Am J Physiol Lung Cell Mol Physiol 289: L429 –L437, 2005;
doi:10.1152/ajplung.00046.2005.
Inducible binding of PU.1 and interacting proteins to the Toll-like
receptor 4 promoter during endotoxemia
Tetyana V. Pedchenko, Gye Young Park, Myungsoo Joo, Timothy S. Blackwell, and John W. Christman
Division of Allergy, Department of Medicine, Pulmonary and Critical Care Medicine, Vanderbilt
University Medical Center; and Department of Veterans Affairs, Nashville, Tennessee
Submitted 24 January 2005; accepted in final form 5 May 2005
PU.1 interacting proteins
or endotoxin, the major structural component of
the outer wall of gram-negative bacteria, is a potent activator of
macrophages. Recognition of gram-negative endotoxin by the
Toll-like receptor 4 (TLR4) complex leads to rapid secretion of
inflammatory cytokines and chemokines that mediate the
pathophysiology of severe sepsis and the acute respiratory
distress syndrome. TLR4 is a pattern recognition molecule on
the surface of macrophages that is essential, but not entirely
sufficient, for mediation of the endotoxin response (5). Several
laboratories have shown that mice that lack functional TLR4
are highly resistant to endotoxin (16, 37, 49). Monocytes/
macrophages are intrinsically more responsive to endotoxin than
neutrophils, basophils, or eosinophils (43) due to abundant expression of TLR4. It is possible that modulation of TLR4 gene
expression in pulmonary macrophages is a critical determinant of
the intensity and/or duration of the response to endotoxin.
Both human and mouse TLR4 gene contain multiple purinerich sequence motifs that are recognized by transcription factors of Ets family, including myeloid-specific factor PU.1.
Several lines of evidence suggest that PU.1 is critical for both
BACTERIAL LPS
Address for reprint requests and other correspondence: J. W. Christman,
Section of Pulmonary, Critical Care, and Sleep Medicine, Univ. of Illinois at
Chicago, 840 S. Wood St., Chicago, IL 60612 (e-mail: [email protected]).
http://www.ajplung.org
macrophage maturation and transcriptional regulation of TLR4
in macrophages. Mice homozygous for a disruption in the PU.1
DNA binding domain prematurely die of severe sepsis because
of loss of myeloid lineages (24, 25). Although PU.1 is involved
in multiple myeloid lineages, including macrophages, B-lymphocytes, and neutrophils, only high levels promote differentiation to fully competent mature macrophages (8). A recent in
vitro study that employed an EMSA in RAW cells indicates
that PU.1 protein binds to a cognate sequence in the TLR4
promoter, and mutation of this site impairs expression of a
promoter-reporter construct (40). Finally, granulocyte/macrophage colony-stimulating factor (GM-CSF) null macrophages
that lack detectable TLR4 are induced to express abundant
TLR4 when transfected with PU.1 (46), indicating that induction of PU.1 may mediate the effects of GM-CSF on mature
macrophage function.
PU.1 is involved in combinatorial regulation of gene expression through interaction with coregulatory partners. Two of
them, Pip/interferon regulatory factor (IRF)4 and interferon
consensus sequence-binding protein (ICSBP)/IRF8, are members of the IRF family, which are specifically expressed in
immune cells. Both factors could be recruited to a composite
Ets/IRF DNA binding site in target genes through interaction
with PU.1 (7, 10, 23, 26). Functional studies show that phosphorylation of PU.1 at serine/threonine residues can enhance
PU.1-dependent transcription in endotoxin-stimulated cells by
promoting interaction between PU.1 and PU.1 interacting proteins (PIP). Formation of a PU.1/PIP transcriptional complex is
dependent on conformational changes in the PEST domain of
PU.1 that is mediated by serine phosphorylation (34, 40). PU.1
is potentially phosphorylated on five separate serine residues
(Ser41, 45, 132, 133, and 148) that are within consensus sites
for casein kinase II (CKII); however, phosphorylation of
Ser148 appears to regulate transaction (21) and is necessary for
maximal expression of the human TLR4 promoter. Based on
this literature, we hypothesized that PU.1/PIP binding to the
TLR4 promoter regulates TLR4 gene and protein expression in
the pulmonary macrophages during endotoxemia. In these
studies, we employed a novel chromatin immunoprecipitation
(ChIP) assay to investigate PU.1/PIP interactions with the
murine TLR4 promoter in RAW cells and in whole lung tissue
in response to treatment with endotoxin.
MATERIALS AND METHODS
Cell culture. A murine macrophage cell line RAW 264.7 (American Type Culture Collection, Rockville, MD) was maintained in
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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Pedchenko, Tetyana V., Gye Young Park, Myungsoo Joo, Timothy S. Blackwell, and John W. Christman. Inducible binding of PU.1
and interacting proteins to the Toll-like receptor 4 promoter during
endotoxemia. Am J Physiol Lung Cell Mol Physiol 289: L429 –L437,
2005; doi:10.1152/ajplung.00046.2005.—We hypothesized that PU.1
and PU.1 interacting proteins (PIP) binding to the Toll-like receptor 4
(TLR4) promoter is involved in endotoxin-induced upregulation of TLR4
gene expression. Our results employing chromatin immunoprecipitation
assays indicate that PU.1 binds to the murine TLR4 promoter both in
macrophage cells and, most importantly, in whole lung tissue. Treatment
of RAW 264.7 cells with endotoxin induced the association of PU.1 and
the TLR4 promoter in a time-dependent manner, and this was closely tied
to interactions between the TLR4 promoter and the PIP interferon
regulatory factors (IRF)4 and IRF8. PU.1 binding was related to increases
in steady-state TLR4 mRNA and total TLR4 protein in RAW cells.
