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Gene Expression by TNF-Dependent NF-
B Activation1
* Department of Obstetrics and Gynecology, Nagasaki University School of Medicine, and Nagasaki University Medical Skill Junior College, Nagasaki, Japan; and
Division of Cytokine Signaling, Department of Molecular Microbiology and Immunology, Nagasaki University Graduate School of Medical Sciences, Nagasaki, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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(MIP-3), also designated as
liver and activation-regulated chemokine (LARC), Exodus, or CCL20, is a
recently identified CC chemokine that is expected to play a crucial
role in the initiation of immune responses. In this study, we describe
that MIP-3 expression is under the direct control of NF-
B, a key
transcription factor of immune and inflammatory responses.
Overexpression of the p65/RelA subunit of NF-B significantly
increased the MIP-3 mRNA level. MIP-3 transcription was
stimulated by TNF, and this stimulation was inhibited by an NF-B
inhibitor, I-B superrepressor. Analysis of the human MIP-3
promoter demonstrated a functional NF-B site responsible for its
expression. We also show that MIP-3 expression is induced in
LPS-treated mouse livers that were primed with Propionibacterium
acnes, which developed massive liver injury with infiltration
of inflammatory cells. This induction was fully dependent on the TNF
signaling cascade, because it was not observed in the livers of
TNFR1-deficient mice. Furthermore, pretreatment with gliotoxin, an
inhibitor of NF-B activity, abrogated the P.
acnes/LPS-induced MIP-3 expression of wild-type mice. These
results clearly demonstrate that MIP-3 gene expression is dependent
on NF-B activity in vitro, and indicate that the TNFR1-mediated TNF
signaling cascade that leads to NF-B activation plays an essential
role in MIP-3 expression in the murine liver injury
model. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B (1).
NF-B is a ubiquitously expressed transcription factor, usually found
as a heterodimer of p50 and p65 (Rel A). Without stimulation, the
heterodimer is associated with a member of the inhibitory I-B family
of proteins and is sequestered in the cytoplasm. Among the I-B
family proteins, the best-characterized molecule is I-B.
Treatment of cells with various stimuli, including mitogens, cytokines,
viruses, bacterial LPS, radiation, and some anticancer drugs, leads to
the phosphorylation of I-B on serines 32 and 36. The
phosphorylated I-B is then rapidly ubiquitinated and subsequently
degraded by the 26S proteasome. The released NF-B then translocates
into the nucleus, where it binds to specific NF-B elements and
activates many important genes (1).
Recently, a wide variety of transcriptional targets of NF-B have
been reported. The group of NF-B-regulated genes that encode
proteins playing important roles in inflammation and immune responses
includes proinflammatory cytokines, such as IFN-
, IL-2, IL-6, and
TNF (1, 2, 3); chemokines, such as monocyte chemoattractant
protein-1, IL-8, RANTES, and macrophage-inflammatory protein
(MIP)3-1 (4, 5, 6, 7);
and genes related to Ag presentation, such as MHC molecules, the
peptide transporter TAP1, and the protease low molecular weight protein
2 (8, 9, 10). Many cell adhesion molecules, such as VCAM-1,
ICAM-1, E-selectin, and the matrix-degrading proteases matrix
metalloproteinase-9 and urokinase-type plasminogen activator,
are also regulated by NF-B (1, 11, 12, 13). NF-B has
also been implicated in the regulation of cell proliferation. Recently,
NF-B was found to stimulate the transcription of cyclin D1, a key
regulator of G1 checkpoint control, providing
evidence for a direct link between NF-B activity and cell cycle
regulation (14). A requirement of NF-B for preventing
apoptosis induced by TNF, ionizing radiation, and anticancer drugs has
also been shown (15, 16, 17). The candidate genes are
c-inhibitor of apoptosis, X-linked inhibitor of apoptosis,
members of the Bcl-2 family A1 and Bcl-xL,
immediate early response gene X-1L, A20, and manganese superoxide
dismutase (18, 19).
