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and IFN-
in Macrophages1
,
,
Departments of
* Medicine,
Biochemistry and Molecular Biology, and
Critical Care Medicine, and
Immunology Research Group, University of Calgary, Calgary, Alberta, Canada
 |
Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and IFN-
, are critical in the regulation of
Bf gene expression in macrophages. In this study,
we investigated the mechanisms of Bf gene regulation by
TNF- and IFN- in murine macrophages. Northern analysis revealed
that Bf mRNA expression was synergistically up-regulated
by TNF- and IFN- in MH-S cells. Truncations of the 5'
Bf promoter identified a region between -556 and -282 bp
that mediated TNF- responsiveness as well as the synergistic effect
of TNF- and IFN- on Bf expression. Site-directed
mutagenesis of a NF-
B-binding element in this region (-433 to -423
bp) abrogated TNF- responsiveness and decreased the synergistic
effect of TNF- and IFN- on Bf expression. EMSAs
revealed nuclear protein binding to this NF-B
cis-binding element on TNF- stimulation. Supershift
analysis revealed that both p50 and p65 proteins contribute to
induction of Bf by TNF-. An I-B dominant negative
mutant blocked Bf induction by TNF- and reduced the
synergistic induction by TNF- and IFN-. In addition, the
proteasome inhibitor MG132, which blocks NF-B induction, blocked
TNF--induced Bf promoter activity and the synergistic
induction of Bf promoter activity by TNF- and IFN-.
LPS was found to induce Bf promoter activity through the
same NF-B cis-binding site. These findings suggest
that a NF-B cis-binding site between -433 and -423
bp is required for TNF- responsiveness and for TNF-- and
IFN--stimulated synergistic responsiveness of the Bf
gene. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although Bf is primarily synthesized by the liver
(15, 16, 17), it has also been shown to be produced at low
levels by a wide variety of extrahepatic cells, including
macrophage/monocytes (18, 19), fibroblasts
(20), epithelial cells, and endothelial cells (21, 22). Macrophages are an important extrahepatic site of Bf
synthesis (15, 18, 23, 24) which may contribute
significantly to the local concentration of Bf at sites of inflammation
(24). At these local sites of inflammation, inflammatory
cytokines, particularly IFN- and TNF-, regulate Bf
expression (15, 18, 23). Although a number of studies have
documented Bf induction by proinflammatory cytokines (e.g., IFN- and
TNF-) (15, 18, 23, 24, 25), there is relatively little
known about the mechanism of Bf induction by these
cytokines. Understanding the mechanism of Bf regulation is
the first step in potentially manipulating alternative complement
activation in sepsis.
TNF- and IFN- are mediators of the immune response, potentiating
signal transduction pathways leading to gene induction. Recent work
done in our laboratory has detailed the molecular regulation of
Bf by IFN- in macrophages (15). We have
mapped the IFN--responsive region of the murine Bf
promoter to between -154 bp and -53 bp 5' to the transcription
initiation site. Moreover, we have revealed that either an IFN-
activation site (GAS) or an IFN-stimulated response element (ISRE)
cis-binding element is required and that both GAS and ISRE
sites act in an additive fashion to induce Bf gene
transcription by IFN-. Further, we demonstrated that Stat1 and
IFN-regulatory factor-1 (IRF-1) take part in Bf induction by
IFN- in macrophages. Although it is clear that TNF- acts
synergistically with IFN- to induce Bf in primary
macrophages (23), little is known of the molecular
mechanism of Bf induction by TNF- and the synergistic
effect of TNF- and IFN-. In the current study, 6.7 kb of the
5'-untranslated region of the Bf gene was analyzed to map
both the region responsible for TNF- responsiveness and for
IFN-/TNF--stimulated synergistic responsiveness in macrophages.
We show that the TNF--responsive region of the murine Bf
promoter occurs between -556 bp and -282 bp 5' to the transcription
initiation site. Mutational analysis reveals that a NF-B
cis-binding sequence at -433 to -423 bp is required for
both the induction of Bf promoter activity by TNF- and
the synergistic induction of Bf promoter activity by TNF-
and IFN-. A nuclear binding complex containing p50 and p65 proteins
binds to this cis element on the Bf promoter
after TNF- simulation in macrophages. LPS and IL-1
also induced
Bf expression in MH-S cells using this same NF-B site. We
also demonstrate that TNF- signaling to Bf induction is
an I-B phosphorylation dependent process.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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TNF- and IFN- were from R&D Systems (Minneapolis, MN).
Pfu DNA polymerase was purchased from Stratagene (La Jolla,
CA), and restriction enzymes were from New England Biolabs (Beverly,
MA). Anti-p65, anti-p50, and isotype-matched control Abs were from
Santa Cruz Biotechnology (Santa Cruz, CA).
