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INTRODUCTION |
Peptidoglycan (PGN),1 a
polymer of alternating GlcNAc and MurNAc cross-linked by short
peptides, is present in all bacterial cell walls and is the major cell
wall component of Gram-positive bacteria (1). PGN, like
lipopolysaccharide (LPS, the major cell wall component of Gram-negative
bacteria), can induce all major signs and symptoms associated with
bacterial infections, including fever, inflammation, leukopenia
followed by leukocytosis, hypotension, decreased appetite, decreased
peripheral perfusion, malaise, sleepiness, and arthritis (2-4). PGN,
together with lipoteichoic acid, also induces circulatory shock and
multiple organ failure (5). These clinical manifestations are due to the activation of host cells, such as macrophages, and release of
cytokines and other pro-inflammatory molecules (2-6) from activated
cells. Both PGN (3, 7-10) and LPS (3, 11-15) activate macrophages
through the pattern recognition receptor CD14. However, there are
several differences in the interaction of PGN and LPS with CD14 (8, 10,
16).
TNF-
(6, 16), IL-1 (7), and IL-6 (7, 16) are the main cytokines
released from PGN-activated macrophages. However, the signal
transduction pathways that culminate in transcriptional activation of
these cytokine genes are so far unknown. The promoters for these
cytokine genes contain binding sites for various transcription factors,
including NF-
B, AP-1, and CREB. We have previously shown activation
of NF-
B by PGN (8), but it is not known if PGN induces activation of
any other transcription factors. It is also not known which
transcription factors are required for PGN-induced activation of
cytokine genes.
The CREB/ATF family of transcription factors are leucine zipper
proteins that bind to the cAMP response element (CRE) with the
consensus sequence, 5'-TGACGTCA-3' (17). CREB, the most extensively
studied CRE-binding protein, is phosphorylated at serine 133 by protein
kinase A in response to cAMP, and this leads to transcriptional
activation (17) of genes whose promoters contain the CRE sequence.
There are other signaling pathways that lead to phosphorylation and
activation of CREB, such as calmodulin kinase, which phosphorylates
CREB in response to increased intracellular Ca2+ (17), or
RSK2 which is activated by mitogen-activated protein (MAP) kinases
(18). ATF-1, another member of this family of transcription factors,
has significant sequence similarity to CREB, including a
phosphorylation domain (17). ATF-1 forms heterodimers only with CREB;
however, the other ATF proteins can also form heterodimers with
specific members of the AP-1 family of transcription factors. In
addition, different heterodimers may bind variant CRE sequences, thus
increasing the number of potential regulatory mechanisms.
The AP-1 family of transcription factors consists of the Jun and
Fos families of proteins that bind the
12-O-tetradecanoylphorbol-13-acetate response element
(TPA-RE), 5'-TGACTCA-3' and induce transcription in response to
many different stimuli, including phorbol esters (19). These proteins
bind DNA as dimers, which are formed through leucine zippers. The Jun
proteins can bind DNA as homodimers or as heterodimers with Fos
proteins; however, the Fos proteins can only bind DNA as heterodimers
(19). In addition to forming heterodimers within the AP-1 family, Jun
proteins can heterodimerize with certain members of other transcription
factor families, such as ATF and C/EBP
(19). Different dimers can
bind different sequences, e.g. Jun-Jun and Jun-Fos dimers
preferentially bind TPA-RE, while Jun-ATF dimers prefer to bind the CRE
sequence (19).
The objective of this study was to: (i) determine if PGN activates the
transcription factors CREB/ATF and AP-1, (ii) identify the specific
members of these two families of transcription factors that are
activated by PGN, and (iii) determine if this activation is
CD14-dependent.
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EXPERIMENTAL PROCEDURES |
Materials--
Soluble PGN (sPGN), a soluble polymeric form of
PGN, was isolated from Staphylococcus aureus Rb grown in the
presence of penicillin (20) and purified by vancomycin affinity
chromatography, and its purity has been previously described (21). sPGN
contained <24 pg of endotoxin/mg, as determined by the
Limulus lysate assay (21). LPS from Salmonella
minnesota Re 595 (ReLPS, a minimal naturally occurring endotoxic
structure of LPS), obtained by phenol-chloroform-petroleum ether
extraction, was purchased from Sigma. All other chemicals were from
Sigma, unless otherwise indicated.
Cells--
Murine macrophage RAW264.7 cell line, obtained from
ATCC (Rockville, MD), was cultured in Dulbecco's modified Eagle's
medium with 10% defined fetal calf serum (HyClone, Logan, UT;
endotoxin content <6 pg/ml). Human monocytic THP-1 cell line, obtained
from ATCC, was cultured in RPMI 1640 with 10% defined fetal calf
serum. For each experiment, THP-1 cells were allowed to differentiate for 72 h in the presence of 100 nM
1
,25-dihydroxyvitamin D3 (Biomol, Plymouth Meeting, PA).