Although many genes have been found, as described above, it is evident
that not all of the NF-B response genes have been identified at
present. Therefore, we performed a subtractive hybridization approach
to identify NF-B response genes. One of the clones obtained was the
CC chemokine MIP-3, also called liver and activation-regulated
chemokine, Exodus, or CCL20, which was recently identified as a
chemoattractant factor for certain dendritic cells and T cells
(20, 21, 22, 23, 24). Because little is known about the
molecular mechanism of MIP-3 transcription, we next examined the
role of NF-B in its expression in vitro and in vivo. In this study,
we provide lines of evidence that MIP-3 gene expression is under the
control of NF-B activity in vitro. We also found that MIP-3
expression is induced in Propionibacterium acnes/LPS-treated
mouse livers, which develop massive liver injury that pathologically
mimics fulminant hepatitis in humans (25). Finally,
functional analysis of the human MIP-3 promoter revealed an NF-B
binding site responsible for its induction.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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HeLa cells and HT1080 cells were grown in DMEM supplemented with
10% FCS and 100 U/ml penicillin-streptomycin. The HISR, a clone
of HeLa cells expressing the I-B superrepressor (I-BSR),
was established by transfection of pcDNA3-I-BSR. A control cell
clone with the stably integrated pcDNA3 vector alone was also
established and maintained, as described. The medium, FCS, and G418
were purchased from Life Technologies (Rockville, MD). Human TNF
and LPS (from Escherichia coli O127:B8) were purchased from
Sigma (St. Louis, MO). P. acnes (ATCC 11827) was grown and
heat killed, as previously described (26).
DNA constructs and transfection
cDNA cloning of I-B was done by RT-PCR and was confirmed
by DNA sequencing. Site-directed mutagenesis was performed, using the
QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA), to
generate pcDNA3-I-BSR bearing a serine-to-glycine substitution at
residue 32 and a serine-to-alanine substitution at residue 36 of human
I-B. These mutations were confirmed by DNA sequencing. The
pNF-B luciferase reporter plasmid was purchased from Stratagene.
Mouse MIP-3 cDNA was generated by RT-PCR, cloned into the pCR2.1
vector (Invitrogen, San Diego, CA), and confirmed by DNA sequencing. To
isolate the MIP-3 regulatory sequences lying upstream of the coding
region, PCR amplification of human genomic DNA was performed on the
basis of the information about the genomic sequences of MIP-3 on
chromosome 2 (GenBank accession AC027560 and AC073065). The amplified
products were directionally cloned into pGL2-basic (Promega, Madison,
WI), and DNA sequencing was performed. Site-directed mutagenesis was
performed, as described above, to inactivate the putative NF-B site
on the promoter. All transfections were conducted with the FuGENE 6
reagent (Roche, Indianapolis, IN).
Northern blot analysis
Total RNA was isolated with the ISOGEN (NipponGene, Tokyo,
Japan) reagent, according to the manufacturer’s protocol. Aliquots of
RNA (10 µg) were fractionated in 1% agarose-formaldehyde gels,
transferred onto Gene Screen Plus hybridization transfer membranes (NEN
Life Science Products, Boston, MA), and immobilized with a UV
cross-linker. cDNA fragments were labeled with the Redi-Prime II
labeling kit (Amersham Pharmacia Biotech, Uppsala, Sweden) in the
presence of [-32P]dCTP. The hybridization
signals were visualized by the image analyzer BAS 5000 system (Fuji
Film, Tokyo, Japan).
Western blot analysis
Cells were washed with TBS and resuspended in buffer A (10 mM
HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.5 mM PMSF). After an
incubation on ice for 15 min, Nonidet P-40 was added to a final
concentration of 0.5%, and then the cells were centrifuged at 12,500
rpm for 30 s at 4°C. The supernatant containing the cytoplasmic
fraction was recovered. The protein from the cytoplasmic extracts was
fractionated on 10% SDS-polyacrylamide gels, transferred onto
Immobilon-P membranes (Millipore, Bedford, MA), and incubated with the
anti-I-B Ab (Santa Cruz Biotechnology, Santa Cruz, CA),
followed by an incubation with HRP-conjugated goat anti-rabbit IgG
(Amersham Pharmacia Biotech). The blots were visualized with the ECL
detection system (Amersham Pharmacia Biotech).