Cell culture
All experiments were performed using MH-S cells (a murine alveolar macrophage cell line) from American Type Culture Collection (Manassas, VA). MH-S cells were maintained in RPMI 1640 (Life Technologies, Gaithersburg, MD) supplemented with 10% FBS (Life Technologies), 100 U/ml penicillin, 100 µg/ml streptomycin, 1 mM sodium pyruvate, and 0.58 µM 2-ME.
Isolation of total cellular RNA and Northern blot analysis
MH-S cells were plated at a density of 5 x
105 cells/35-mm dish 24 h before stimulation
with different concentrations of TNF- and/or IFN- for defined
time periods. Total RNA was isolated using TRIzol (Life Technologies)
as described by the manufacturer’s protocol. Northern blot analysis
was conducted as previously described (26). Briefly, the
isolated RNA was separated by agarose gel electrophoresis, transferred
to a Nytran membrane, and probed for mRNA using a
32P-labeled murine Bf cDNA probe, and
radioactive bands were developed by autoradiography. The probe was
labeled with [-32P]dCTP using a random
primer labeling kit (Life Technologies).
Subcloning of pGL3-Bf reporter gene
The vector containing Bf promoter was a gift from Drs. G. Garnier and H. Colten (Washington University School of Medicine, St. Louis, MO). Various Bf promoter-pGL3 chimeric reporter constructs were generated by restriction enzyme digestion followed by fragment ligation to pGL3 basic vector (Promega, Madison, WI).
Site-directed mutagenesis
The NF-B-binding elements on the Bf promoter
(-433 to -423 and -525 to -516) were mutated using PCR
site-directed mutagenesis as previously described (27, 28). Primers containing 5'-437CACACGGAATTTCC-424
and 5'-525GGGAATCCCC-516 were designed to mutate the
NF-B sites (mutated nucleic acids are underlined). The mutants were
generated by PCR overlap extension method using the pGL3-Bf (-556)
construct as template DNA. After PCR, the products containing the
mutants were gel isolated, digested with appropriated restriction
enzymes, and ligated into the pGL3-basic vector (Promega). The
mutations were confirmed by direct sequencing at the University of
Calgary Core DNA Services Laboratory Facility (Calgary, Canada).
Transient transfection
Transient transfections were performed by electroporation of
MH-S cells with 1.5 µg of pGL3-Bf reporter vector and 0.5 µg of
LNC-Gal (a constitutively expressing
-galactosidase-containing vector from Dr. S. Robbins, University of
Calgary). The cells were cultured for 96 h to allow expression of
the transgene. Transfectants were treated with medium alone (control),
or medium in the presence of cytokine(s) or LPS for an additional
6 h. Cell lysates were analyzed for luciferase and
-galactosidase activities. Luciferase activity was measured using a
luminometer (Monolight 2010; Analytical Luminescence Laboratory, San
Diego, CA). Measurements were taken for 30 s after mixing of the
lysate and luciferin assay reagent (Promega) as recommended by the
manufacturer’s protocol. -Galactosidase was assayed in cell lysates
using a -galactosidase enzyme assay system kit (Promega).
-Galactosidase was used as an internal control for normalizing
variability in reporter luciferase activity due to transfection
efficiency or variability in the cell extract preparation. Each
transfection was performed in triplicate, and at least three
independent experiments were conducted with each construct.
Adenoviral construction and transduction of MH-S cells
The cDNA encoding I-B (wild-type (WT)) or I-B (dominant
negative (DN)) (I-B S32A/S36A mutant from Dr. M. J. Tremblay,
Laval University, Laval, Canada) were cloned into the adenoviral
plasmid pACCMV.pLpA (29), cotransfected with pJM17 into
HEK 293 cells, and plaque purified as previously described
(30). Plaques were screened for rAdI-B(WT) and
rAdI-B(DN) using PCR and primers encoding the I-B insert.
Adenovirus vector particle number was determined by
OD260 (31). MH-S cells were first
transfected with pGL3-Bf (-556). After 72 h, cells were
transduced with AdI-B(WT), AdI-B(DN), or vehicle alone. After
24 h, the cells were treated as indicated.
Preparation of nuclear extracts
MH-S cells were treated with medium alone (control) or TNF-
and/or IFN-. After stimulation, cells were washed with cold PBS and
lysed in 400 µl of lysis buffer (10 mM HEPES, 10 mM KCl, 0.1 mM EDTA,
0.625% Nonidet P-40, 2 mM DTT, 1 mM PMSF, 2 µg/ml leupeptin, and 20
µg/ml aprotinin) on ice. After 15 min, the cells were scraped, and
lysates were centrifuged at 20,000 x g for 30 s
at 4°C to collect nuclei. The supernatant was discarded, whereas
cellular debris and nuclei in the pellet were resuspended in 50 µl of
extraction buffer (20 mM HEPES, 0.42 M NaCl, 5 mM EDTA, 10% glycerol,
5 mM DTT, 1 mM PMSF). Samples were incubated for 30 min at 4°C with
constant agitation. The samples were then centrifuged at 20,000 x
g for 10 min at 4°C. Protein concentrations were
quantified using a Bio-Rad protein assay as described by the
manufacturer’s protocol (Bio-Rad, Hercules, CA). The supernatant was
stored in aliquots at -70°C.