Phosphorylation of ATF-1 and CREB and Western
Blots--
RAW264.7 cells were seeded at 0.35-0.4 × 106/ml in 24-well plates (2 ml/well) and cultured for
16-20 h. THP-1 cells were seeded at 0.15 × 106/ml in
24-well plates (2 ml/well) and allowed to differentiate as above for
72 h. All cells were activated with the stimulants indicated under
"Results" and then washed and lysed as before (6). In some
experiments, sPGN and ReLPS were incubated with 5 µg/ml polymyxin B
for 30 min and then added to cells. In other experiments, THP-1 cells
were incubated with 10 µg/ml anti-CD14 monoclonal antibodies, MY4
(Coulter, Hialeah, FL) or MEM18 (Sanbio-Monosan, Uden, The
Netherlands), or the isotype control IgG2b (clone MPC-11; Coulter) for
30 min at 37 °C before stimulation. Cell lysates were separated on
12% SDS-PAGE and transferred to Immobilon P (6). Phosphorylation of
ATF-1 and CREB was determined by Western blotting with 0.5 µg/ml
rabbit anti-pCREB antibody (Upstate Biotechnology, Inc., Lake Placid,
NY), and detected by the ECL system. This antibody specifically
recognizes the phosphorylated forms of both ATF-1 and CREB. Control
blots were done with 0.5 µg/ml rabbit anti-CREB (Upstate
Biotechnology) or rabbit anti-ATF-1 monoclonal antibodies (Santa Cruz
Biotechnology, Santa Cruz, CA), which recognize both phosphorylated and
nonphosphorylated forms of CREB or ATF-1, respectively.
Activation of AP-1 and Western Blots--
Cells were cultured,
activated, and lysed as described for ATF-1/CREB. Phosphorylation of
c-Jun protein was detected using a monoclonal antibody generated
against a c-Jun peptide containing phosphorylated serine 63 (Santa Cruz
Biotechnology). Nonphosphorylated c-Jun, Jun B, Jun D, and c-Fos were
also detected by Western blots using antibodies from Santa Cruz Biotechnology.
Enzyme Digestions--
30-µg aliquots of sPGN biosynthetically
labeled with [14C]alanine (20) were digested for 72 h at 37 °C with 1 mg/ml affinity-purified lysostaphin or 1 mg/ml
lysozyme (Grade I from chicken egg, from Sigma), or buffer alone (as a
control), and were dialyzed four times (10-12-kDa cut-off) against
Dulbecco's phosphate-buffered saline at 4 °C (10). The extent of
digestion was determined by measuring the amount of 14C
remaining in the samples after dialysis.
Phosphatase Treatment--
Stimulated and control RAW264.7 cells
were lysed in three different buffers depending on the phosphatase
treatment (22). The first group of cells was lysed in the same buffer
as above with 0.8% Nonidet P-40 for control samples. The second group
of cells was lysed in 50 mM Tris, pH 7.5, 1 mM
MgCl2, 0.8% Nonidet P-40, and protease inhibitors, and
digested with 250 units/ml alkaline phosphatase for 15 min at 30 °C.
The third group of cells was lysed in 50 mM Tris, pH 7.5, 1 mM MnCl2, 1 mM dithiothreitol, with
0.8% Nonidet P-40, 1.0 nM okadaic acid, and protease
inhibitors, and digested with 2.9 units/ml protein phosphatase 2A
(Calbiochem, San Diego, CA) for 20 min at 37 °C. The final Nonidet
P-40 concentration in all samples was 0.6%. The samples were separated
on 12% SDS-PAGE and analyzed for phosphorylated and nonphosphorylated
c-Jun by Western blots.
Electrophoretic Mobility Shift Assays--
Cells were cultured
as described above for Western blots, and nuclear extracts were
prepared as described previously (8). 5 µg of nuclear protein were
first incubated with different antibodies as indicated under
"Results," for 20 min at 22 °C. The nuclear proteins were then
incubated with 32P-labeled oligonucleotide containing
consensus CRE (17) or TPA-RE (19) binding sites
(AGAGATTGCCTGACGTCAGAGAG and AGCTTGATGACTCAGCCG, respectively), for 30 min at 22 °C. Anti-Jun antibody was from Santa
Cruz Biotechnology, and all other antibodies used in these experiments
were as described above. In some experiments, the samples were
preincubated with an excess of unlabeled specific or nonspecific
oligonucleotide for 20 min at 22 °C. All samples were then separated
on 5% nondenaturing polyacrylamide gels, and the DNA-protein complexes
were visualized by autoradiography.