Endotoxemia in animals caused the similar inducible interaction between
PU.1 and IRF4 and the TLR4 promoter in lung tissue of mice that was
treated with a single intraperitoneal injection of endotoxin. PU.1 binding
to the TLR4 promoter was not enhanced in the lung tissue of endotoxinresistant C3H/HeJ mice in response to endotoxemia. Transient transfection studies in RAW cells indicate that inducible binding of PU.1 to the
TLR4 promoter is abrogated by a Ser148 to Ala mutation in PU.1. These
data suggest that induction of PU.1/PIP binding to the TLR4 promoter is
involved in endotoxin response in vivo and may mediate transcriptional
changes in TLR4 gene expression.
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Flow cytometric analysis. RAW 264.7 cells (⬃106/sample) were
incubated in serum-depleted medium overnight and stimulated with 1
␮g/ml of highly purified endotoxin for the different periods of time.
Cells were washed twice with cold 0.1% NaN3-PBS and harvested.
After being suspended in 1 ml of cold 0.1% NaN3-PBS, each sample
was divided in two 500-␮l aliquots. One aliquot was used to detect the
cell surface expression of TLR4 and the other one to detect total
cellular expression. To evaluate the cell surface TLR4/MD-2 expression, cells were suspended in 250 ␮l of 1% FBS-0.1% NaN3-PBS and
incubated with 2.5 ␮l of TLR4/MD-2 phycoerythrin-conjugated antibody (MTS510, Santa Cruz) on ice for 45 min. To detect total
cellular expression of TLR4/MD-2 complex, samples were fixed with
1% paraformaldehyde-PBS and permeabilized with 0.1% saponin
(wt/vol)-1% FBS-0.1% NaN3-PBS buffer. After being incubated with
antibody, cells were washed and resuspended in 1% paraformaldehyde-0.1% NaN3-PBS. Cells incubated with an isotype-matched irrelevant antibody (mouse IgG2a) were used as a control. Staining with
7-amino-actinomycin D (cat. no. A-1310, Molecular Probes) was
performed to exclude dead cells. Thereafter, cells were subjected to
flow cytometric analysis on a BD FACScan analyzer (BD Biosciences).
Western blotting for TLR4 in membrane fraction of RAW cells.
Cells were incubated in serum-depleted medium overnight and treated
with 1 ␮g/ml of LPS for 2 h. Cells were washed with cold PBS and
lysed in lysis buffer (20 mM Tris, 5 mM EDTA, protease inhibitors
cocktail added) by freezing-thawing (3 cycles) using dry ice. After
sonication, total cell lysate was ultracentrifuged at 100,000 g for 1 h
at 4°C. The supernatant was collected as cytosol fraction. The pellet
was resuspended in lysis buffer with 1% Nonidet P-40 added. Supernatant was collected as a membrane fraction after additional centrifugation at 100,000 g for 1 h at 4°C. Proteins of membrane fraction were
separated by SDS-PAGE and were transferred to polyvinylidene difluoride membrane. The membrane was probed with anti-TLR4 antibody
(H-80, Santa Cruz). Protein detection was carried out using ECL system.
ChIP. ChIP was performed by using a modified protocol from
Upstate Biotechnology (Lake Placid, NY). Cells were grown to
60 –70% confluence in 10-cm dishes. Cells were then cultured in the
serum-depleted media overnight before endotoxin stimulation. Proteins were cross-linked to DNA by adding the formaldehyde directly
to culture medium to a final concentration of 1% for 10 min. After
being rinsed three times with ice-cold PBS, the cells were resuspended
in SDS-lysis buffer (50 mM Tris 䡠 HCl, pH 8.1, 10 mM EDTA, 1%
SDS, and protease inhibitors). Sonication of cell lysate was performed
three times for 12 s each, followed by centrifugation at 4°C for 10
min. Supernatants were collected and diluted 1:10 with dilution buffer
(16.7 mM Tris 䡠 HCl, pH 8.1, 1.2 mM EDTA, 167 mM NaCl, 0.01%
SDS, 0.1% Triton X-100, and protease inhibitors). A portion of
diluted supernatant (1%) was kept to estimate the amount of DNA
present in different samples. It is referred to as “input” sample. After
preclearing the extracts with 60 ␮l of salmon sperm DNA-saturated
protein A- or G-Sepharose beads 50% slurry (Zymed, San Francisco,
CA) for 30 min at 4°C, the immunoprecipitation was carried out
overnight at 4°C with 2–5 ␮g of specific antibody. The antibodies
used were: rabbit-polyclonal ␣-PU.1 (Santa Cruz, CA), goat polyclonal ␣-IRF4/Pip, ␣-IRF8/ICSBP (Santa Cruz, CA), mouse M2
␣-FLAG (Sigma), or irrelevant IgG (Santa Cruz, CA). After immunoprecipitation, 40 ␮l of salmon sperm DNA-saturated protein A- or
G-Sepharose beads 50% slurry were added, and samples were incubated for 1 h followed by brief centrifugation. After washing the
antigen, we eluted complexes with 200 ␮l of elution buffer (1% SDS
and 0.1 M NaHCO3). Elutes along with previously saved input
samples were incubated overnight at 65°C in the presence of 0.2 M
NaCl to reverse formaldehyde cross-linking. The samples were treated
with 10 ␮g of proteinase K at 45°C for 1 h. DNA was recovered and
purified with a DNA clean-up kit (Qiagen). Both the immunoprecipitated samples and the input samples were processed in the same way.