EMSA
An oligonucleotide with the potential NF-B-binding element of
the Bf promoter region (5'-TTCACACGGAATTTCCCAGT-3') was
synthesized by Life Technologies. Using T4 kinase in a total reaction
volume of 30 µl, 10 pM concentrations of the sense and antisense
oligonucleotides were 32P end labeled for 30 min.
Free nucleotide was removed using Qiaquick nucleotide removal columns
(Qiagen, Chatsworth, CA). The oligonucleotide was recovered in 45 µl
double-distilled H2O, to which NaCl was added to
a final concentration of 50 mM. The probe was heated to 80°C, and
strand annealing was performed at 50°C for 30 min followed by 25°C
for 30 min. Nuclear protein (2 µg), poly(dI-dC) (3.3 µg), and 5x
incubation buffer (5 mM EDTA, 500 mM NaCl, 50 mM Tris-Cl, 5 mM
MgCl2; 5 µl) were added to a total volume of 27
µl double-distilled H2O. After incubating for
20 min at room temperature, 3 µl of labeled probe, 
100 fmol, were
added to each tube. Binding was allowed to occur for 20 min. Samples
were resolved through a 6% acrylamide, 0.5x Tris-buffered EDTA
gel. Gels were dried, and radioactive bands were quantified by
PhosphorImager analysis using a Storm PhosphorImager (Molecular
Dynamics, Sunnyvale, CA). Supershift assays were performed by
incubating nuclear extracts with anti-p65 Ab, anti-p50 Ab, or
isotype-matched control Ab in the presence of all the components of the
reaction described above for 20 min at room temperature following
addition of the probe.
Statistics
Student’s t test was used to determine whether
differences existed between experimental mean values. A value of
p 
0.05 was considered significant. All statistical
analysis was done using StatView software (SAS Institute, Cary,
NC).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IFN- induce Bf mRNA expression
synergistically in macrophages
We have previously shown that IFN- alone was sufficient to
induce Bf mRNA expression in macrophages (15).
We questioned whether TNF- alone or in combination with IFN-
would induce a higher level of Bf mRNA expression than
IFN- alone. Incubation of MH-S cells (a murine alveolar macrophage
cell line) with TNF- alone resulted in a small induction of
Bf mRNA (Fig. 1
A;
the relative densitometry values of lanes 1, 2, and
3 are 1, 1.85, and 1.31, respectively). However, incubation
of MH-S cells with TNF- and IFN- resulted in synergistic
expression of Bf mRNA, compared with TNF- or IFN-
alone (Fig. 1A; the relative densitometry values of
lanes 4, 5, 6, and 7 are 1.46, 2.33, 4.32, and
4.75, respectively). In the presence of IFN-, TNF- produced a
dose-dependent increase in the level of Bf mRNA (Fig. 1A). A time course of Bf mRNA expression in the
presence of TNF- (1 ng/ml) and IFN- (10 ng/ml) is shown in Fig. 1B. Costimulation resulted in a detectable increase of
Bf mRNA expression in as early as 4 h, which continued
to increase from 8 h through 24 h before beginning to
decrease by 36 h. As an internal control, 28S rRNA was used
(15), showing equal RNA loading on the gels. Thus,
costimulation with TNF- and IFN- results in a dose- and
time-dependent synergistic increase in Bf mRNA induction in
murine macrophages.
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responsiveness of the Bf gene
Recently, we have identified that ISRE and GAS
cis-elements, located between -154 and -53 bp of
Bf promoter, are responsive regions for Bf
induction by IFN-. We questioned which portion of the Bf
promoter conferred TNF- responsiveness and
TNF-/IFN--synergistic responsiveness. To identify the region of
the Bf promoter that mediates TNF-- and
TNF-/IFN--stimulated Bf promoter induction in
macrophages, we analyzed sequential 5' Bf promoter deletion
fragments. As shown in Fig. 2, a series
of Bf promoter deletion fragments ranging from -6670 to
-282 bp were cloned into to the pGL3-luciferase reporter vector.