Transfection and Chloramphenicol Acetyltransferase (CAT) and
Luciferase Assays--
RAW264.7 cells were cultured at 0.35-0.4 × 106/ml in six-well plates (2 ml/well) for 16-20 h and
transfected with 200 µg/ml DEAE-dextran and DNA (the amount of DNA
used was optimized for different plasmids and is indicated in the
figure legends). The following plasmids were used for transfections:
(
71)Som-CAT (23), A-CREB (24), pAP1-Luc (Stratagene, La Jolla,
CA), 2xAP1-Luc (25),
73Col-Luc (26),
60Col-Luc (26), A-Fos (27),
and A-ATF1, which was constructed as described previously (24). Cells
were allowed to recover for 24-48 h and then were left unstimulated or
were stimulated as described in the figure legends. Lysates were
prepared and were assayed for luciferase activity using the Luciferase
Reporter kit (Promega, Madison, WI) or for CAT activity as described
(22).
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RESULTS |
sPGN Induces Phosphorylation of ATF-1 and CREB--
Activation of
the transcription factor CREB is regulated by phosphorylation at serine
133. ATF-1, also a CRE-binding protein, has extensive homology to CREB,
including a conserved phosphorylation site. We tested if sPGN induces
phosphorylation of CREB and ATF-1 in RAW264.7 cells, using an antibody
that specifically recognizes both phosphorylated CREB and
phosphorylated ATF-1. sPGN induced rapid and transient
dose-dependent phosphorylation of both ATF-1 and CREB (Fig.
1, A and D) with
similar kinetics. The control stimulant, ReLPS, also induced
phosphorylation of ATF-1 and CREB with kinetics similar to those seen
with sPGN. The phosphorylation of ATF-1 was stronger than
phosphorylation of CREB for both stimulants. Identical samples analyzed
with antibodies that recognize both phosphorylated and
nonphosphorylated CREB (Fig. 1B) and ATF-1 (Fig.
1C) showed equal amounts of CREB and ATF-1 protein present in all lanes.

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Fig. 1.
sPGN and ReLPS induce phosphorylation of
ATF-1 and CREB: time kinetics, dose response, and effect of polymyxin
B. RAW264.7 cells were stimulated with 10 µg/ml sPGN or 10 ng/ml
ReLPS for the indicated times, and total cell lysates were subjected to
SDS-polyacrylamide gel electrophoresis and analyzed by Western blots
for: phosphorylation of ATF-1 and CREB using an anti-phosphorylated
CREB Ab (A), total (phosphorylated and nonphosphorylated)
CREB using an anti-CREB Ab (B), or total ATF-1 protein using
an anti-ATF-1 Ab (C). Cells were stimulated with the
indicated concentrations of sPGN or ReLPS for 30 min. Cells stimulated
in the presence or absence of polymyxin B were stimulated with 10 µg/ml sPGN or 10 ng/ml ReLPS. Total cell lysates were analyzed for
phosphorylation of ATF-1 and CREB (D) or total CREB
(E). pCREB, pATF-1, CREB, and ATF-1 are indicated by
arrows; all other bands are nonspecific. The results are
from one of three similar experiments.
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sPGN-induced phosphorylation of ATF-1 and CREB was not inhibited by
polymyxin B (an antibiotic that binds LPS and inhibits its biologic
effects), unlike the induction by ReLPS, which was almost completely
inhibited (Fig. 1D). This confirms that the sPGN-induced
effect was not due to endotoxin contamination. In control experiments,
the amounts of total (phosphorylated and nonphosphorylated) CREB (Fig.
1E) and ATF-1 (not shown) proteins were the same in all samples.
Composition of CREB/ATF Complexes That Bind CRE Consensus
Sequence--
To determine if CREB and ATF-1 bind to the CRE sequence
and to determine the composition of the CRE-binding complexes,
supershift assays were performed with an oligonucleotide containing the
CRE consensus sequence and antibodies to specific proteins in the CREB/ATF and AP-1 families of transcription factors. The AP-1 family
was included because Jun proteins also bind CRE sequence.
Nuclear extracts from sPGN- or ReLPS-stimulated RAW264.7 cells
contained proteins that bind to the CRE consensus site (the protein-DNA
complex ran higher than the free oligonucleotide) (Fig.
2A). There was no difference
in the binding of proteins to the CRE site between stimulated and
control cells (Fig. 2A, compare lanes
1 for Nil, sPGN, and ReLPS samples). This is characteristic of the protein complex that binds CRE sequence, where activation does
not result in a change of the binding of the proteins, but induces
phosphorylation of the already bound proteins (17).

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Fig. 2.
