate
cooperative DNA binding not only with other STAT family members (e.g.,
STAT-1/STAT-2 and STAT-1/STAT-3; see Refs.
10,
14, and
16) but also with other
proteins and transcription factors, including transcriptional activator
specificity protein (SP)-1 and CCAAT enhancer binding protein
(22,
23). Recently, physical
association between STAT-3 and c-Jun on the
2-macroglobulin
enhancer element has been shown to yield maximal enhancer functions
(48).
Based upon this knowledge, we hypothesized that cooperative interaction
between AP-1 and STAT-1 pathways may be important in the activation of
IFN-
-activated genes, such as NOS2. Here, we show that c-Fos
rapidly interacts with STAT-1 after IFN-
activation and the
c-Fos/STAT-1 complex binds to the GAS element in close proximity to AP-1 sites
located in a 665-bp region at 4.9 kb upstream of the transcription start site.
Taken together, our findings support a physical interaction between c-Fos and
STAT-1 and suggest a role for c-Fos and STAT-1 in transcriptional activation
of NOS2 gene.
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MATERIALS AND METHODS
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NOS2 luciferase reporter constructs. A series of deletion
constructs containing the NOS2 gene 5'-flanking region cloned
into the pGL-3-basic luciferase reporter gene vector (Promega, Madison, WI),
and named pGL3-8296, -7196, -6251, -5574, -4909, -4711, -4060, -3137, and
-336, or the pGL38296 full-length NOS2 promoter that had
undergone oligonucleotide-directed mutagenesis of AP-1-binding sites were used
in experiments (3,
26). All constructs were
subjected to digestion with restriction enzymes and sequence analysis to
verify the 5'-end of the insert.
Cell culture. HAEC were isolated from bronchoscopic brushing of
the airway, or from surgical specimens of tracheas and main-stem bronchi, and
cultured as previously described
(11,
41). Primary cultures of
passage 03 were used in experiments. The epithelial nature of primary
and cultured cells was confirmed by immunocytochemical staining, as previously
described (12). A549 cells, an
epithelial cell line derived from lung adenocarcinoma, were cultured in MEM
(Invitrogen, Carlsbad, CA) with 10% heat-inactivated FCS, or 24 h before
cytokine stimulation with 1% FCS. A STAT-1-deficient fibrosarcoma cell line,
U3A, was maintained in DMEM (Invitrogen) with 10% FCS
(1,
7). 293T cells, a clone of 293
(human embryonic kidney fibroblast cells) that expresses the Simian virus 40
large-T antigen, were maintained in DMEM (Invitrogen) with 10% FCS. Human
IFN-
was a gift from Genentech (South San Francisco, CA) or was
purchased from R&D Systems (Minneapolis, MN). Recombinant human
interleukin (IL)-1
and tumor necrosis factor (TNF)-
were
purchased from Biosource (Camarillo, CA).
Transient transfection and luciferase assay. With the use of
Lipofectamine Reagent (Invitrogen), 40% confluent HAEC and 30% confluent A549
cells in six-well plates were transfected with various NOS2
luciferase reporter constructs. Transfections were performed using equal
amounts of DNA, as previously described
(3). After adding the DNA with
Lipofectamine to each well and incubating for 4 h for HAEC and 10 h for A549,
the medium was replaced with normal growth medium for HAEC and MEM with 1% FCS
for A549 cells. After (24 h) transfection, cells were exposed to cytokine
mixture (CK). Later (24 h), cells were washed in PBS, harvested after the
addition of 250 µlof1x Passive Lysis Buffer, freeze-thawed two times,
and centrifuged (12,000 g, 2 min). Supernatants were assayed for
firefly luciferase activity using the Dual-Luciferase Reporter Assay (Promega)
in which luciferase activities are normalized by dual (Renilla)
luciferase assay (Promega). In separate experiments, pCMV-
-Gal
(Invitrogen) was used to determine the percentage of cells transfected.
Relative luciferase activity is reported as means of values from more than
three independent experiments, each performed in triplicate.