Reporter assays
To analyze the NF-B activity, cells seeded into six-well
plates were transfected with 1 µg pNF-B luciferase reporter
plasmid and 1 ng pRL-actin luciferase (27) as an
internal control. The total amount of transfected DNA was kept at 2
µg by supplementation with pcDNA3. To analyze the MIP-3 promoter
activity, HeLa cells were cotransfected with 1 µg pGL2 reporter
plasmid containing either the wild-type or mutant MIP-3 promoter and
1 ng pRL-actin luciferase, together with 1 µg pcDNA3 or
pcDNA3-I-BSR. Twenty-four hours after transfection, cells were
treated with or without TNF for 3 h, and then extracts were
prepared and the luciferase activity was determined with the
dual-luciferase reporter assay system (Promega), according to the
manufacturer’s protocol. The luciferase activity was normalized with
the Renilla luciferase activity from the internal control.
Subtractive hybridization
Total RNA was prepared from both HeLa cells transiently expressing p65 and HISR. Poly(A)+ RNA was purified from total RNA using an mRNA purification kit (Amersham Pharmacia Biotech). Purified mRNA (2 µg) was reverse transcribed and subjected to subtractive hybridization with a PCR-Select cDNA subtraction kit (Clontech, Palo Alto, CA). After hybridization, differential transcripts were selectively amplified, and were ligated with the pCR2.1 vector using the Topo-TA cloning kit (Invitrogen). Partial cDNA sequences were determined and compared with the entries in the GenBank database using the BLAST SEARCH program. For subsequent Northern blot analysis, radiolabeled probes were generated from the subtracted cDNAs. Total RNA from HeLa cells transiently expressing p65 and from HISR was fractionated and transferred to the membranes, and Northern blot analyses were performed using these probes.
In vivo MIP-3 expression
Healthy 6- to 8-wk-old TNFR1-/- mice (28) and age-matched C57BL/6 control mice received an i.p. injection of 10 mg P. acnes suspended in 300 µl PBS. Seven days later, the mice received an i.p. injection of 20 µg LPS in 300 µl PBS. In some cases, the mice were pretreated with 20 µg gliotoxin (29, 30) (Sigma) by i.p. injection 3 h before the LPS injection. Control mice were treated with 300 µl PBS only. Ninety minutes or three hours after the LPS injection, the mice were sacrificed and total RNA was prepared from the liver for Northern blot analysis. At least three mice were examined for each set of experiments. The animal protocols described above were approved by the Nagasaki University Animal Research Center Committee.
Nuclear extract preparation
Nuclear extracts were prepared according to the method of Zhou et al. (31). Briefly, tissues were homogenized in 1 ml ice-cold lysis buffer (10 mM HEPES, pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 0.5 mM DTT, 0.5 mM PMSF, supplemented with a protease inhibitor mixture tablet (one tablet per 10 ml; Roche)). The homogenates were kept on ice for 15 min, and then 25 µl 10% Nonidet P-40 was added. After a brief vortexing, they were incubated on ice for 20 min and centrifuged at 12,500 rpm for 30 s at 4°C. The pellets of nuclei were resuspended in 200 µl extraction buffer (20 mM HEPES, pH 7.9, 400 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, and 0.5 mM PMSF, supplemented with a protease inhibitor mixture tablet (one tablet per 10 ml)) and were kept on ice for 30 min. The nuclear suspension was centrifuged at 12,500 rpm for 15 min at 4°C to collect the supernatants containing nuclear proteins.
To prepare the nuclear extracts from HeLa cells, 2 x 106 cells were washed and resuspended in buffer A, and then cytoplasmic fractions were removed, as described above, in the Western blot analysis. The pellets of nuclei were resuspended in 50 µl buffer C (20 mM HEPES, pH 7.9, 400 mM NaCl, 1 mM EDTA, 1 mM DTT, 1 mM PMSF) at 4°C for 15 min. The nuclear suspension was centrifuged at 15,000 rpm for 5 min at 4°C to collect the supernatants containing nuclear proteins.