CREB and ATF-1 bind to CRE consensus sequence
in sPGN- or ReLPS-stimulated cells. A, RAW264.7 cells
were stimulated with 10 µg/ml sPGN or 10 ng/ml ReLPS for 30 min and
nuclear extracts were preincubated with the indicated Abs for 20 min,
followed by a 30-min incubation with a radioactively labeled
oligonucleotide containing a CRE consensus sequence. The DNA-protein
complexes were separated on nondenaturing polyacrylamide gel. The
arrows indicate the shift in the DNA-protein complexes upon
Ab binding. B, specificity of binding to CRE sites was
demonstrated using an excess of unlabeled specific (CRE) and
nonspecific (NS) oligonucleotides. The results are from one
of two similar experiments.
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The protein-DNA complexes with nuclear extracts from sPGN- or
ReLPS-stimulated cells were supershifted by antibodies against CREB-1,
pCREB, and ATF-1, but not against ATF-2, Jun, and c-Fos (Fig.
2A). This indicates that the protein dimer that binds CRE consensus sequence in sPGN- or ReLPS-stimulated RAW264.7 cells consists
of ATF-1 and CREB. There were no qualitative differences in the
specific transcription factors that bind the CRE site between stimulated and nonstimulated cells; however, there was a difference in
the amount of CREB and pCREB in the bound complex between stimulated and nonstimulated cells (Fig. 2A). The amount of pCREB
increased upon stimulation by sPGN or ReLPS, as the antibodies against
pCREB caused a complete shift of the protein-oligonucleotide complex from the stimulated cells (compare the pCREB lanes between stimulated and control in Fig. 2A). These results confirm that upon
stimulation DNA-bound CREB undergoes phosphorylation, which has been
shown to activate CREB.
The specificity of binding to the CRE sequence was confirmed using
excess unlabeled specific and nonspecific oligonucleotides (Fig.
2B). In nuclear extracts from both sPGN- or ReLPS-stimulated cells, the binding of proteins to the oligonucleotide carrying CRE
sequence was inhibited by an excess of unlabeled specific oligonucleotide (CRE), but not by a nonspecific
(NS) oligonucleotide with no CRE sequence (Fig.
2B). These data indicate that the proteins that bind the CRE
oligonucleotide specifically recognize the CRE sequence.
sPGN Induces Transactivation of a CREB-regulated Gene That Is
Inhibited by Dominant Negative CREB and Dominant Negative
ATF-1--
To determine if sPGN- or ReLPS-induced phosphorylation of
CREB and ATF-1 results in functional activation of these transcription factors, we transfected RAW264.7 cells with the plasmid
(
71)Som-CAT or empty vector (CMV) and tested for chloramphenicol
acetyltransferase (CAT) activity in the transfected cells after
stimulation with sPGN or ReLPS. The plasmid
(
71)Som-CAT contains
71 to +53 bp of somatostatin promoter fused to the CAT gene, and this
plasmid has been shown previously to be regulated by CREB (23). Both sPGN and ReLPS induced 14.1 ± 2.5-fold and 13.4 ± 2.1-fold
(means ± S.E., n = 6) increase, respectively, in
CAT activity in transfected macrophage cells (Fig.
3A). RAW264.7 cells
transfected with the empty vector showed no induced CAT activity upon
stimulation (Fig. 3A).

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Fig. 3.
sPGN and ReLPS induce transactivation of a
CREB-regulated gene, and this induction is inhibited by dominant
negative CREB (A-CREB) and dominant negative ATF-1 (A-ATF1).
A, RAW264.7 cells were transfected with 1.0 µg/ml
CREB-regulated CAT plasmid, ( 71)Som-CAT, or empty vector, pUC18;
24 h after transfection, cells were stimulated with 10 µg/ml
sPGN or 10 ng/ml ReLPS for 36 h or left unstimulated. Cell lysates
were prepared, and aliquots were assayed for CAT activity. The results
are means of duplicate samples from one out of six similar experiments.
B, cells were co-transfected with 1.0 µg/ml
( 71)Som-CAT and 2.0 µg/ml A-ATF1, or A-CREB, or empty vector
(CMV), or 2.0 µg/ml each of A-ATF1 and A-CREB or 4.0 µg/ml CMV and
were stimulated and assayed as in A. The percent of empty
vector = (mean cpm stimulated with dominant negative mean
cpm unstimulated with dominant negative) × 100/(mean cpm stimulated
with empty vector mean cpm unstimulated with empty vector). The
results are means ± S.E. from three experiments.
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To determine if the induced CAT activity in sPGN- or ReLPS-stimulated
cells was due to ATF-1 and/or CREB, we co-transfected RAW264.7 cells
with
(
71)Som-CAT and A-ATF1 (a dominant negative inhibitor of
ATF-1), A-CREB (a dominant negative inhibitor of CREB), or the empty
CMV. These dominant negative proteins were constructed by fusing an
acidic extension at the N terminus of the leucine zipper domain. This
acidic region of the recombinant protein binds the basic region of the
wild type protein, and the basic region is thus no longer available for
binding to DNA. Both dominant negative ATF-1 and dominant negative
CREB, individually or in combination, inhibited the sPGN- or
ReLPS-induced activation of CAT reporter gene (Fig. 3B).