The antisense phosphorothioated oligodeoxynucleotide
(5'-CCGAGAACATCATCGTGGCG-3') was directed against the translation
initiation site of c-Fos mRNA
(40). Corresponding sense
oligodeoxynucleotide (5'-CGCCACGATGATGTTCTCGG-3') was used as a
control. With the use of Lipofectamine reagent, A549 cells were cotransfected
with a 8,296-bp full-length NOS2 promoter and the c-Fos antisense
phosphorothioated oligodeoxynucleotide or sense oligodeoxynucleotide and then
incubated in the presence or absence of CK for 6 or 12 h. Cells were
harvested, and supernatants were assayed for luciferase activity.
With the use of Lipofectamine Reagent (Invitrogen), 293T cells or A549
cells were transfected with 2 µg DNA containing various expression vectors,
HA-tagged STAT-1 (HA-STAT-1; see Ref.
34), HA-tagged TAK1 (HA-TAK1;
see Ref. 37), or pRSV-c-Fos
(33). The medium was replaced
with 1% FCS after 10 h transfection. After transfection (24 h), cells were
exposed to CK for 24 h, and then cells were washed in PBS and harvested.
EMSA. Whole cell extract (WCE) was prepared as previously
described (11,
41). For nuclear extract, the
cell suspensions were centrifuged and resuspended in 0.4 ml ice-cold buffer
[10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.5 mM PMSF, and 1
mM DTT] by gentle pipetting in a yellow tip, and then cells were allowed to
swell on ice for 15 min. Subsequently, 25 µl of 10% solution of Nonidet
P-40 was added, vigorously vortexed for 10 s, and then spun for 30 s
in a microfuge. The nuclear pellet was resuspended in 50 µl ice-cold buffer
[20 mM HEPES (pH 7.9), 25% glycerol, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM
PMSF, and 1 mM DTT].
The duplex oligonucleotides used in EMSA
(Table 1) were synthesized by
Operon (Alameda, CA) and then end-labeled with [
-32P]ATP by
polynucleotide kinase (11,
41). For binding reactions,
cell extract was incubated in 20 µl total reaction volume containing 20 mM
HEPES (pH 7.9), 5% glycerol, 50 mM NaCl, 5 mM DTT, 0.1 mM EDTA, 100 µg/ml
BSA, and 2 µg polydeoxyinosinic-polydeoxcytidylic acid (Amersham, Arlington
Heights, IL) for 15 min at room temperature. The 32P-labeled
oligonucleotide (2 x 105 counts/min) was added to the
reaction mixture and incubated for 20 min at room temperature. The reaction
mixture was analyzed by electrophoresis on a 4% polyacrylamide gel in
0.25x buffer containing 12.5 mM Tris, 12.5 mM borate, and 0.5 mM EDTA.
The gels were dried and analyzed by autoradiography. To demonstrate
specificity of binding, competition was performed by adding unlabeled
wild-type and mutated oligonucleotide at a 100-fold molar excess of
32P-labeled oligonucleotide probe in the binding reaction. To
specifically identify AP-1, GAS binding-factor, and NF-
B proteins in
binding complexes, 4 µg rabbit anti-c-Fos, FosB, Fra-1, Fra-2, c-Jun and
JunD, STAT-3, STAT-5, p50 or p65 polyclonal antibody (Ab; Santa Cruz
Biotechnology, Santa Cruz, CA), rabbit anti-STAT-1 polyclonal Ab
(11,
13), or nonimmune rabbit IgG
(Biodesign, Saco, ME) was added to the binding reaction mix and incubated for
30 min at room temperature before adding the 32P-labeled
oligonucleotide. Antibodies used in experiments include anti-c-Fos Ab (rabbit
polyclonal Ab against domain of c-Fos p62 of human origin; Santa Cruz
Biotechnology), and c-Fos(2) Ab (rabbit polyclonal Ab against the amino
terminus of c-Fos p62 of human origin; Santa Cruz Biotechnology).