PCR conditions were as follows: 95°C for 5 min; 36 cycles of 95°C
for 1 min, 54°C for 1 min, and 72°C for 1 min; and final elongation
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DMEM (Cellgro) containing 10% FBS (Hyclone), penicillin/streptomycin (Invitrogen), and 2 mM glutamine (Sigma).
Bone marrow-derived macrophages. Bone marrow was collected
from the femurs of mice ranging from 8 to 16 wk of age with a
25-gauge needle and unlabeled DMEM. Cells were then centrifuged
and suspended in a macrophage differentiation medium. This medium
consisted of DMEM, 10% heat-inactivated FBS, 4 mM L-glutamine
(GIBCO, Grand Island, NY), 100 units of penicillin (GIBCO)/ml, 0.1
mg of streptomycin (GIBCO)/ml, and 10% conditioned medium from
L929 mouse fibroblasts (conditioned L929 medium contains GMCSF, necessary for macrophage differentiation and maturation). After
5–7 days, macrophage precursors divide and differentiate into mature
macrophages. Cells were cultured for 7 days at 37°C and 5% CO2.
After being incubated overnight with medium that contained only 1%
FBS, cells were treated with endotoxin (1 ␮g/ml of highly purified
LPS from Escherichia coli, serotype R515, Alexis Biochemicals).
After treatment, cells were used in ChIP assay as described further.
Animal model. Male and female C57Bl/6, C3H/HeJ, and BALB/c
mice weighing 20 –28 g were used for experiments. Highly purified
endotoxin was given as a single intraperitoneal injection of 2 ␮g/g in
normal saline. Control animals were injected with saline instead of
endotoxin. Mice were killed by CO2 asphyxiation at different times
following challenge. All experiments using animals were approved by
the Vanderbilt University Institutional Animal Care and Use Committee. After perfusion with PBS, lungs were harvested.
Bronchoalveolar lavage. C57Bl/6 mice were killed, tracheas were
cannulated, and lungs were lavaged with sterile pyrogen-free physiological saline that was instilled in two 0.7-ml aliquots and gently
withdrawn with a 1-ml tuberculin syringe. Lung lavage fluid was
centrifuged at 400 g for 10 min to separate cells from supernatant, and
cells were suspended in serum-free RPMI culture medium. After
being cultured for 1 h at 37°C and 5% CO2, the adherent cells’
representative alveolar macrophages were washed with PBS and
harvested for the total RNA isolation.
Plasmids and transfection. RAW 264.7 cells (0.5 ⫻ 106 cells) were
transiently transfected with plasmids by GenePORTER 2 (Gene
Therapy Systems, San Diego, CA) as specified by the manufacturer.
The PU.1 and PU.1S148A plasmids were kindly provided by Dr. M. L.
Atchison and were described previously (37). To distinguish between
endogenous and transfected proteins, the PU.1 was tagged with FLAG
as described previously (18). Each transfection was normalized with
appropriate empty vector plasmids. The expression of FLAG-tagged
PU.1 and its mutant was evaluated with ␣-PU.1 antibody (Santa Cruz
Biotechnology) as well as M2 antibody (Sigma). Transfected cells
were incubated for 14 –16 h at 37°C and 5% CO2. For the endotoxin
treatment, the cells were maintained further without serum overnight
and subsequently treated with 1 ␮g/ml of endotoxin in PBS (Sigma)
for different time points. Cells were then harvested and lysed.
RT-PCR. Total RNA was isolated from RAW 264.7 cells and
alveolar macrophages (bronchoalveolar lavage cells) using RNeasy
Mini Kit (QIAGEN, Valencia, CA) and from a whole lung using
TRIzol reagent (Invitrogen, Carlsbad, CA) following the manufacturer’s protocols. RT-PCR was performed with Titan One Tube
System (Roche, Indianapolis, IN). Isolated total RNA (50 ng) was
used for reverse transcription followed by 30 cycles of amplification
(94°C for 15 s, 52°C for 40 s, and 68°C for 60 s). Amplification of
TLR4 gene product was performed with the following primers:
5⬘-TTC CTG TTC TAG TCT TCA GT-3⬘ (sense) and 5⬘-GTT GAA
GCT CAG ATC TAT GT-3⬘ (antisense). Mouse hypoxanthine guanine phosphoribosyl transferase (HPRT) gene was amplified as an
internal control using 25 ng of template RNA, the same RT-PCR
conditions, and the following primers: 5⬘-GTT GGA TAC AGG CCA
GAC TTT GTT G-3⬘ (sense) and 5⬘-GAG GGT AGG CTG GCC
TAT AGG CT-3⬘ (antisense). PCR products were separated on 1%
agarose gel and stained with ethidium bromide. The TLR4 and HPRT
primers produce products of 398 and 352 bp, respectively.