Gel shift analysis
To examine the NF-B activity in the mouse livers, an aliquot
of the nuclear extracts (5 µg) was incubated in a reaction buffer (10
mM HEPES, pH 7.9, 100 mM NaCl, 0.4 mM MgCl2, 0.3
mM EDTA, 0.5 mM DTT, 0.5 mM PMSF, 10% glycerol, and 100 µg/ml
poly(dI-dC)). In some cases, a 20-fold molar excess of unlabeled
double-stranded oligonucleotide was added as a competitor. After a
20-min incubation on ice, 
10 kcpm 32P
end-labeled double-stranded oligonucleotide containing a consensus
NF-B-binding sequence (5'-GGGCTGGGGAATCCCGCTAA-3') was added to the
reaction, which was then incubated for an additional 30 min at room
temperature. The same oligonucleotide in the unlabeled form was used as
the wild-type competitor. The mutant competitor sequence was
5'-GGGCTGTGGAATCACGCTAA-3'. In some cases, the reactions were further
incubated with anti-p65 or anti-p50 Abs (Santa Cruz
Biotechnology) at room temperature for 30 min. To examine the binding
of NF-B to the putative NF-B site of the MIP-3 promoter, a gel
shift analysis was performed using nuclear extracts from TNF-treated or
untreated HeLa cells with the same protocol as described above. The
labeled double-stranded oligonucleotide was derived from the sequence
of the MIP-3 promoter containing the putative NF-B site
(5'-CACATGGGGTTTTCCCCATTGA-3'). The same oligonucleotide in the
unlabeled form was used as the wild-type competitor. The mutant
competitor sequence was 5'-CACATGTTTTTTTCCCCATTGA-3'. In some
cases, the reactions were further incubated with anti-p65 or
anti-p50 Abs.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B activity
To establish a cell line in which the NF-B activity is
impaired, we isolated drug-resistant clones and examined them for
I-BSR expression. One of the clones, designated as HISR, showed
strong and constitutive I-B expression, as revealed by Northern
blot analysis (Fig. 1
A).
I-B protein in control cell line was almost completely degraded
after a 10-min incubation with TNF and returned to the untreated level
by 30 min (Fig. 1B). In contrast, in HISR, the mutant
I-B protein was continuously present after a 60-min incubation
with TNF (Fig. 1B, HISR, I-B SR), along with
the continuous presence of endogenous I-B (Fig. 1B, HISR, I-B). Similar effect of exogenous
I-B on the stability of endogenous I-B was reported
previously (32). Then, we examined NF-B activation in
response to TNF stimulation with an NF-B-dependent luciferase
reporter plasmid, pNF-B luciferase (Fig. 1C), which
contains five tandem repeats of the NF-B site upstream of a basic
promoter element (TATA box) and the luciferase gene. In HISR, the
TNF-induced activation of NF-B was blocked, although not completely,
as compared with the control cells. As the inhibition of NF-B
activation renders cells sensitive to TNF-induced cell death
(15, 16, 17, 18, 19, 33), we then investigated the sensitivity of HISR
to TNF. The HISR became sensitive to TNF, and remarkable cell death was
observed by 12 h, but not control cells (Fig. 1D).
These data demonstrate that the biological activity of NF-B is
specifically impaired in HISR.
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B
Next, we performed cDNA subtractive hybridization between HeLa
cells transfected with the p65/RelA expression plasmid and HISR, to
identify novel NF-B response genes. Using the prepared mRNA from
both cell lines, we performed a Northern blot analysis to verify the
quality of the mRNA samples by confirming that A1 (18), a
direct transcriptional target of NF-B, was specifically detected in
the mRNA sample from HeLa cells expressing p65 (data not shown). The
transcripts were converted to cDNAs by reverse transcription and were
subjected to subtractive hybridization, as previously reported
(34). Differential transcripts were selectively amplified
by suppression PCR and were inserted into cloning vectors.