Immunoprecipitation and Western blot analysis. Extracts were
prepared by lysing the cells in ice-cold buffer containing 50 mM Tris (pH
7.9), 50 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.5% Nonidet P-40, 10%
glycerol, 1 mM PMSF, 5 µg/ml leupeptin, 10 µg/ml pepstatin A, 200 µM
NaOV, and 20 µg/ml aprotinin on ice for 30 min and centrifuging at 13,000
g for 30 min at 4°C. The cleared supernatant containing
400600 µg proteins was incubated with 46 µg antibodies
(anti-c-Fos, c-Jun, STAT-1, STAT-3, STAT-5 Ab, and anti-HA Ab; Upstate
Biotechnology, Lake Placid, NY) or nonimmune rabbit IgG for 2 h at 4°C,
and then 100 µl protein G-Sepharose (Amersham) was added and incubated for
2 h at 4°C. The captured beads were washed and boiled in denaturing
buffer, and the released proteins were analyzed by Western blot. Protein was
separated by electrophoresis on an 8 or 12.5% SDS-polyacrylamide gel and then
electrophoretically transferred to nitrocellulose (Osmonics, Minnetonka, MN).
Signal detection was accomplished with primary rabbit polyclonal Abs, followed
by a secondary anti-rabbit Ab (Amersham).
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RESULTS
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Effect of CK and IFN-
on activity of human NOS2
promoter constructs in HAEC and A549 cells. NOS2 mRNA is induced by
IFN-
in HAEC (12,
42). Here NOS2 protein
expression in HAEC is also induced by IFN-
alone
(Fig. 1). In contrast, NOS2
expression in most cell lines, including A549, requires exposure to
IFN-
in combination with a mixture of cytokines, such as IL-1
and/or TNF-
(3). The
time course of NOS2 protein induction in HAEC by IFN-
is slower than
CK, with equivalent protein present at 48 h. The time course of NOS2
mRNA induction by IFN-
is also delayed compared with CK
(42). The lack of NOS2
induction in U3A, a cell line lacking STAT-1, may be because of the fact that
STAT-1 activation and binding to GAS elements is required for NOS2 expression
in cells (11,
29).
To identify IFN-
-responsive transcriptional elements in the
5'-flanking region of the NOS2 gene, HAEC and A549 cells were
transiently transfected with constructs containing various segments of the
proximal 5'-flanking region of the human NOS2 gene driving
luciferase expression. After transfection, cells were exposed for 24 h to
10,000 U/ml IFN-
or CK containing 10,000 U/ml IFN-
, 0.5 ng/ml
IL-1
, and 10 ng/ml TNF-
or were left untreated. Transfection of
pGL-3-basic reporter constructs lacking promoter served as a negative control.
Efficiency of Lipofectamine transfection in HAEC determined by
-galactosidase expression plasmid (pCMV-
-Gal) was 5 ± 2%
cells/high-power field, whereas 15 ± 5% A549 cells were transfected. As
previously shown (3,
26), the NOS2
promoter (regions containing 5,574 bp or greater up to 8,296 bp) was activated
by CK in A549 cells, whereas CK inducibility of constructs in A549 cells was
lost with the 4,909-bp or shorter regions of the 5'-flanking promoter
region construct (Fig.
2A). In contrast, full-length or deletion constructs of
the NOS2 promoter were not activated by IFN-
alone in A549
cells (degree of induction compared with unstimulated A549 cells of the
5,574-bp promoter construct: CK induced 7 ± 1, IFN-
induced 1.4
± 0.2; n
3).
Similar to A549 cells, induction of the NOS2 promoter by CK in
HAEC also occurred with 5,574-bp or longer constructs
(Fig. 2B). However,
IFN-
alone activated the NOS2 promoter (regions containing
5,574 bp or greater up to 8,296 bp) in HAEC (degree of induction compared with
unstimulated HAEC of the 5,574-bp promoter construct: CK induced 2.4 ±
0.3, IFN-
induced 2.5 ± 0.2; n
3). CK or
IFN-
inducibility of constructs in HAEC was lost with the 4,909-bp or
shorter regions of the 5'-flanking promoter region construct, indicating
that the 665-bp region upstream of the 4,909-bp fragment contained important
transcription regulatory elements (degree of induction compared with
unstimulated HAEC of the 4,909-bp promoter construct: CK induced 1.2 ±
0.3, IFN-
induced 1.4 ± 0.3; n
3). In HAEC
incubated with CK or IFN-
alone, the luciferase activities increased at
24 h and were highest at 48 h (Fig.