This inhibition was specific for A-ATF1 and A-CREB, as the empty vector
did not inhibit sPGN- or ReLPS- induced CAT activity. These results
provide evidence that sPGN and ReLPS induce functional activation of
the transcription factors ATF-1 and CREB.
sPGN and ReLPS Induce Phosphorylation of c-Jun and Protein
Synthesis of JunB and c-Fos--
We next determined if sPGN and ReLPS
induce activation of the transcription factor AP-1. The AP-1
transcription factor family consists of proteins which include c-Jun,
JunB, JunD, and c-Fos. This transcription factor binds to the
12-O-tetradecanoylphorbol 13-acetate response element, usually as a
Jun-Fos heterodimer. c-Jun is activated by phosphorylation of serine 63 and serine 73. To test if sPGN activates c-Jun, RAW264.7 cells were
stimulated with sPGN or ReLPS and phosphorylation of c-Jun was measured
using an antibody specific to phosphorylated serine 63 and the adjacent c-Jun sequence. sPGN consistently induced rapid and transient dose-dependent phosphorylation and hyperphosphorylation of
c-Jun (Fig. 4, A and
C). ReLPS induced phosphorylation of c-Jun similar to that
seen with sPGN (Fig. 4, A and C). Stripping and
reprobing the blots with anti-c-Jun antibody, which recognizes both
phosphorylated and nonphosphorylated protein, revealed that sPGN and
ReLPS stimulation also causes a modest increase (1.5-2 times) in the
total (phosphorylated and nonphosphorylated) amount of c-Jun protein
(Fig. 4, B and D). This is not unexpected as
activated c-Jun protein induces transcription of the c-Jun gene
(19).

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Fig. 4.
sPGN and ReLPS induce phosphorylation of
c-Jun: time kinetics, dose response, effect of polymyxin B, and
elimination by phosphatase. RAW264.7 cells were stimulated with 10 µg/ml sPGN or 10 ng/ml ReLPS for the indicated times, and total cell
lysates were analyzed by Western blots for: phosphorylated c-Jun
(A) or total (phosphorylated and nonphosphorylated) c-Jun
(B). Cells were stimulated as in Fig. 1D, and
cell lysates were analyzed for phosphorylated c-Jun (C) or
total c-Jun protein (D). E, cells were stimulated
as in A for 30 min and cell lysates were treated with
alkaline phosphatase (AP) or protein phosphatase 2A
(PP2A) or not treated with any enzyme and analyzed for
phosphorylation of c-Jun by Western blots. F, the blot shown
in E was stripped and reprobed with anti-c-Jun Ab. The
results are from one of two similar experiments.
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sPGN-induced phosphorylation of c-Jun was not inhibited by polymyxin B,
in contrast to the phosphorylation induced by ReLPS, which was
completely inhibited by polymyxin B (Fig. 4C). These data
confirm that sPGN-induced activation of RAW264.7 cells and phosphorylation of c-Jun is due to PGN and not due to an endotoxin contaminant in our sPGN preparation. In control experiments, the amount
of c-Jun protein did not change with polymyxin B treatment, for either
stimulant (Fig. 4D).
To confirm that the anti-phosphorylated c-Jun antibody recognizes
phosphorylated c-Jun, samples were treated with different phosphatases
and then analyzed by Western blot. Cell lysates from both sPGN- and
ReLPS-stimulated cells, treated with alkaline phosphatase, a
nonspecific phosphatase, did not show any binding to the
anti-phosphorylated c-Jun antibody (Fig. 4E). Furthermore,
protein phosphatase 2A, a serine phosphatase, strongly diminished
binding to the anti-phosphorylated c-Jun antibody (Fig. 4E).
However, the binding to the anti-c-Jun antibody was not eliminated or
reduced by either phosphatase treatment (Fig. 4F). These
results confirmed that the anti-phosphorylated c-Jun antibody was
indeed specific for the phosphorylated c-Jun.
JunB, JunD, and c-Fos are other members of the AP-1 family of
transcription factors. sPGN consistently induced a
dose-dependent increase of JunB and c-Fos, but not of JunD
protein synthesis in RAW264.7 cells (Fig.
5). ReLPS also induced increases in JunB and c-Fos proteins, but not of JunD protein (Fig. 5). The up-regulation of JunB by sPGN was not inhibited by polymyxin B, in contrast to
ReLPS-induced increase in JunB protein, which was completely inhibited
by polymyxin B (Fig. 5B).

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Fig. 5.
sPGN and ReLPS induce synthesis of JunB and
c-Fos proteins. RAW264.7 cells were stimulated as in Fig.