Transiently transfected MH-S cells were treated with medium alone or
with TNF- (10 ng/ml) and/or IFN- (10 ng/ml) for 6 h, and
then cell lysates were analyzed for luciferase activity. A 6-h time
point was chosen because this time point showed maximal luciferase
activity in a time course analysis of IFN--stimulated MH-S cells
transfected with a Bf promoter-luciferase chimeric construct
(data not shown). Fig. 2 shows that TNF- treatment of MH-S cells
transfected with pGL3-Bf (-6670) resulted in a 3.2-fold induction of
luciferase activity compared with that in untreated cells. The pGL3-Bf
(-4254) and pGL3-Bf (-556) constructs induced 2.7- and 2.8-fold
increase in luciferase activity, respectively, when stimulated with
TNF- alone. The construct containing -282 bp of the Bf
promoter (pGL3-Bf (-282)) was unresponsive to TNF- stimulation,
indicating a TNF--responsive region is located between -556 and
-282 bp. The synergistic increases in luciferase activity on
stimulation with TNF- and IFN- were found to be 7.4-, 7.6-, and
6.4-fold increased with pGL3-Bf (-6670), pGL3-Bf (-4254), and pGL3-Bf
(-556), respectively (Fig. 2). As with TNF- alone, the -282
Bf fragment showed no synergistic response in the presence
of TNF-/IFN-. These data reveal that the region between -556 and
-282 bp of the Bf promoter is necessary for both the
TNF- responsiveness of the Bf gene and the synergistic
induction seen with TNF- and IFN- costimulation.
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B cis-element between -433 and -423 bp
is required for expression of Bf in response to TNF-
Deletion mapping of the Bf promoter indicated that the
region between -556 and -282 is necessary for TNF- responsiveness
of the Bf promoter. Therefore, this region was examined by
web-based computer analysis (using TESS, MatInspector, and TFSEARCH)
for potential transcription factor-binding sites. Fig. 3 reveals that a number of potential
transcription factor cis-binding elements are located in
this region including two potential NF-B cis-binding
sites located between -525 and -516 bp and between -433 and -423
bp. To identify whether these sites may be involved in TNF-
responsiveness of the Bf promoter, the potential B
cis-binding sequences were mutated by overlapping PCR
site-directed mutagenesis. The WT reporter plasmid, pGL3-Bf (-556),
was used to construct plasmids containing a mutation in each of the
B cis-elements. Mutations were confirmed by direct
sequencing. The WT construct and mutants were transiently transfected
into MH-S cells and stimulated with or without TNF- (10 ng/ml)
and/or IFN- (10 ng/ml) for 6 h. We found that the potential
NF-B-binding site between -525 and -516 bp was not required for
Bf induction by TNF- because mutation
(5'-GGGAATCCCC-3' to 5'-GTCCATCCCC-3') of this
site did not significantly change induction of Bf promoter
activity by TNF- (Fig. 4A).
However, mutation of the NF-B site between -433 and -423 bp
(5'-CACACGGAATTTCCC-3' to
5'-CACAATCAATTTCCC-3') virtually abolished TNF--induced
luciferase activity in transfected macrophages (Fig. 4A).
This mutant also decreased the synergistic effect of TNF- and
IFN- (p = 0.03) but, as expected, had no
effect on the IFN--mediated response (Fig. 4B). Mutation
of NF-B (-423), GAS, as well as ISRE cis elements
virtually abrogated TNF- and IFN- as well as synergistic
responsiveness of the Bf promoter (Fig. 4B).
These data indicate that the NF-B cis-binding element at
-433 to -423 bp and not -525 to -516 bp, is required for
Bf induction by TNF-. The NF-B cis-binding
element (-433 to -423 bp) is also important for the synergistic
effect of TNF-/IFN- costimulation.
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-stimulated macrophages induce a nuclear binding complex
containing p50 and p65 subunits of NF-B to a B
cis-element of the Bf promoter
To further investigate the potential role of the B-like
cis-binding element of the Bf promoter, we
examined whether the B cis-binding site located at -433
to -423 bp is a target for trans-acting factor binding.
EMSA were conducted on unstimulated and TNF--stimulated (10 ng/ml)
MH-S cells using a 32P-labeled DNA probe of the
Bf promoter region containing the B-like region of the Bf
promoter. As shown in Fig. 5
(lanes 1 and 2), a DNA-protein complex was
induced after 30 min of stimulation with TNF-. Formation of the
nuclear binding complex was strongly inhibited when a 25-fold molar
excess of unlabeled competitor DNA was included (Fig. 5, A,
lane 5, and B, lane 3). Addition of an
excess amount of a mutant Bf B cis-element did
not inhibit nuclear binding to the Bf B site (Fig. 5, A, lane 6, and B, lane 4).
Costimulation with TNF-/IFN- exhibited the same level of binding
activity as that seen in cells stimulated with TNF- alone (data not
shown). Importantly, there was no induction of NF-B binding to the
B cis-element at -525 to -516 bp in these
TNF--stimulated MH-S macrophages (data not shown). These experiments
reveal that TNF- stimulation of MH-S macrophages induced the binding
of nuclear protein(s) that are specifically directed to the B
cis-element of the Bf promoter located at -433
to -423 bp.