REGULATION OF TLR4 BY PU.1/PIP
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at 72°C for 7 min. PCR for the input was performed with five times
less template amount. The PCR products were analyzed on 1%
agarose gel or 8% polyacrylamide gel. Primers for the promoter
region of the mouse TLR4 gene were 5⬘-CAC AAG ACA CGG CAA
CTG AT-3⬘ (sense) and 5⬘-TCG CAG GAG GGA AGT TAG AA-3⬘
(antisense). This primer set flanks the TLR4 promoter sequence region
⫺359 to ⫺35 containing putative PU.1 binding sites (40, 41) and is
predicted to yield a 325-bp PCR product.
Lung ChIP assay. The lungs were perfused with 15 ml of PBS
through the heart with incision in the left ventricle. The tissue was
then fixed with 2 ml of 2% formaldehyde delivered to the lung
intratracheally. The lungs were dissected and incubated in 1% formaldehyde for ⬃10 min. After that, the lungs were harvested and
transferred into cold PBS solution. After being washed in PBS, the lung
tissue (⬃60 – 80 mg) was homogenized with 1 ml of SDS lysis buffer
(with protease inhibitors cocktail) using Polytron homogenizer. The
lysate was incubated on ice for 20 min and sonicated (12 s ⫻ 4 times).
Thereafter, the ChIP assay was performed as described for the cells.
Coimmunoprecipitation of PU.1 and IRF4. After the PU.1 immunoprecipitation step in ChIP assay described above, coimmunoprecipitated protein was analyzed by IRF4 immunoblotting. Samples
(A/antibody/proteins/DNA complex) were boiled for 10 min with
Laemmli buffer, resolved on 10% gel, and an immunoblot procedure
was performed using anti-IRF4 antibody (M-17).
Statistical analysis. For comparison among groups, unpaired t-tests
and unpaired ANOVA tests were used (with the assistance of InStat;
GraphPad Software, San Diego, CA). P values ⬍ 0.05 were considered significant.
RESULTS
TLR4 mRNA and protein expression in macrophages in
response to treatment with endotoxin. Initially, we determined
the time course for the expression of steady-state TLR4 mRNA
in RAW 264.7, a macrophage-like cell line, in response to in
vitro treatment with endotoxin. TLR4 mRNA was detected by
RT-PCR and normalized to HPRT mRNA expression. As
shown in Fig. 1A, treatment of RAW cells with endotoxin
resulted in a substantial increase in steady-state TLR4 mRNA
between 2 and 4 h. Increased TLR4 mRNA was detected as
early as 30 min of endotoxin treatment, increased until 4 h, and
decreased steadily before approaching preinduction level at
24 h following stimulation.
We next determined whether the TLR4 mRNA expression
profile in RAW cells treated with LPS reflects response of
pulmonary macrophages to endotoxemia in vivo. Mice were
injected intraperitoneally with endotoxin, and TLR4 mRNA
was measured in bronchoalveolar lavage cells collected from
mice at 4 and 24 h following the injection (Fig. 1B). Expression
of mRNA was increased by 4 h in adherent bronchoalveolar
lavage cells, and it was decreased by 24 h following treatment
with endotoxin.
To investigate TLR4 protein production, first we have
showed the decrease of TLR4 protein level in the RAW cell
membrane fraction after 2 h of treatment with LPS (Fig. 2A).
Then we cultured RAW cells with endotoxin for various
periods of time and evaluated both cell surface and total cell
expression of TLR4/MD-2 complex using fluorescent antibody
and fluorescence-activated cell sorting (FACS) analysis. The
TLR4/MD-2 antibody was selected because a specific murine
TLR4 antibody could be obtained that measures the functional
protein complex capable to bind LPS. Our results demonstrate
a time-dependent decrease of TLR4/MD-2 expression on the
RAW cell surface in response to treatment with endotoxin,
AJP-Lung Cell Mol Physiol • VOL
Fig. 1. Endotoxin stimulation modulates Toll-like receptor 4 (TLR4) expression in RAW cells and bronchoalveolar lavage (BAL) cells. TLR4 mRNA
expression was detected by RT-PCR and normalized to hypoxanthine guanine
phosphoribosyl transferase (HPRT) mRNA using densitometric analysis (bottom panels). A: RAW cells were treated with purified endotoxin (1 ␮g/ml) for
indicated periods of time (n ⫽ 4 –5 at each time point). B: BAL was done on
mice at 4 and 24 h following intraperitoneal injection of 2 ␮g/g of endotoxin
(n ⫽ 3 separate experiments with 1 animal at each time point). BAL cells were
enriched for a macrophage population by selective adherence at 1 h. Top panels
show 1 representative experiment; bottom panels show the densitometric
analysis for TLR4 mRNA after normalization to HPRT mRNA. Results are
expressed as means ⫾ SD; *P ⬍ 0.05, **P ⬍ 0.01 vs. untreated control.
with a return to the untreated control level by 8 h (Fig. 2A). In
contrast, FACS analysis of permeabilized cells showed upregulation of total antigenic TLR4/MD-2 (Fig. 2B), which remains
elevated at the 8-h time point. Presumably, the difference
between intracellular pool and presented at cell surface pool of
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REGULATION OF TLR4 BY PU.1/PIP
TLR4/MD-2 is the result of TLR4 internalization in response
to interaction with endotoxin. We interpret these data as
indicating that there is a small increase in total antigenic
TLR4/MD-2 in macrophages in response to treatment with
endotoxin that is not available on the cell surface.