Radiolabeled probes were generated from the subtracted cDNAs, and
Northern blot analysis was performed to screen for the cDNA clones
whose expression was specifically up-regulated in the p65 transfectant.
Among the 131 nonredundant cDNA clones, 17 clones showed clear
p65-dependent expression. As shown in Table I, 7 clones had coding sequences that
were previously reported as transcriptional targets of NF-B. Another
7 clones were identical to expressed sequence tag clones. One clone had
no significant homology with entries in the expressed sequence tag
database, but corresponded to a human BAC clone. The remaining 2
clones were identical to genes whose coding amino acids and functions
had been characterized, but not reported as NF-B-regulated genes,
and one of them is the recently identified CC chemokine, MIP-3.
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gene expression in an NF-B-dependent manner
It has been reported that MIP-3 expression could be induced by
some proinflammatory cytokines (20, 21). However, the
precise transcriptional regulation of the MIP-3 gene has not been
revealed, and most importantly, it is not known whether NF-B is
involved in the expression. As clearly demonstrated in Fig. 2, ectopic expression of p65 can induce
MIP-3 expression in HeLa cells. We also confirmed that TNF
stimulation induced MIP-3 expression in HeLa cells and HT1080 cells,
but not in Jurkat cells (data not shown).
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expression is induced in wild-type, but not in
TNFR1-/-, mouse livers treated with LPS after
P. acnes priming
To investigate MIP-3 expression and its NF-B dependence in
vivo, we used a mouse hepatitis model, in which priming with P.
acnes and subsequent LPS injection generates intrahepatic TNF. The
produced cytokines may subsequently activate NF-B in the liver.
MIP-3 expression was not detected in the livers of control mice
(Fig. 3, lane 1), or those
treated with P. acnes alone (Fig. 3, lane 2) or
LPS alone (Fig. 3, lanes 5 and 6). However, the
LPS challenge after P. acnes priming clearly induced
MIP-3 expression in the wild-type mouse livers in a time-dependent
fashion (Fig. 3, lanes 3 and 4). It was reported
that cytokine-induced NF-B activation was impaired in
TNFR1-/- mouse livers (35), and
LPS challenge after P. acnes priming caused few pathological
changes (25). As shown in Fig. 3, there was no induction
of MIP-3 in the TNFR1-/- mouse livers,
either in the presence or absence of P. acnes/LPS treatment
(Fig. 3, lanes 5–8). These results indicate that
TNF/TNFR1-mediated signaling is required for the induction of MIP-3
in P. acnes/LPS-treated mouse livers, and suggest that
NF-B activation plays a critical role in the MIP-3
expression.
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B activation, abolishes the
induction of MIP-3 expression in P. acnes/LPS-treated
mice livers
To confirm the dependence of MIP-3 expression on NF-B
activity in vivo, we examined the effects of gliotoxin on the MIP-3
induction. The effect of gliotoxin was reported to be NF-B specific
because the toxin did not affect activation of the transcription factor
NF-AT or of STAT, or the activity of the constitutively DNA-binding
transcription factors Oct-1 and CREB (29). Although the
mechanism by which gliotoxin inhibits NF-B activation remains
unknown, it was demonstrated to inhibit the degradation of I-B
(30). P. acnes-primed wild-type mice were
pretreated with gliotoxin, and then received an LPS injection. To
examine the NF-B activity, a gel shift analysis was performed with
the liver nuclear extracts, using a radiolabeled NF-B consensus
probe. The LPS challenge after P. acnes priming induced the
DNA-protein complex in the nuclear extracts from wild-type mouse livers
(Fig. 4, A and B,
lanes 1 and 2). The complex formation was
disturbed by the wild-type competitor of the NF-B oligonucleotide,
but not by the competitor with mutations in the consensus NF-B
sequence (Fig. 4B, lanes 3 and 4). The
complex was either eliminated or supershifted in the presence of p65 or
p50 Abs (Fig. 4B, lanes 5 and 6). It
is important to note that NF-B activation was not induced by
P. acnes/LPS treatment in TNFR1-/-
mouse livers (Fig. 4A, lane 5). In addition,
pretreatment with gliotoxin, before the LPS injection, abrogated the
induction of NF-B activation (Fig. 4A, lane
3). Northern blot analysis revealed that gliotoxin pretreatment
dramatically inhibited the MIP-3 expression following P.
acnes/LPS treatment (Fig. 5).