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B:B nuclear binding complex may consist of a variety of
hetero- or homodimers, however, p50 and p65/Rel proteins have been
shown to constitute important NF-B subunits in most cell types
(32). Therefore, we examined whether p50 and p65 proteins
were involved in the binding complex that occurs on the B site at
-423 to -433 bp of the Bf promoter. As shown in Fig. 5
, B (lane 5) and A
(lane 3), incubation with anti-p50 Ab and
anti-p65 Ab, respectively, resulted in supershifting of the B
binding complex. In contrast, incubation with isotype-matched control
Ab had no effect on the formation or mobility of the NF-B complex
(Fig. 5A, lane 4). We also used both anti-p50
and anti-p65 Abs together which completely supershifted the NF-B
band (data not shown). Although several forms of NF-B dimers exist,
these results indicate the involvement of both the p50 and p65 subunits
of NF-B in binding the B element at -433 to -423 bp of the
Bf promoter after TNF- stimulation in macrophages.
TNF- mediates Bf induction in an I-B
phosphorylation-dependent manner
NF-B is normally retained in the cytoplasm by I-B, the
inhibitor of NF-B. After cellular stimulation, the I-B proteins
are phosphorylated on serine residues (S32, S36) and rapidly degraded
by proteasomes, allowing the nuclear translocation of the NF-B
dimer. To further determine the role of NF-B in TNF--induced
Bf expression, we tested whether overexpression of a DN
I-B mutant (I-B(DN), S32A/S36A) could inhibit TNF--stimulated
Bf promoter activity. We used an I-B(DN) construct to
block I-B phosphorylation and subsequent proteasome-mediated
degradation of I-B. MH-S cells were cotransfected with pGL3-Bf
(-556) and I-B(DN) or I-B(WT). After 96 h, transfected
cells were treated with TNF- and/or IFN- for an additional 6
h. As shown in Fig. 6A,
I-B(DN), but not I-B(WT), completely abolished TNF--induced
Bf promoter activity. I-B(DN) also inhibited the
synergistic effect of TNF-/IFN- on Bf promoter
activation. However, as expected, neither I-B(WT) nor I-B(DN)
show any effect on IFN--stimulated Bf induction. Similar
results were obtained when the I-B(WT) construct and I-B(DN)
construct were transduced into MH-S cells using adenoviral gene
transfer methods. We constructed an adenovirus containing the WT
I- (AdI-B(WT)) and an adenovirus containing the DN I-B
construct (AdI-B(DN)). MH-S cells were first transfected with the
pGL3-Bf (-556) promoter-reporter construct and then transduced with
either AdI-B(WT) or AdI-B(DN) or incubated with vehicle alone
followed by stimulation with IFN-, TNF-, or both as indicated
(Fig. 6B). Although the vehicle alone may have had a small
stimulatory response (likely secondary to the hyperosmolar effect of
the glycerol in the vehicle), the data clearly show that
TNF--mediated Bf gene induction was inhibited when the
MH-S cells were transduced with the vector AdI-B(DN). The
IFN-/TNF--stimulated synergistic response was also inhibited, but
not completely so, in the AdI-B(DN)-transduced cells. These data
demonstrate that TNF--mediated Bf induction depends on
the phosphorylation and degradation of I-B.
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B is mediated by proteasomes
(33), we also investigated whether the proteasome
inhibitor MG132 could affect TNF--induced Bf induction.
pGL3-Bf (-556) promoter-reporter-transfected MH-S cells were
preincubated for 30 min with 1.0 µM MG132 and then treated with
different cytokines for 6 h. As shown in Fig. 7, 1 µM MG132 completely blocked the
activation of Bf induced by TNF-. Interestingly, MG132
also reduced IFN--stimulated Bf induction (Fig. 7A). MG132 at 1 µM does not change cell viability as
measured by transcriptional and translational cell function, because
relative -galactosidase activities were unchanged with MG132
treatment (Fig. 7B). These data suggest that proteasomes are
involved in TNF--stimulated Bf induction in macrophages
and proteasome function may also affect IFN--mediated gene
regulation in macrophages.
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and LPS induced Bf promoter activity
In addition to TNF-/IFN-, there are other inflammatory
stimuli that affect Bf induction in macrophages. Among them, IL-1
and LPS are known to induce Bf production in some cell types (20, 34). Because LPS and IL-1 have been shown to activate
NF-B, we questioned whether LPS and IL-1 used the same mechanism
of induction of Bf as TNF-. As shown in Fig. 8A, both IL-1 and LPS
stimulated Bf induction in MH-S cells. Although
significantly different from control, IL-1-stimulated MH-S cells do
not induce Bf promoter activity as extensively as TNF-,
IFN-, and LPS. Moreover, mutation of the B site between -433 and
-423 bp of the Bf promoter significantly reduced the
induction of Bf by LPS (Fig. 8B) and IL-1
(data not shown). These data demonstrate that many factors (including
IL-1, TNF-, IFN-, and LPS) can induce Bf production
in macrophages and that TNF-, LPS, and IL-1 appear to use the
same mechanism to induce Bf promoter activity in
macrophages, that is primarily through the -433 to -423 B
cis-binding site of the Bf promoter.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IFN- in murine macrophages.
Bf mRNA expression is synergistically up-regulated by
TNF- and IFN- in MH-S cells. Detailed studies of the
Bf promoter demonstrated that the region between -556 and
-282 bp mediates TNF- responsiveness as well as the synergistic
effect of TNF- and IFN- on Bf promoter activation.