Endotoxin stimulation results in an inducible interaction
between PU.1 and TLR4 promoter in vivo. Because our initial
data indicated that endotoxin resulted in an increase in both
TLR4 mRNA and TLR4/MD-2 protein complex production in
RAW cells, we employed a novel ChIP to evaluate an interaction between PU.1 and the TLR4 promoter. RAW cells were
treated with endotoxin for various lengths of time and fixed
with formaldehyde to cross-link protein to DNA to determine
binding of specific transcription factors to the TLR4 promoter
that regulate gene expression. A ChIP assay was performed,
using ␣-PU.1 antibody for DNA-protein complex immunoprecipitation and the primers, flanking both potential PU.1 binding
sites on murine TLR4 promoter, for PCR amplification. Immunoprecipitation with an irrelevant immunoglobulin, instead
of specific antibody, was used as a control for the assay
specificity. A weak band was detected initially indicating a
basal interaction between PU.1 and TLR4 promoter in the
absence of stimulation (Fig. 3A). However, there was a marked
increase in the interaction between PU.1 and the TLR4 promoter, and this was sustained at 4 h.
AJP-Lung Cell Mol Physiol • VOL
To reveal changes in binding of PU.1 to the TLR4 promoter
that result from endotoxemia, we adapted the ChIP assay to
determine the interaction between PU.1 and the TLR4 promoter in whole lung tissue. Mice were injected with 2 ␮g/g of
endotoxin intraperitoneally, and the protein-DNA cross-link in
the lung tissue was performed by intratracheal formaldehyde
delivery to the lung at different periods of time following
injection. Lung was harvested, and whole lung homogenate
was processed further similar to cell lysate before immunoprecipitation with ␣-PU.1 and detection of the TLR4 promoter by
PCR. These data show (Fig. 3B) that there is slight binding of
PU.1 to the TLR4 promoter in the lung tissue of untreated
animals. Similar to the finding in RAW cells, endotoxin treatment markedly induced PU.1 binding activity in a time-dependent manner. The maximum activation was observed at 2 h. By
16 h following endotoxin injection, binding activity returned to
basal level. Although this assay is done with whole lung tissue,
the interaction between PU.1 and the TLR4 promoter is specific for PU.1 containing cells that in lung are presumed to be
macrophages.
PU.1 interaction with TLR4 promoter is a part of lung
response to endotoxin. Data shown in Fig. 3 indicate that the
interaction between PU.1 and the TLR4 promoter was induced
in a time-dependent manner following treatment with endotoxin. An inducible interaction between PU.1 and the TLR4
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Fig. 2. TLR4 protein and TLR4/MD-2 protein complex presentation on the RAW cell
surface is decreased but total cellular TLR4/
MD-2 is increased in response to treatment
with the endotoxin. A: RAW 264.7 macrophages were cultured with purified endotoxin (1 ␮g/ml) for 4 h. After that, TLR4
expression was analyzed by Western blot
analysis in the membrane fraction of RAW
cells. Total HeLa cell extract served as a
positive control for ␣-human TLR4 antibody
(1 representative Western blot is shown). B:
RAW 264.7 macrophages were cultured
with purified endotoxin (1 ␮g/ml) for indicated periods of time, and both cell surface
and total cell expression of TLR4/MD-2
complex were evaluated using phycoerythrin-conjugated MTS510 antibody and
fluorescence-activated cell sorting analysis.
Dotted lines depict staining with irrelevant
IgG2a; thick lines depict staining for untreated cells; thin lines depict staining for
endotoxin-treated cells. Two independent
experiments were performed with the same
result; the representative one is shown.
REGULATION OF TLR4 BY PU.1/PIP
promoter could have a physiological role in regulating TLR4
gene expression during endotoxemia. To confirm that this
interaction between PU.1 and the TLR4 promoter is induced by
treatment with endotoxin, we examined PU.1 binding to the
TLR4 promoter in whole lung tissue from C3H/HeJ mice
treated with endotoxin. These mice are known to carry the
point mutation (proline to histidine at position 712) in the
intracellular domain of TLR4 receptor, which makes them
unresponsive to endotoxin (16, 34). We employed the lung
tissue ChIP assay to investigate the interaction of PU.1 and
TLR4 promoter in this animal model. Before this experiment,
we demonstrated by Western blot analysis that TLR4 mutation
does not affect total PU.1 protein level (data not shown). Next,
we performed ChIP analysis on a whole lung from both
wild-type and TLR4 mutant mice (Fig. 4). PU.1 binding to
TLR4 promoter was assessed at 2 h of endotoxin stimulation
since this was shown to be a peak of activation in previous
experiments (Fig. 3B). This ChIP assay did not detect a change
in the interaction between PU.1 and the TLR4 promoter in the
lung tissue from the C3H/HeJ mice in response to treatment
with endotoxin. In contrast, there was a substantial increase in
the interaction between PU.1 and TLR4 promoter in the lung
tissue of the wild-type mice. These data support the conclusion
that the interaction between PU.1 and the TLR4 promoter is a
part of response to treatment with endotoxin in vivo.