Therefore, we concluded that MIP-3 expression is dependent on
NF-B activation in vivo, and that the TNFR1-mediated TNF signaling
cascade that leads to NF-B activation is essential for MIP-3
expression in the liver injury model.
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promoter contains an NF-B site responsible
for its TNF-inducible expression
To verify a direct role of NF-B in the regulation of MIP-3
expression, we isolated and characterized the regulatory sequences of
the human MIP-3 gene. The cloned 5'-upstream sequence of MIP-3
gene is identical to the reported sequence, except for one base
substitution (A to T) at position -142 relative to the first
nucleotide in the human MIP-3 cDNA (GenBank/EMBL/DDBJ accession
D86955). Because repeated amplifications of five independent clones
revealed the same substitution, we regarded it as a personal
polymorphism. Using the reporter plasmid (Fig. 6A), the MIP-3 promoter
activity was assayed with or without TNF stimulation. As shown in Fig. 6B, the -874/+58 reporter construct showed minimal basal
activity on its own. TNF stimulation enhanced the construct’s
expression by 20-fold, and this enhancement was completely inhibited by
cotransfection of the I-BSR expression plasmid (Fig. 6B). This result further supports the NF-B-dependent
expression of the MIP-3 gene with TNF stimulation. Alignment of the
MIP-3 promoter sequences revealed a putative NF-B binding site on
the lower strand, at a position between -82 and -91 relative to the
first nucleotide in the human MIP-3 cDNA sequence (GGGGTTTTCC).
Site-directed mutagenesis was performed to introduce the mutation into
the consensus NF-B site between -82 and -91 (GTTTTTTTCC; -874/+58
mB). With the luciferase assay, this mutation was found to
decrease the basal activity of the MIP-3 promoter. Furthermore, this
mutation greatly impaired the TNF inducibility of the MIP-3 promoter
activity (Fig. 6B). In a gel shift assay using a probe
containing the NF-B sequence found in the MIP-3 promoter,
TNF-induced NF-B activity was readily detected (Fig. 6C,
lane 2), which was not competed by the mutant competitor,
including the same B mutation introduced in the promoter assay (Fig. 6C, lane 4). These findings demonstrate that the
human MIP-3 promoter contains a functional NF-B site between -82
and -91 that is responsible for its expression, and that MIP-3 is a
chemokine under the direct control of NF-B.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B and the inflammatory mediator MIP-3, which
both play important roles in the development of inflammation. After
identifying MIP-3 as a new transcriptional target of NF-B/p65, we
further performed experiments both in vitro and in vivo to clarify the
regulatory mechanism. Functional analysis of the MIP-3 promoter
revealed an NF-B site, at a position between -82 and -91, which is
responsible for the TNF-dependent induction (Fig. 6
). Although
inactivation of the consensus NF-B site greatly reduced the gene
expression, a marginal response to TNF stimulation still remained.
Because cotransfection of the I-BSR expression plasmid with the
NF-B site mutant reporter (-874/+58 mB) completely abolished the
response to TNF stimulation (data not shown), an unidentified NF-B
element(s) together with the element we identified may regulate
MIP-3 expression cooperatively or synergistically. Our preliminary
data from the deletion analysis of the MIP-3 promoter indicate that,
in addition to the -82/-91 NF-B site, the region from -258 to
-163 is required for the maximal induction of the reporter gene by TNF
stimulation. In this region, an NF-B-like sequence was found on the
lower strand, at a position between -210 and -219 (GGGAGAATCC).