Mutation of a potential B cis-binding element in this
region (-433 to -423) abrogated TNF- responsiveness and decreased
the synergistic effect of TNF- and IFN- on Bf promoter
activity. EMSAs revealed nuclear protein binding to this
NF-B-binding element on TNF- stimulation. Supershift analysis
revealed that both p50 and p65, protein members of the NF-B family,
contribute to induction of Bf by TNF-. Experiments using
a DN I-B construct revealed that TNF- mediates Bf
promoter activity in an I-B phosphorylation-dependent manner. Thus,
activation of NF-B is a critical component of transcriptional
induction of the Bf gene by TNF- in macrophages. Finally,
using site-directed mutagenesis of the Bf promoter, we show
that TNF-, IL-1, and LPS use the same B cis-binding
site to induce Bf promoter activity in macrophages.
Macrophages play an important role in initiating and regulating immune
and inflammatory responses (35). Macrophages produce
proinflammatory cytokines including TNF- and IFNs, which in turn
induce Bf production (18, 23). IFN- plays an important
role in mediating inflammation, stimulating the production of Bf and
potentiating the effects of other proinflammatory cytokines, such as
TNF- and IL-1, and bacterial cell wall products, such as LPS. We
have previously shown that Stat1 and IRF-1 are involved in IFN-
signaling and that both GAS and ISRE cis-elements are
required for the induction of Bf in macrophages by IFN-
(15). However, the mechanisms of TNF--stimulated
Bf induction and cooperation of IFN- and TNF- in
Bf induction have never been detailed. The main purpose of
this study was to extend previous studies: 1) to conduct a detailed
examination of TNF--stimulated Bf induction; 2) to
examine the cooperation of IFN- and TNF- on Bf gene
expression; and 3) to elucidate the involvement of potential factor(s)
in the signaling pathway(s) to Bf induction by IFN- and
TNF-.
Many studies have shown that TNF- stimulation activates NF-B
(35). The NF-B family consists of several subunits,
including p65, c-Rel, Rel B, p100, p105, p50, and p52, which form
homodimeric or heterodimeric nuclear binding complexes (36, 37). The complex composed of p50 and p65 is the most common
active NF-B dimer found in mammalian cells (32, 38).
Our data confirmed that the regulation of Bf by TNF-,
like that of many other genes, uses both p50 and p65 subunits of
NF-B. There are two potential NF-B-binding sites between the
transcription initiation site and -556 bp of the Bf
promoter: one of them located between -525 and -516 bp; and the other
between -433 and -423 bp. We found that the potential NF-B-binding
site between -525 and -516 bp was not required for Bf
induction by TNF- because mutation of this site did not
significantly change the induction of Bf by TNF-. This is
in contrast to reports by Nonaka et al. (34, 39), who
described this region as required for IL-1 induction of Bf
in murine L(tk-) cells and human HepG2 cells.
Our investigations have shown that the NF-B-binding site between
-433 and -423 bp is required not only for TNF--, but also for LPS-
and IL-1-stimulated Bf gene expression in MH-S cells
(Figs. 4 and 8 and data not shown). The differences between our
results and the results of Nonaka et al. may be explained by the use of
different NF-B sites in different cell types. However, Nonaka et al.
used only truncation fragments and EMSAs, not mutants of the NF-B
site, to prove its requirement for IL-1-stimulated Bf
gene expression (39).
TNF- appears to act by inducing the release and degradation of
I-B from NF-B:I-B trimer complex subsequent to the
phosphorylation of serine residues on I-B. The phosphorylated I-B
is released from NF-B and activated NF-B is then rapidly
translocated from the cytoplasm to the nucleus to bind to the
Bf promoter on the -423 to -433 B
cis-element in macrophages (33, 35). In this
study, we have found that Bf induction by TNF- was
inhibited by MG132 which is known to inhibit NF-B activation. This
supports the concept that Bf induction by TNF- is
dependent on the activation of NF-B, which requires I-B
phosphorylation and ubiquitin-mediated proteasomal degradation. In
addition to MG132, we have also used the proteasome inhibitor
pyrrolidinedithiocarbamate which also blocks Bf induction by
TNF-/IFN- (data not shown). Like MG132, inhibition of
Bf induction by TNF- is more sensitive to
pyrrolidinedithiocarbamate than induction by IFN-. Activation of
NF-B by TNF- is also blocked by overexpression of I-B(DN) in
macrophages. However, IFN--stimulated Bf promoter
activation is not blocked by I-B(DN). Therefore, the signaling
pathways of TNF- and IFN- to Bf are quite different.
Because MG132 inhibited both TNF-- and IFN--stimulated
Bf induction in viable cells, our data suggest that
ubiquitin-mediated proteasomal degradation may be involved in both
TNF- and IFN- signaling whereas I-B phosphorylation is
required only for TNF- signaling. Other reports also show the
involvement of proteasomes in IFN-mediated gene induction (40, 41).