3). Thus IFN-
or CK similarly induced the NOS2
promoter in HAEC but not in A549 cells. Yet the important cytokine-responsive
elements for IFN-
or CK activation of the promoter in both A549 cells
and HAEC were located within the 665-bp region between -5,574 and -4,909
bp.
Effect of mutation in AP-1-binding sequence of NOS2 gene in HAEC and
A549 cells. Previous studies have demonstrated that the GAS element in
NOS2 promoter is essential for IFN-
activation
(8,
9,
45). However, prior work has
also emphasized the importance of AP-1-binding sites in activation of
NOS2 promoter in response to cytokine combinations
(3,
26). Multiple cytokine binding
sites are present in the nucleotide sequence from -5,574 to -4,909 bp of the
NOS2 promoter (Fig.
4). Based on the consensus sequence TTN5AA
(14), this region contains two
putative GAS, two consensus-binding sites (TGANTCA) for AP-1
(3,
35), and two consensus-binding
sites (GGGRNWYYCC) for NF-
B
(3,
39).
To determine whether AP-1 sites are important in induction of NOS2
in HAEC by IFN-
alone, 8,296-bp full-length NOS2 promoter
bearing three-base mutations in the AP-1-binding sites termed mAP-1
(26) was studied. HAEC and
A549 cells transfected with mutated NOS2 promoter (mAP-1) were
exposed to CK or IFN-
for 24 h. As previously shown, A549 cells
transfected with the AP-1 mutated construct had a marked decrease in
luciferase activity with CK (Fig.
2A; see Ref.
26). Compared with the
wild-type promoter construct, HAEC transfected with mutant AP-1 constructs had
decreased induction by CK and, unexpectedly, by IFN-
(Fig. 2B). These
results suggested that CK or IFN-
induction of the human NOS2
promoter requires AP-1 regulatory region(s).
STAT-1 and c-Fos interaction in IFN-
induced NOS2 GAS
activation. To determine which transcription factors bind to the GAS in
the region from -5,574 to -4,909 bp of the NOS2 promoter, DNA-protein
interactions were investigated by EMSA using extracts from HAEC or A549 cells
after exposure to IFN-
for 30 min. With the use of oligonucleotides
bearing both the sequences of upstream AP-1 (AP-1u) and GAS elements in the
NOS2 promoter (Table
1), WCE from HAEC had binding activity at baseline that increased
by exposure of cells to IFN-
(Fig.
5). Antibodies to c-Fos or c-Jun produced supershift of the
complex, indicating that these proteins are present in the DNA-protein
complexes. With the use of an oligonucleotide only bearing the sequence of the
GAS element in the NOS2 promoter
(Table 1), DNA binding activity
was present in HAEC WCE (Fig.
6A) or A549 cell nuclear extract (data not shown) exposed
to IFN-
but not in nonstimulated cells. Antibodies against c-Fos or
STAT-1 supershifted DNA-protein bands, indicating that both proteins are
present in the binding complex. On the other hand, antibodies against STAT-3,
STAT-5, c-Jun (Fig. 6), FosB,
Fra-1, Fra-2, or JunD (data not shown) did not produce a supershift of the
binding complex, indicating that no other members of the AP-1 complex (FosB,
Fra-1, Fra-2, c-Jun, or JunD) interact with STAT-1. NF-
B binding sites
overlap GAS in the NOS2 promoter. To determine whether NF-
B
binding occurs in this region, EMSA was performed on WCE of
TNF-
-stimulated A549 cells using an oligonucleotide containing the
NOS2 NF-
B binding sequence that overlaps GAS
(Table 1). WCE from CK- or
IFN-
-exposed cells contained binding activity to GAS that was the
result of STAT-1 and AP-1 binding but not the result of NF-
B
(Fig. 6B). Notably,
competitive binding of STAT-1 and NF-
B has been demonstrated in the
region of the GAS site such that STAT-1 precludes NF-
B binding
(8).