4A for A, C, and D or as in
Fig. 4C for B and analyzed for total protein
using an anti-JunB Ab (A and B), anti-JunD Ab
(C), or anti-cFos Ab (D). JunB runs as a doublet
as shown previously (30). JunB, JunD, and c-Fos are
indicated by arrows; all other bands are nonspecific. The
results are from one of two similar experiments.
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c-Jun, JunB, and c-Fos Bind to the TPA-RE Consensus Sequence in
sPGN-stimulated Cells--
To identify the specific members of the
AP-1 family of transcription factors that bind the TPA-RE consensus
sequence, gel shift assays were performed. The binding of proteins to
an oligonucleotide with the TPA-RE sequence showed no differences
between sPGN- or ReLPS-stimulated and unstimulated RAW264.7 cells (Fig.
6; gel shift assays with unstimulated
lysates are not shown). Using a supershift assay and a series of
antibodies that recognize different members of the AP-1 and CREB family
of transcription factors, we determined that in both sPGN- and
ReLPS-stimulated cells (Fig. 6A) and in unstimulated cells
(data not shown) c-Jun, JunB, and c-Fos bind to the TPA-RE sequence.
The specificity of the oligonucleotide that was used in the supershift
assays was confirmed by inhibition of binding of nuclear proteins by an
excess of unlabeled specific oligonucleotide, but not by a nonspecific
oligonucleotide with no TPA-RE sequence (Fig. 6B).

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Fig. 6.
c-Jun, JunB, and c-Fos bind to the TPA-RE
consensus sequence in sPGN- and ReLPS-stimulated cells.
A, RAW264.7 cells were stimulated and assayed as in Fig. 2,
with a radioactively labeled oligonucleotide containing a TPA-RE
consensus sequence. The DNA-protein complexes were separated on
nondenaturing polyacrylamide gel. The band was shifted by JunB and
c-Fos Abs and eliminated with c-Jun Abs. B, specificity of
binding to the TPA-RE site was demonstrated using an excess of
unlabeled specific (TPA-RE) and nonspecific (NS)
oligonucleotides. The results are from one of two similar
experiments.
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sPGN Induces Transactivation of an AP-1-regulated Gene That Is
Inhibited by Dominant Negative c-Fos--
To determine if sPGN induced
functional activation of AP-1, we transfected RAW264.7 cells with the
plasmid pAP1-Luc, which has seven AP-1 binding sites upstream of the
luciferase gene, or
60Col-Luc, which has no AP-1 binding sites. Cells
transfected with pAP1-Luc showed an average 9.0 ± 0.6-fold and
9.4 ± 1.4-fold (means ± S.E., n = 3)
increase in inducible luciferase activity when stimulated by sPGN or
ReLPS, respectively (Fig. 7A).
Cells transfected with the control plasmid
60Col-Luc showed no
luciferase activity in the absence or presence of the stimulants (Fig.
7A). We also confirmed activation of AP-1 by sPGN or ReLPS
using two additional plasmids:
73Col-Luc, which has
73 to +63 bp of
the collagenase promoter fused to the luciferase gene, and 2xAP1-Luc, which has two AP-1 binding sites upstream of the luciferase gene (data
not shown). These results demonstrate that sPGN and ReLPS induced
functional activation of the transcription factor AP-1.

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Fig. 7.
sPGN and ReLPS induce transactivation of an
AP-1-regulated gene, and this induction is inhibited by dominant
negative c-Fos. A, RAW264.7 cells were transfected with
0.5 µg/ml pAP1-Luc plasmid or the empty vector 60Col-Luc; 48 h
after transfection, cells were stimulated with 10 µg/ml sPGN or 10 ng/ml ReLPS for 5 h or left unstimulated. Cell lysates were
prepared, and aliquots were assayed for luciferase activity. The
results are means of duplicate samples from one out of three similar
experiments. B, RAW264.7 cells were co-transfected with 0.5 µg/ml pAP1-Luc plasmid and 1.0 µg/ml dominant negative A-Fos, or
empty vector (CMV). Cells were stimulated and assayed as in
A. The percentage of control vector was calculated as in
Fig. 3. The results are means ± S.E. from three
experiments.
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To confirm that the sPGN- or ReLPS-induced luciferase activity was due
to AP-1, we co-transfected RAW264.7 cells with pAP1-Luc and A-Fos, a
dominant negative mutant of c-Fos, or empty vector (CMV). A-Fos
heterodimerizes with Jun proteins in an AP-1 complex and inactivates
their ability to bind DNA. A-Fos, but not the empty vector, inhibited
both sPGN- and ReLPS-induced increases in luciferase activity (Fig.