Both TNF- and IFN- are common and important acute phase inducers
of complement genes (42). Studies have shown that high
levels of Bf induction in macrophages require two signals, a
priming signal (e.g., IFN-) and a triggering signal (e.g., TNF-)
(18, 23). MH-S cells treated with TNF- and IFN-
result in activating and binding of NF-B, Stat1, and IRF-1 to their
mutually exclusive cis-elements on the Bf
promoter. The molecular mechanisms involved in the synergistic effect
are multifactorial. The synergistic effect of TNF- and IFN- on
the Bf promoter may involve a complex interaction between
the transcription factors activated by the two cytokines
(35) and/or the ability of IFN- to induce the
expression of TNF- receptors (43). TNF-/IFN- may
also induce other cytokines or act in an autocrine manner to increase
their effect. Another potential mode of activation may be through the
interaction of transcription factors to enhance both DNA binding
affinity and protein complex stability (44, 45). Mutation
of ISRE/GAS and the B cis sites virtually abolished
either IFN-- or TNF--mediated Bf promoter activation;
however, it could not completely abolish the induction of Bf
promoter when both IFN- and TNF- were used together as
stimulants. Similarly, I-B(DN) completely blocked the responsiveness
to TNF- alone; however, it only partially blocked the induction
mediated by cotreatment with IFN- and TNF- (Fig. 6). This
suggests that Bf promoter activation may occur without
binding to these cis sites or by activating and binding of
other transcription factors to secondary sites (other than NF-B
sites). There are no significant differences in the pattern of DNA
binding complexes to the B site in nuclear extracts between cells
treated with IFN- and TNF- in combination and cells treated with
TNF- alone (data not shown). It is possible that the B-binding
factors may interact with other proteins in vivo that cannot be
detected in vitro by EMSA. Several reports do indicate the potential
cooperation between IFN--induced proteins (e.g., IRF-1) and
TNF--activated proteins (e.g., NF-B or AP-1) on several other
genes (46, 47, 48, 49, 50). Although the data presented here do show
that IFN- and TNF- act synergistically to induce Bf,
it is not entirely clear how IFN- cooperates with TNF- signaling
to synergistically induce gene expressions. Our model system of
Bf promoter activation also does not show the same
synergistic induction as one can see using Northern analysis,
suggesting that other mechanisms of activation of the Bf
promoter may be operating either up or down stream of -556 to +105 bp
of Bf promoter in the synergistic response. Further, Fig. 2
supports the possibility of a minor contribution by (a) factor(s)
acting on a cis-element(s) upstream of -556 bp of the
Bf promoter with TNF- and IFN- costimulation. However,
our data do show that NF-B acting on the kB cis-binding
site at -423 to -433 is crucial for the synergistic effect of
Bf induction by TNF- and IFN-.
In addition to TNF-/IFN-, there are several factors that may
affect Bf induction. Among them, IL-1 (34)
and LPS (20) are known to induce Bf. However,
the magnitude of the effect of IL-1 on Bf promoter
activity is not as large as IFN-, TNF-, and LPS in MH-S
macrophages (Fig. 8). The response to LPS was not increased by
cotreatment with TNF- (data not shown), indicating that LPS and
TNF- may share the same or similar pathway. We also found that
mutation of the NF-B site between -433 and -423 bp significantly
reduced Bf promoter activation by LPS and IL-1 (Fig. 8 and data not
shown). Thus, the importance of the B cis site between
-433 and -423 bp is not limited to TNF- stimulation. This
B-binding site also participates in LPS and IL-1-stimulated
Bf gene induction in macrophages.
It has been observed that it is difficult to transfect DNA into
macrophages using conventional methods (51). We find that
the transfection efficiencies are 10% when MH-S cells are
transfected with a green fluorescent protein vector by electroporation
or by liposomal methods. However, DNA transfer efficiencies can reach
>80% using adenoviral vectors in human macrophages (52, 53). Fig. 6 shows that transducing the I-B(DN) construct into
macrophages inhibits TNF- and IFN-/TNF--stimulated
Bf induction. The hyperosmolar vehicle (glycerol) used in
the adenoviral experiments may itself induce an inflammatory response
as evidenced by elevated baseline luciferase activity, however this is
inhibitable by the I-B(DN) construct. Thus, viral mediated
I-B(DN) gene transfer may be an efficient strategy to control
Bf induction in macrophages.
In summary, we have demonstrated that TNF- and IFN-
synergistically induce Bf expression in macrophages. A B
cis-binding site at -433 to -423 bp is required for
TNF--stimulated Bf promoter induction as well as for the
synergistic induction in the presence of TNF- and IFN-. This site
also appears to mediate LPS- and IL-1-induced Bf promoter
activity in macrophages. The synergistic effect highlights the
importance of cytokine interactions amplifying their biological effects
during inflammation. A clear understanding of the molecular mechanisms
that regulate Bf expression may lead to the development of
novel treatments for inflammatory stimulated complement-mediated host
damage seen in sepsis and septic shock.