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Fig. 6. NOS2 GAS binding activity in HAEC and A549 cells treated with
IFN- . A: WCE from nonstimulated HAEC (lanes 1) or
HAEC stimulated with IFN- for 30 min (lanes 210) was
analyzed by EMSA using oligonucleotide bearing the sequence of the
NOS2 GAS sequence motif. The specificity of the binding complex
(arrow a) was assessed by the addition of a 100-fold molar excess of
unlabeled wild-type (lane 4) or mutant GAS oligonucleotides (lane
3) before incubation with the labeled probe. Anti-c-Fos, c-Jun, STAT-1,
STAT-3, STAT-5, or NF- B p65 polyclonal Ab was added to binding
reactions to identify proteins in the binding complex. IFN- led to
binding activity (arrow a) that was supershifted by anti-STAT-1
(lane 7, arrow b) or anti-c-Fos Ab (lane 5, arrow c).
Similar results were obtained in 3 separate experiments. B: WCE from
HAEC stimulated with CK for 30 min (lanes 13), IFN- for
30 min (lanes 46), or TNF- for 30 min (lanes
79) was analyzed by EMSA using the NOS2 GAS. CK or
IFN- led to a prominent binding complex (arrow). STAT-1 and c-Fos were
both present in the complex, as shown by supershift with anti-STAT-1
(lanes 3 and 6) and anti-c-Fos Ab (lanes 2 and
5). Similar results were obtained in a minimum of 3 separate
experiments.
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To investigate AP-1 activation and which transcription factors bind to the
AP-1 site, EMSA was also performed using oligonucleotide containing only the
AP-1u binding sequence (Table
1). Basal AP-1 binding activity in nonstimulated A549 was low and
significantly increased by exposure of cells to CK for 3 h
(Fig. 7A). Anti-c-Fos,
Fra-2, c-Jun, or JunD led to significant supershift of the complex. No
supershift was detected with FosB or Fra-1
(Fig. 7A). WCE from
HAEC had basal binding activity that was not appreciably increased by exposure
to CK (Fig. 7B) or
IFN-
(data not shown). Antibodies to c-Fos or c-Jun supershifted the
complex, indicating that c-Fos and c-Jun are activated and bind to the AP-1
element even in the absence of cytokine stimulation in HAEC. This basal AP-1
activation may explain, in part, why IFN-
alone is sufficient to
activate NOS2 in HAEC, whereas multiple cytokines are required in
A549 cells. With the use of an oligonucleotide containing only the downstream
AP-1 (AP-1d) sequence, binding activity determined by EMSA was much weaker
with AP-1d than with AP-1u (Fig.
7C). This indicated that the AP-1u is more relevant to
promoter activation.

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Fig. 7. Identification of the NOS2 AP-1-binding proteins in A549 and HAEC
by EMSA. A: EMSA of WCE from nonstimulated A549 (lane 1) or
A549 stimulated with CK for 3 h (lanes 28) was analyzed using
the radiolabeled NOS2 AP-1u oligonucleotide. Antibodies were added to
reactions to identify binding proteins as indicated. Anti-c-Fos (lane
3), Fra-2 (lane 6), c-Jun (lane 7), or JunD (lane
8) led to significant supershift of the complex. No supershift was
detected with FosB or Fra-1. The autoradiograph is representative of 2
independent experiments. B: EMSA of WCE from nonstimulated HAEC
(lane 1) or HAEC stimulated with CK for 3 h (lanes
27) using radiolabeled AP-1u sequence. The specificity of the
binding complex (arrow) was assessed by the addition of a 100-fold molar
excess of unlabeled wild-type (lane 4) or mutant AP-1u (lane
3) oligonucleotide before the labeled probe. Anti-c-Fos, c-Jun, or STAT-1
polyclonal Ab was added to reactions to identify binding proteins. Anti-c-Fos
(lane 5), anti-c-Jun (lane 6), and perhaps anti-STAT-1
(lane 7) led to supershift of the complex. The autoradiographs are
representative of 3 experiments. C: NOS2 AP-1u and AP-1d
binding activation in A549 cells by EMSA. WCE from nonstimulated A549
(lanes 1 and 4) or A549 stimulated with CK for 3 h
(lanes 23 and 56) was analyzed by EMSA using
radiolabeled oligonucleotide AP-1u or downstream (d) AP-1. Anti-c-Fos Ab was
added to reactions to identify binding protein.