PU.1 phosphorylation at Ser148 is critical for the interaction with TLR4 promoter. The activation of PU.1 in response to
endotoxin is presumed to be regulated at the posttranslational
level, since there were no changes in PU.1 protein levels in
response to treatment with endotoxin (data not shown). Stimulation of RAW 264.7 cells with endotoxin has been shown to
induce PU.1 phosphorylation at Ser148 through the induction
AJP-Lung Cell Mol Physiol • VOL
Fig. 4. TLR4 mutation abolishes endotoxin-induced activation of PU.1 binding in vivo. Top: PU.1 binding to the TLR4 promoter was evaluated by ChIP
assay in the lung of TLR4 mutant (mTLR4; C3H/HeJ) and wild-type (WT;
BALB/c) mice at 2 h after intraperitoneal injection with 2 ␮g/g of endotoxin.
Bottom: densitometric analysis for PU.1 IP samples after normalization to
corresponding DNA input samples. One out of two independent experiments is
shown.
of CKII, resulting in enhanced transactivation function (21).
Based on these data, we expected that endotoxin could increase
TLR4 gene expression by inducing phosphorylation of Ser148,
and, therefore, PU.1 activity. To address this possibility, RAW
cells were transiently transfected with plasmids expressing
either mutant PU.1S148, where Ser148 is replaced with alanine
preventing phosphorylation, or wild-type PU.1. To distinguish
between transfected and endogenous PU.1, constructs were
FLAG-tagged. Equivalent efficiency of mutant and wild-type
PU.1 transfection was confirmed by Western blotting analysis
using anti-FLAG antibody M2 showing equal expression of the
transgene (data not shown). Next, transfected RAW cells were
treated with endotoxin for 2 h, and ChIP assay was performed
using M2 anti-FLAG antibody for immunoprecipitation. As
shown in Fig. 5, lack of Ser148 phosphorylation abolished
endotoxin-inducing PU.1 binding to TLR4 promoter. This
finding suggests that endotoxin induces a PU.1 posttranslational modification related to phosphorylation of Ser148 that is
Fig. 5. S148A mutation in the PEST domain of PU.1 abrogates binding to
TLR4 promoter. RAW cells were transiently transfected with plasmids expressing either mutant FLAG-tagged PU.1S148A or wild-type FLAG-tagged
PU.1. After endotoxin stimulation (1 ␮g/ml), ChIP analysis was performed
using anti-FLAG antibody. One of 3 independent experiments with a similar
result is shown.
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Fig. 3. PU.1 binding to the TLR4 promoter is enhanced in response to
endotoxin in both RAW macrophages and whole lung. A: RAW cells were
stimulated with 1 ␮g/ml of endotoxin for the indicated periods of time, and
chromatin immunoprecipitation (ChIP) assay was performed using ␣-PU.1
antibody for DNA-protein complex immunoprecipitation (IP) or using irrelevant IgG as a control. PCR amplification was done with the primers, flanking
putative PU.1 binding sites on murine TLR4 promoter. One of the 3 independent experiments is shown. B: C57BL/6 mice were injected with 2 ␮g/g of
endotoxin intraperitoneally, and the protein-DNA cross-link in the lung tissue
was performed by intratracheal formaldehyde delivery to the lung at different
periods of time following endotoxin injection. After that, the lung tissue was
subjected to ChIP analysis as described above (n ⫽ 5 at each time point).
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Fig. 6. Endotoxin stimulation recruits PU.1-interactive protein (Pip/IRF4) to
TLR4 promoter in the murine lung. C57BL/6 mice were treated intraperitoneally with 2 ␮g/g of endotoxin for the indicated periods of time, and after
intratracheal formaldehyde delivery, the lung tissue was harvested and subjected to ChIP analysis (n ⫽ 4 –5). The IP of protein-DNA complex was
performed with ␣-IRF4 antibody. PCR amplification was done with the set of
primers flanking the putative PU.1/IRF composite site. A representative result
is shown.
AJP-Lung Cell Mol Physiol • VOL
Fig. 7. IRF4 interacts with PU.1 in vivo during endotoxemia. C57BL/6 mice
were treated intraperitoneally with 2 ␮g/g of endotoxin for 2 h, and after
intratracheal formaldehyde delivery, the lung tissue was harvested and subjected to ChIP analysis (n ⫽ 4 –5). The bone marrow-derived macrophages
were isolated and also subjected to ChIP analysis. The IP of protein-DNA
complex was performed with ␣-PU.1 antibody, followed by Western blot
analysis with ␣-IRF4 antibody. IP with corresponding Ig instead of ␣-PU.1
antibody was used as a negative control. BMDC, bone marrow-derived cells.
posite sites. The inducible binding activity was demonstrated
for all three factors (Fig. 7), which suggests that endotoxin
signaling involves formation of complex of PU.1/IRF4/IRF8
for TLR4 transcriptional regulation in macrophages. The possibility of PU.1/IRF complex formation on the TLR4 promoter
in vivo was supported by demonstrating PU.1 and IRF4 coimmunoprecipitation from bone marrow-derived cells and from
whole lung tissue of animals treated with LPS. After PU.1
immunoprecipitation step in ChIP assay, the samples were
subjected to Western blot analysis with IRF4 antibody (Fig. 8).