7B). These results confirm that sPGN and ReLPS induce a
functionally active AP-1, and that c-Fos and Jun form the active AP-1 complex.
sPGN-induced Phosphorylation of CREB and c-Jun in Human Monocytes
Is CD14-dependent--
Since we have previously shown that
sPGN-induced activation of NF-
B is mediated through the membrane
receptor CD14 (8), we now determined if sPGN-induced phosphorylation of
CREB and c-Jun also requires CD14. We first tested if sPGN induced
phosphorylation of CREB and c-Jun in the human monocyte cell line
THP-1. THP-1 cells activated with sPGN and ReLPS showed phosphorylation
of CREB (Fig. 8A). THP-1 cells
differed from the mouse RAW264.7 cells, in that they did not have
detectable levels of ATF-1, which in RAW264.7 cells was present in high
amounts (Fig. 1C) and was strongly phosphorylated upon
stimulation of the cells (Fig. 1A). Total amount of CREB did
not change upon cell activation by sPGN or ReLPS (Fig.
8B).

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Fig. 8.
sPGN and ReLPS induce phosphorylation
of CREB and c-Jun in human THP-1 cells, and this phosphorylation is
inhibited by anti-CD14 antibodies. THP-1 cells were differentiated
with 100 nM vitamin D3 for 72 h and then stimulated
with 10 µg/ml sPGN or 10 ng/ml ReLPS (A-F) for the
indicated times (A-D). In E and F,
differentiated cells were preincubated for 30 min with 10 µg/ml
anti-CD14 antibodies, MY4 or MEM18, or control IgG, and then stimulated
for 30 min. Cell lysates were analyzed with antibodies to:
phosphorylated CREB (A and E), total
(phosphorylated and nonphosphorylated) CREB (B),
phosphorylated c-Jun (C and F) and total
(phosphorylated and nonphosphorylated) c-Jun (D). pCREB,
pc-Jun, CREB, and c-Jun are indicated by arrows; all other
bands are nonspecific. The results are from one of two similar
experiments.
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sPGN or ReLPS also induced phosphorylation of c-Jun in THP-1 cells
(Fig. 8C) and an increase in the total amount of c-Jun protein (Fig. 8D). The increase in c-Jun protein may be due
to the activated c-Jun itself, which is known to induce transcription of the c-Jun gene (19).
Anti-CD14 monoclonal antibodies, MY4 and MEM18, inhibited both sPGN-
and ReLPS-induced phosphorylation of CREB (Fig. 8E) and c-Jun (Fig. 8F). These data confirm that sPGN and ReLPS
activate cells through CD14 and that CD14 is required for both sPGN-
and ReLPS-induced phosphorylation of CREB and c-Jun.
Lysostaphin and Lysozyme Reduce sPGN-induced Phosphorylation of
ATF-1, CREB, and c-Jun--
To confirm the identity of sPGN as the
activating molecule that induces phosphorylation of ATF-1, CREB, and
c-Jun, sPGN was digested with lysostaphin or lysozyme, enzymes that
specifically degrade PGN. Digestion with both enzymes reduced
sPGN-induced phosphorylation of ATF-1 and CREB (Fig.
9A) and c-Jun (Fig.
9C), and this reduction was proportional to the extent of
digestion of sPGN (Fig. 9E). As expected, the total amount
of CREB (Fig. 9B) and c-Jun (Fig. 9D) proteins
remained the same in all treated and untreated groups.

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Fig. 9.
Lysostaphin and lysozyme treatments reduce
the capacity of sPGN to induce phosphorylation of ATF-1/CREB and
c-Jun. [14C]Ala-labeled sPGN was digested with 1 mg/ml lysostaphin or lysozyme for 72 h or left undigested, and
then dialyzed against Dulbecco's phosphate-buffered saline. Aliquots
of dialyzed sPGN were used to stimulate RAW264.7 cells
(A-D) at 2 µg/ml or to measure the remaining
14C in nondialyzable (undigested macromolecular) sPGN
(E). Cell lysates were analyzed with antibodies to:
phosphorylated CREB (A), total CREB (B),
phosphorylated c-Jun (C), and total c-Jun
(D).
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DISCUSSION |
Our results demonstrate that: (i) PGN induces phosphorylation and
functional activation of the transcription factors ATF-1 and CREB and
these proteins bind DNA as a dimer; (ii) PGN induces phosphorylation of
c-Jun, protein synthesis of JunB and c-Fos, and functional activation
of the transcription factor AP-1; and (iii) PGN-induced activation of
CREB/ATF and AP-1 is mediated through CD14. This is the first study to
demonstrate activation of these transcription factors by PGN or by any
other component of Gram-positive bacteria.