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Effect of c-Fos antisense phosphorothioated oligodeoxynucleotide on CK
induction of human NOS2 promoter. To further test whether c-Fos is
important in induction of NOS2, A549 cells were cotransfected with
the 8,296-bp full-length NOS2 promoter and the c-fos
antisense phosphorothioated oligodeoxynucleotide. Compared with the sense
oligodeoxynucleotide and no oligo, CK induction of the NOS2 promoter
was decreased significantly in A549 cells exposed to CK for 6 or 12 h
(P < 0.05; Fig. 8),
which indicates the essential role of c-Fos in NOS2 induction.
However, STAT-1 and c-Fos overexpression did not produce any significant
increase in NOS2 expression in A549 and 293T cells (data not
shown).

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Fig. 8. Effect of c-fos antisense phosphorothioated oligodeoxynucleotide
on CK induction of human NOS2 promoter transfected with 8,296-bp
full-length NOS2 promoter. A549 cells were cotransfected the c-Fos
antisense phosphorothioated oligodeoxynucleotide (AS), sense
oligodeoxynucleotide (S), or no oligo (N) and then cultured in the presence or
absence of CK for 6 or 12 h. Data are expressed as the degree of induction
compared with unstimulated A549 cells transfected with full-length
NOS2 promoter (n = 2). P < 0.05 vs. N and S.
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Interaction between c-Fos and STAT-1 proteins. In the context that
STAT-1 and c-Fos are present in the binding complexes with the GAS sites, we
investigated the interactive binding of c-Fos and STAT-1, and whether c-Fos
interaction with STAT-1 requires DNA-binding, using coimmunoprecipitation and
Western blot analysis. The cell lysate of HAEC exposed to IFN-
(400
µg total protein) was imunoprecipitated with an anti-c-Fos polyclonal Ab.
The immunocomplex was resolved on an 8% SDS-polyacrylamide gel, and the
immunoblot was probed with an anti-STAT-1 polyclonal Ab. Cell lysates of HAEC
and A549 cells immunoprecipitated with anti-STAT-1 polyclonal Ab and probed
with anti-STAT-1 polyclonal Ab served as a positive control, and the
STAT-1-deficient fibrosarcoma cell line, U3A, served as a negative control.
The time course (024 h) and the dose response to IFN-
were
evaluated. STAT-1 coimmunoprecipitated with c-Fos by 30 min after IFN-
exposure (Fig. 9A). An
IFN-
dose of 100 U/ml was necessary to detect STAT-1
coimmunoprecipitation with c-Fos (Fig.
9B). Lack of coimmunoprecipitation at baseline suggests
that STAT-1 activation/phosphorylation is required for c-Fos/STAT-1
interaction.

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Fig. 9. Interaction of c-Fos and STAT-1. A: STAT-1 coimmunoprecipitated
with c-Fos from HAEC lysate after IFN- exposure. Lysates from
nonstimulated HAEC (lane 1) or HAEC stimulated with IFN-
(lanes 25) were immunoprecipitated using anti-c-Fos Ab, run on
8% gel, and immunoblotted with anti-STAT-1 antibody. Immunoprecipitation with
anti-STAT-1 and immunoblot with anti-STAT-1 of HAEC lysate (lane 6)
or A549 lysate (lane 7) served as a positive control, whereas U3A
cells that lack STAT-1 expression were a negative control (lane 8).