DISCUSSION
We found that treatment with endotoxin accentuates PU.1
binding to the endogenous TLR4 promoter in vivo in a timedependent manner in RAW cells, and this is associated with
Fig. 8. IRF8 associates with TLR4 promoter in vivo. Bone marrow-derived
macrophages were isolated from C57BL/6. Formaldehyde cross-linking was
performed on cells treated with endotoxin for 2 h or were left untreated.
Nuclear extract from each sample was divided in 3 aliquots, and each aliquot
was used for ChIP assay with either PU.1 or IRF4 or IRF8 IP followed by PCR
amplification of TLR4 promoter sequence containing PU.1/IRF composite site.
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crucial for mediating the interaction between PU.1 and the
TLR4 promoter in RAW cells.
IRF4 is involved in TLR4 transcriptional regulation in vivo
during endotoxemia. There is growing information indicating
the importance of PU.1 phosphorylation for its interaction with
PIP, especially with members of the IRF family. It has been
shown that short region of TLR4 promoter (only 75 bp) was
sufficient in transient transfection to induce maximal luciferase
activity in THP-1 cells, and it contains composite interferon
response factor/Ets motif (40). Both the human and murine
TLR4 promoters contain composite IRF/PU.1 binding motif
(40). For the murine TLR4 promoter disrupting the PU.1binding sites by site-directed mutagenesis completely abolished (distal site) or strongly reduced (proximal site) basal
luciferase activity (41). All these data, together with our
finding about the important role of Ser148 phosphorylation for
PU.1 interaction with endogenous TLR4 gene promoter,
prompted us to address two questions: does IRF4 bind to the
murine TLR4 promoter in vivo and, if so, is it involved in
endotoxin signaling? To test the ability of IRF4 to bind
elements within TLR4 promoter, we used lung tissue ChIP
assay. Mice were treated intraperitoneally with 2 ␮g/g of
endotoxin for indicated periods of time, and after formaldehyde fixation, lungs were harvested from control and treated
animals and then subjected to ChIP analysis. The immunoprecipitation of protein-DNA complex was done with anti-IRF4
antibody. PCR amplification was done with the same set of
primers used for PU.1 ChIP assay because they also flanked the
possible PU.1/IRF composite sites. These data show that there
is an inducible interaction between IRF4 and the TLR4 promoter, with peak activity at 2 h following endotoxin injection,
returning to the basal, almost undetectable level, at 24 h (Fig. 6).
PU.1 can also interact with another member of the IRF
family, IRF8 (ICSBP), at IRF/PU.1 composite element (9, 11,
22, 23, 26). IRF4 has been reported to interact physically with
IRF8/ICSBP. To examine the possibility of formation of heterocomplex for TLR4 transcriptional regulation in macrophages during endotoxemia, we assessed binding of all three
factors, PU.1, IRF4, and IRF8, to the TLR4 in bone marrowderived macrophages. Formaldehyde cross-linking was performed on cells treated with endotoxin for 2 h or were left
untreated. Nuclear extract from each sample was divided in
three aliquots and subjected to ChIP assay with either PU.1 or
IRF4 or IRF8 immunoprecipitation followed by PCR amplification of TLR4 promoter sequence containing PU.1/IRF com-
REGULATION OF TLR4 BY PU.1/PIP
AJP-Lung Cell Mol Physiol • VOL
in lung macrophages, although we cannot exclude these data
may also reflect an interaction in B-lymphocytes since small
numbers may also be present in lung tissue.
The interaction between PU.1 and the TLR4 promoter was
enhanced by treatment with endotoxin. When the endotoxin
signaling through TLR4 receptor was impaired (in C3H/HeJ
mice), there was no enhancement of PU.1 binding in response
to treatment with endotoxin. PU.1 is potentially phosphorylated on five serine residues (41, 45, 132, 133, and 148) that are
within consensus sites for CKII. Treatment of the RAW macrophages with endotoxin induces phosphorylation of Ser148
through induction of CKII, resulting in enhanced transactivating activity (21). Although it would be expected from these
data that endotoxin could increase TLR4 gene expression by
inducing phosphorylation of Ser148 and enhancement of PU.1
trans-activation function, this has not yet been reported. After
performing transient transfection with plasmid vectors that
express either wild-type or mutant PU.1 (Ser148 is replaced by
alanine), we subjected RAW cell lysates to ChIP assay. The
prevention of Ser148 phosphorylation abrogated PU.1 binding
activity in endotoxin-stimulated macrophages. Therefore, we
showed that phosphorylation of PU.1 is involved in the process
of recruitment to endogenous TLR promoter by endotoxin
stimulation.
Functional studies showed that phosphorylation of PU.1 can
enhance PU.1-depended transcription in endotoxin-stimulated
cells, presumably by promoting interaction between PU.1 and
other transcriptional regulatory factors (17, 21). Furthermore,
phosphorylation of Ser148 in PU.1 PEST domain is known to
be important for physical interaction with IRF4, also called
Pip, an essential cofactor in B cells (10, 32, 36) and macrophages (22, 42). IRF was recognized as a part of ternary
complex between PU.1 and a domain of the Ig light chain
enhancer (34). PU.1 and IRF4 can bind as a complex to a
composite PU.1/IRF4 DNA-binding element, which has been
identified in a number of genes (9, 11, 15, 22, 31, 36). EMSA
analysis revealed that murine macrophages contained both
IRF4/PU.1 and ICSBP/PU.1 complexes (22). PU.1 and ICSBP,
also known as IRF8, were reported to participate in the basal
regulation of human TLR4 in myeloid cells (40).