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Acknowledgments |
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B
constructs. We also thank Connie Mowat for technical assistance. |
Footnotes |
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2 Address correspondence and reprint requests to Dr. Brent W. Winston, Departments of Medicine, Critical Care Medicine and Biochemistry and Molecular Biology, University of Calgary, Health Sciences Center, Room 1843, 3330 Hospital Drive N.W., Calgary, Alberta, Canada T2N 4N1. E-mail address: bwinston{at}ucalgary.ca
3 Abbreviations used in this paper: Bf, complement factor B; ISRE, IFN-stimulated response element; GAS, IFN- activation site; WT, wild type; DN, dominant negative; IRF-1, IFN-regulatory factor-1.
Received for publication December 26, 2001. Accepted for publication July 1, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
regulation of the complement factor B gene in macrophages. Eur. J. Immunol. 31:3676.[Medline]
1-antitrypsin, C8, and factor B. Clin. Immunol. Immunopathol. 16:84.[Medline]
interferon and lipopolysaccharide for synthesis of factor B, but not C2, in human fibroblasts. J. Exp. Med. 167:1. induces synthesis of complement alternative pathway proteins by human endothelial cells in culture. J. Exp. Med. 168:1917. by interferons: implications for the pleiotropic activities of TNF. J. Clin. Invest. 93:1661.
B activity. Annu. Rev. Immunol. 18:621.[Medline]
B1 precursor protein and the activation of NF-B. Cell 78:773.[Medline]
B-like hepatoma nuclear factor. Mol. Cell. Biol. 10:6283.B signal transduction pathway: introduction. Oncogene 18:6842.[Medline]
B transcription factors: structural views. Oncogene 18:6845.[Medline]
B signaling pathways in mammalian and insect innate immunity. Genes Dev. 15:2321.-regulated genes. Eur. J. Biochem. 267:4011.[Medline]
induces cell surface expression for both types of tumor necrosis factor receptors. FEBS Lett. 312:87.[Medline]
gene expression requires the assembly of an enhanceosome. Cell 83:1091.[Medline]
B. Mol. Cell. Biol. 13:7191. and tumor necrosis factor- in transcriptional activation is mediated by cooperation between signal transducer and activator of transcription 1 and nuclear factor B. J. Biol. Chem. 272:14899.B and interferon regulatory factor 1 physically interact and synergistically induce major histocompatibility class I gene expression. J. Interferon Cytokine Res. 15:1037.[Medline]
B as a transcriptional activator of vascular cell adhesion molecule 1. Mol. Cell. Biol. 15:2558.[Abstract]
B during activation of inducible nitric oxide synthase transcription. J. Mol. Biol. 289:459.[Medline]
B into human macrophages: lipopolysaccharide-induced, but not zymosan-induced, proinflammatory cytokines are inhibited, but IL-10 is nuclear factor-B independent. J. Immunol. 162:2939.This article has been cited by other articles:
|
|
 |
|
  K. Goring, Y. Huang, C. Mowat, C. Leger, T.-H. Lim, R. Zaheer, D. Mok, L. A. Tibbles, D. Zygun, and B. W. Winston Mechanisms of human complement factor B induction in sepsis and inhibition by activated protein C Am J Physiol Cell Physiol, May 1, 2009; 296(5): C1140 - C1150. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. S. Bolger, D. S. Ross, H. Jiang, M. M. Frank, A. J. Ghio, D. A. Schwartz, and J. R. Wright Complement levels and activity in the normal and LPS-injured lung Am J Physiol Lung Cell Mol Physiol, March 1, 2007; 292(3): L748 - L759. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Fairweather, S. Frisancho-Kiss, D. B. Njoku, J. F. Nyland, Z. Kaya, S. A. Yusung, S. E. Davis, J. A. Frisancho, M. A. Barrett, and N. R. Rose Complement Receptor 1 and 2 Deficiency Increases Coxsackievirus B3-Induced Myocarditis, Dilated Cardiomyopathy, and Heart Failure by Increasing Macrophages, IL-1beta, and Immune Complex Deposition in the Heart J. Immunol., March 15, 2006; 176(6): 3516 - 3524. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Schroder, P. J. Hertzog, T. Ravasi, and D. A. Hume Interferon-{gamma}: an overview of signals, mechanisms and functions J. Leukoc. Biol., February 1, 2004; 75(2): 163 - 189. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. P. Sorescu, M. Sykes, D. Weiss, M. O. Platt, A. Saha, J. Hwang, N. Boyd, Y. C. Boo, J. D. Vega, W. R. Taylor, et al. Bone Morphogenic Protein 4 Produced in Endothelial Cells by Oscillatory Shear Stress Stimulates an Inflammatory Response J. Biol. Chem., August 15, 2003; 278(33): 31128 - 31135. [Abstract] [Full Text] [PDF]
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