STAT-1 (arrow) coimmunoprecipitated with c-Fos from HAEC lysate after
IFN- exposure (nonspecific band noted above STAT-1 band). B:
STAT-1 coimmunoprecipitated with c-Fos from lysate of HAEC exposed to a
minimum of 100 U/ml IFN- . Lysates of HAEC exposed to increasing amounts
of IFN- were immunoprecipitated using anti-c-Fos and immunoblotted with
anti-STAT-1 antibody (lanes 25). Immunoprecipitation with
anti-STAT-1 and immunoblot with anti-STAT-1 in A549 lysate derived from
CK-stimulated cells served as a positive control (lane 7), whereas
U3A cells, which lack STAT-1 expression, were a negative control (lane
8). Arrow: STAT-1 coimmunoprecipitated with c-Fos from lysate of HAEC
exposed to a minimum of 100 U/ml IFN- . C: HA-STAT-1
coimmunoprecipitated with c-Fos from 293T cells transfected with HA-STAT-1.
Immunoprecipitation with anti-STAT-1 or anti-HA Ab in 293T cells transfected
with HA-STAT-1 served as positive controls (lanes 3 and 4),
whereas immunoprecipitation with nonimmune rabbit IgG was a negative control
(lanes 5 and 8). Arrow: STAT-1 coimmunoprecipitated with
c-Fos from 293T cell lysate nontransfected (lane 6) or transfected
(lane 2) with HA-STAT-1. D: c-Fos coimmunoprecipitated with
STAT-1. Lysates from A549 stimulated with CK (lanes 13) were
immunoprecipitated using nonimmune rabbit IgG, anti-c-Fos, and STAT-1 Ab, run
on 12.5% gel, and immunoblotted with anti-STAT-1 or anti-c-Fos(2) Ab.
Immunoprecipitation with nonimmune rabbit IgG was a negative control (lane
1). Arrows: STAT-1 coimmunoprecipitated with c-Fos and c-Fos
coimmunoprecipitated with STAT-1.
|
|
To further confirm the interactive binding of c-Fos and STAT-1, 293T cells
were transfected with an expression construct for HA-STAT-1
(34) or as a control with the
expression construct or HA-TAK1, a protein involved in the IL-1 signaling
pathway (37). Lysates from
293T cells stimulated with CK were immunoprecipitated using anti-c-Jun, c-Fos,
HA or STAT-1 Ab or nonimmune rabbit IgG, electrophoresed on 8% gel, and
immunoblotted with anti-HA or STAT-1 Ab. Immunoprecipitation with anti-STAT-1
or anti-HA Ab of lysates from 293T cells transfected with HA-STAT-1 served as
a positive control, whereas immunoprecipitation with nonimmune rabbit IgG was
a negative control. HA-STAT-1 was coimmunoprecipitated with c-Fos in 293T
transfected with HA-STAT-1 (Fig.
9C) but not in lysates from 293T cells nontransfected
(Fig. 9C) or
transfected with HA-TAK1 (data not shown). Endogenous STAT-1
coimmunoprecipitated with c-Fos in 293T cells nontransfected, transfected with
HA-STAT-1 (Fig. 9C),
or transfected with HA-TAK1 (data not shown). Thus either endogenous STAT-1 or
HA-STAT-1 interacts with c-Fos, but not c-Jun. Coimmunoprecipitation of c-Fos
and STAT-1 or HA-STAT-1 from 293T cells transfected with HA-STAT-1 was also
confirmed using different c-Fos antibodies (data not shown). In addition,
c-Fos coimmunoprecipitated with STAT-1 in lysates from A549 cells
(Fig. 9D).