Our data show that endotoxin stimulation recruits endogenous IRF4 (Pip) to TLR4 promoter in murine lung in a
time-dependent manner very similar to PU.1 binding profile.
The difference is that a basal binding activity observed with
ChIP for PU.1 was almost undetectable in the case of IRF4. We
demonstrated by coimmunoprecipitation the interaction between PU.1 and IRF4 in both bone marrow-derived cells and
whole lung tissue during endotoxemia. This finding is in
agreement with a previously suggested mechanism of PU.1/
IRF interaction: IRF4 (Pip) and IRF-8 (ICSBP) bind very
weakly to DNA containing an IRF site but can be recruited via
interactions with other transcriptional factors, such as PU.1 and
Spi-B, to the composite Ets/IRF element (7, 10, 12, 35). Our
data indicate that IRF8 (ICSBP) is also involved in formation
of PU.1/IRF transcriptional complex on TLR4 promoter in
bone marrow-derived macrophages.
In conclusion, our data indicate that the macrophage response to endotoxin involves activation of PU.1 binding to the
TLR4 promoter followed by modulation of both TLR4 mRNA
levels and protein expression. Endotoxin stimulation of macrophages results in phosphorylation of PU.1 followed by in-
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increases in steady-state TLR4 mRNA and in TLR4 protein
production. We also found that two members of IRF family,
IRF4/Pip and IRF8/ICSBP, interact with PU.1 and the TLR4
promoter in response to treatment with endotoxin.
Reports of TLR4 expression during endotoxemia are contradictory, and our results are both consistent with and in
contrast to some reports. For example, endotoxin increases
TLR4 mRNA expression in human monocytes and polymorphonuclear leucocytes (27). In recovered murine alveolar macrophages, endotoxin has been shown to decrease TLR4 mRNA
expression (13), but no change in TLR4 mRNA expression was
reported for alveolar macrophage cell line MH-S (33). The
level of TLR4 mRNA in the mouse whole lung has been shown
to remain unchanged after intranasal instillation of LPS in one
report (33), but it was decreased after intratracheal administration of endotoxin through a mechanism that involves reduced
mRNA stability according to another report (13). There are
also reports that endotoxin treatment reduces the expression of
TLR4 mRNA in RAW 264.7 cells (35, 50).
Previous studies have shown that expression of TLR4 receptor mRNA is not closely related to protein levels (29). After
showing, by Western blot analysis, decreased TLR4 protein
expression in membrane fraction of RAW cells during LPS
treatment, we employed flow cytometry using an antibody
against the TLR4/MD-2 protein complex. TLR4 binds endotoxin only in contact with the accessory protein MD-2. In the
absence of MD-2, TLR4 is retained in Golgi apparatus (20, 28,
47), and MD-2 is required for TLR4 glycosylation at Asn526
and Asn575 and expression on the cell surface (30, 38). Thus
FACS analysis with antibody against TLR4/MD-2 allows evaluation of the biologically active TLR4/MD-2 protein complex.
Our data are consistent with other reports that there is downregulation of TLR4/MD-2 on the cell surface of RAW cells
that are treated with endotoxin (1, 29). In contrast to expression
of antigenic TLR4/MD-2 on the cell surface, we observed a
time-dependent increase in total cellular TLR4/MD-2 expression in RAW cells in response to treatment with endotoxin.
The TLR4 gene has a typical myeloid type promoter, and
full human TLR4 promoter activity is retained by a 75-bp
proximal TLR4 promoter construct that contains four putative
protein binding sites, an inner and outer PU.1 site that flanks an
octamer motif and an interferon factor binding site (40). PU.1
has been shown to be involved in murine macrophage expression of TLR4 (19, 41). The investigation of the role of PU.1 in
gene expression has been mostly carried out by employing an
EMSA and transient transfection assay using promoter-reporter
constructs. In contrast to EMSA, which examines the interaction between the extracted protein and double-stranded DNA
oligonucleotides, ChIP allows investigation of specific interactions between transcriptional factors and cellular DNA in the
context of cells or tissue. We have employed ChIP assay to
explore the interaction between endogenous PU.1, interacting
proteins, and the TLR4 promoter in vivo to capture molecular
events during endotoxemia. To elucidate whether it reflects the
real events occurring in whole organism, we have developed a
novel implication of ChIP assay in an animal model. We
observed that treatment with endotoxin results in an increase in
PU.1 binding to the TLR4 promoter in RAW cells and in lung
tissue. Because PU.1 is predominantly located in macrophages,
our results that employ the ChIP assay in whole lung tissue are
likely reflective of an association of PU.1 with TLR4 promoter
L435
L436
REGULATION OF TLR4 BY PU.1/PIP
teraction with IRF4 via their PEST domains and thus enhances
the transcriptional activation of TLR4 promoter. TLR4 transcriptional regulation in murine lung during endotoxemia involves formation of heterocomplex between PU.1 and IRF
family members. The lung ChIP assay developed in our laboratory is a unique tool to reveal the real-time interactions in an
animal model between transcriptional factors and regulated
gene promoters.
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