Our data also confirm that: (i) CREB and ATF-1 from LPS-activated
nuclear extracts bind the CRE sequence (28); (ii) LPS induces
phosphorylation of CREB (28); (iii) LPS induces increase in JunB
protein, but not JunD (29); and (iv) JunB and c-Jun bind the TPA-RE
sequence (29). In addition, our data demonstrate: (i) that LPS induces
phosphorylation of ATF-1 and c-Jun, and (ii) that LPS-induced
activation of CREB/ATF and AP-1 is CD14-dependent, which
are new findings for LPS.
Our data also demonstrate that LPS induces transcriptional activation:
(i) of a CRE reporter plasmid and that this activation is mediated by a
complex of ATF-1 and CREB, and (ii) of an AP-1 reporter plasmid and
that this activation is mediated by a complex containing c-Fos
proteins. These data are in agreement with other investigators'
results showing that the CRE sites in the promoters of TNF-
(30),
MIP-1
(31), and P-selectin (32) genes are necessary for LPS-induced
transcriptional activity of these genes. Also in agreement with our
findings are the results showing that site-specific mutations in the
CRE site in the IL-1
promoter result in a substantial loss in
transcriptional induction following combined stimulation with LPS,
phorbol myristate acetate, and dibutyryl cAMP (33), and also that
the AP-1 site is required for LPS-induced transcriptional activation of
tissue factor (34) and heme oxygenase (35) genes.
However, the actual protein complex that binds the CRE or AP-1 site and
regulates transcription may be different for different genes,
e.g. the CRE site in the IL-1
promoter binds CREB and ATF-1 (33), while the c-Jun protein binds the CRE site in the human
TNF-
promoter, and the amount of c-Jun bound to this site increases
when cells are stimulated with LPS (30). In addition, different
proteins may have different effects on transcription, e.g.
c-Jun is an effective transcriptional activator, while JunB is not, and
thus JunB may have an inhibitory function (26). These data emphasize
the importance of the CRE and AP-1 sites and the CREB/ATF and AP-1
families of transcription factors in LPS-induced transcriptional
activation of pro-inflammatory genes. These transcription factors and
their binding sites may also play a significant role in PGN-induced
inflammatory response.
Although both PGN (10) and LPS (36) bind to CD14 and activate cells
through CD14 (8), there are several differences in the function of CD14
as the PGN and LPS receptor. In particular, both the binding sites for
PGN and LPS on CD14 and the sites needed for cell activation are
partially similar but partially different (8, 10), and only
LPS-induced, but not PGN-induced cell activation and binding affinity
for CD14 are enhanced by the LPS-binding protein (7, 10). Moreover, PGN
and LPS induce differential activation of MAP kinases, with LPS
strongly inducing all three families of kinases (ERK, JNK, and p38),
but with PGN only inducing ERK and JNK, but not p38 (37). Furthermore,
soluble CD14·LPS complexes activate CD14-negative cells, whereas
soluble CD14·PGN complexes do not (16).
Despite these differences, in this study we did not detect any
differences between PGN and LPS in the activation of CREB/ATF and AP-1
transcription factors. Therefore, our current and previous (8) results
indicate that transcription factors NF-
B, CREB/ATF-1, and AP-1 are
either induced by CD14-dependent signal transduction pathways that are common for PGN and LPS, or that different pathways activated by PGN and LPS converge to activate these three families of
transcription factors. Such a convergence of initially different signal
transduction pathways to activate the same transcription factors has
been demonstrated in the activation of cells through cytokine
receptors, e.g. IL-1 and TNF-
(38).
The signal transduction pathway(s) through which PGN and LPS activate
CREB/ATF and AP-1 are still not clear. Activation of AP-1 is consistent
with the strong activation of JNK and ERK1 and ERK2 by both PGN and LPS
(37), since JNK can activate c-Jun and both JNK and ERK can induce
c-Fos through activation of ternary complex factor/Elk-1 (39). The
possible mechanism of activation of ATF-1 and CREB are less clear,
since in other systems the main mechanism of activation of ATF-1 and
CREB is through protein kinase A, but we could not show any activation
of protein kinase A by PGN or LPS (37). Other possible mechanisms of
activation could involve calmodulin kinase or the MAP kinases ERK1 and
ERK2 (17, 18).
The functional significance of the activation of NF-
B, CREB/ATF, and
AP-1 for the induction of cytokine genes by PGN is still not clear. As
seen for LPS, these transcription factors are required for the
induction of specific genes coding for pro-inflammatory molecules, and
our preliminary data indicate that NF-
B, CREB, and ATF-1, but not
AP-1, are required for PGN-induced transcriptional activation of
TNF-
.2 These transcription
factors are also likely to play a role in the induction of several
other pro-inflammatory molecules, such as cytokines, chemokines, and
adhesion molecules in PGN-activated cells.
In summary, we demonstrate that activation of macrophages by PGN leads
to the functional activation of the transcription factors CREB/ATF and
AP-1 and that this activation is CD14-dependent.