These results confirm that c-Fos interacts with STAT-1, indicating that
activation/phosphorylation of STAT-1 is necessary for c-Fos interaction.
 |
DISCUSSION
|
---|
In this study, we demonstrate the physical interaction between c-Fos and
STAT-1, which participate in NOS2 gene transcriptional activation
after IFN-
activation. Fos and Jun family proteins usually function as
dimeric transcription factors that bind to AP-1 regulatory elements
[TGA(C/G)TCA] in the promoter of numerous genes, including NOS2
(2,
35,
43). Jun proteins can form
stable homodimers or heterodimers with Fos proteins, but Fos proteins do not
form stable homodimers. Fos-Jun heterodimers bind DNA more stably than Jun
homodimers (2,
28,
35,
43). Thus c-Fos
heterodimerization with Jun family members enhances association of Jun
proteins to DNA. Although other Fos family members may be capable of
substituting functionally for c-Fos in c-Fos-deficient mice, our studies show
that only c-Fos physically associates with STAT-1 after IFN-
activation
(17). Notably, AP-1 is
activated in unstimulated HAEC in culture, with no appreciable increase in
activation after stimulation with CK or IFN-
. This suggests that basal
AP-1 activation is sufficient to allow subsequent activation of NOS2
gene expression by IFN-
alone in the HAEC, a unique feature of these
cells (12,
42).
Previous studies indicate that genistein, a tyrosine kinase inhibitor of
the JAK-STAT-1 pathway, abolishes induction of NOS2 by IFN-
in
airway epithelial cells (11),
and tyrophostin A25, a pharmacological inhibitor of Jak 2 kinase, inhibits
cytokine-induced NOS2 expression in a dose-dependent manner in A549
cells (8). These data suggest
that the JAK-STAT pathway is involved in regulating cytokine or
IFN-
-induced NOS2 expression in lung epithelial cells.
Furthermore, cotransfection with the dominant-negative STAT-1 expression
vector significantly inhibits cytokine-induced NOS2 reporter
expression (8), implicating
STAT-1 as a positive regulator of NOS gene transcription in these
cells.
Cooperative DNA binding of proteins usually involves regions in close
proximity, which functionally represent a composite regulatory element
(2,
48). In this study, the 100-bp
region encompassing the GAS and AP-1 site of NOS2 promoter may serve
as composite binding elements. These closely located sites support that
heterodimeric c-Fos interaction with STAT-1 in binding complexes on DNA
elements is important for maximal gene activation. Experimental support of
this is provided by the decreased IFN-
inducibility of the
NOS2 promoter containing mutated AP-1 sites. Definitive evidence of
physical association between c-Fos and STAT-1 is provided by
coimmunoprecipitation of endogenously expressed or exogenously expressed
factors from cells after exposure to IFN-
. We speculate that STAT-1
binding may be facilitated on the GAS element through interaction with c-Fos,
i.e., STAT-1 may more firmly associate with GAS, in part through interaction
with c-Fos. In support of this concept, previous studies have shown that
low-affinity and low-specificity Smad-family DNA binding proteins rely on
interactions with other DNA-binding proteins, including Jun, to target them to
specific regulatory DNA elements
(2). Similarly, a previous
study of IFN-
induction of intercellular adhesion molecule-1 in primary
human airway cells has shown that STAT-1 activation and DNA binding require
the transcriptional activator SP-1
(23). Furthermore,
transcription factor TFII-I can form protein-protein complexes with STAT-1,
STAT-3, and serum response factor, which enhances the response of the
c-fos promoter (19).
Taken together, pairing of STAT-1 and c-Fos to the promoter provides maximal
activation of NOS2 expression in cells.
 |
ACKNOWLEDGMENTS
|
---|
We are indebted to Andrew Larner for the gift of the HA-STAT-1 expression
construct, Xiaoxia Li for the HA-TAK1 expression construct, F. Dong for
pRSV-c-Fos expression construct, and to Xiaoxia Li and Donna Driscoll for
helpful suggestions to the manuscript.
This work was supported by National Heart, Lung, and Blood Institute Grant
HL-60917.
 |
FOOTNOTES
|
---|
Address for reprint requests and other correspondence: S. C. Erzurum,
Cleveland Clinic Foundation, Lerner Research Institute, 9500 Euclid Ave./NB40,
Cleveland, OH 44195 (E-mail:
erzurus{at}ccf.org).
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.
 |
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