Deletion of this region moderately reduced the TNF response of the
promoter (data not shown). This suggests that the NF-B-like element
between -210 and -219 possesses an additional, but not essential,
role in the regulation of MIP-3 transcription, even though the
sequence deviates from the consensus NF-B sequence. Thus, the TNF
responsiveness of MIP-3 expression may be dignified by collaborative
action of the strong and the weak NF-B response elements.
A number of in vivo studies have highlighted the importance of
cytokine/chemokine biology in both acute and chronic liver disease
(36, 37). In addition, because MIP-3 was first
identified as a novel inducible CC chemokine expressed mainly in the
liver (20), we reasoned that the liver would be the best
organ to examine MIP-3 induction in vivo. It is known that P.
acnes priming and LPS challenge in mice cause massive liver
injury, which consists of priming and eliciting phases
(25). P. acnes priming induces mononuclear cell
infiltration into the liver lobules and granuloma formation
(25). The subsequent LPS injection elicits acute and
massive hepatic injury, with a concomitant release of various
cytokines, such as IL-1, IL-6, IL-8, IL-12, IL-18, TNF, and IFN-
(38, 39, 40, 41, 42). Among these, TNF is a crucial factor to mediate
hepatocyte apoptosis and hepatic necrosis, because
TNFR1-/- mouse livers showed few pathological
changes after the LPS challenge (data not shown) (25). In
accordance with these pathologies, MIP-3 expression was induced by
the LPS challenge after P. acnes priming in the wild-type
mouse livers, but not in the TNFR1-/- mouse
livers (Fig. 3).
We propose in this study that the difference between the wild-type and
TNFR1-/- mice in MIP-3 induction may
originate from the two pathogenesis steps. The first one is the
NF-B-independent intrahepatic granuloma formation in the priming
phase. TNFR1-/- mice showed no apparent
granuloma formation after priming with P. acnes (data not
shown) (25). A significant reduction in the number of
granulomas was also seen in IFN--/- or
M-CSF-/- mice (42, 43). As
neither IFN- nor M-CSF is an activator of NF-B, the
granuloma formation is probably independent of the NF-B activity. In
addition, without P. acnes priming, the LPS challenge could
not induce MIP-3 even in wild-type mice (Fig. 3). From these
results, together with a previous report that the number and the size
of the granulomas paralleled the levels of TNF, IL-12, and IL-18
(41), it seems that the intrahepatic granuloma formation
induced by P. acnes is essential for MIP-3 induction. The
second step in the pathogenesis is NF-B-dependent activation of
responsible genes, including MIP-3, in the eliciting phase. Although
it has been reported that the P. acnes/LPS treatment
produces various cytokines, as described above, our results with the
TNFR1-/- livers clearly indicate that NF-B
is induced exclusively by TNF in this model. It was reported that TNFR2
can mediate TNF signaling and activate NF-B (44); this
result showed that TNFR2 did not compensate for the TNFR1 defect in the
activation of NF-B in MIP-3 induction. This may be due to the
cell type specificity of TNFR2-mediated NF-B activation. A previous
report described that TNFR2 has only a minor role in the liver injury
by the injection of mice with bacterial LPS (45).
In conclusion, the current study demonstrates that MIP-3 gene
expression is induced by the TNF-activated transcription factor
NF-B. This implies the possibility that some of the various
biological effects of TNF are mediated by MIP-3 via NF-B
activation.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Toshifumi Matsuyama, Division of Cytokine Signaling, Department of Molecular Microbiology and Immunology, Nagasaki University Graduate School of Medical Sciences, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan. E-mail address: tosim{at}net.nagasaki-u.ac.jp
3 Abbrevation used in this paper: MIP, macrophage-inflammatory protein.
Received for publication September 26, 2001. Accepted for publication March 15, 2002.
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References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
B and IB proteins: new discoveries and insights. Annu. Rev. Immunol. 14:649.[Medline]
promoter in primary macrophages. J. Immunol. 147:1694.[Abstract]
B. Proc. Natl. Acad. Sci. USA 92:3632.B sites and NF-B/Rel subunit specificity. J. Biol. Chem. 272:31092.B. Mol. Cell. Biol. 13:7191.B and IFN-regulatory factor transcription factors. J. Immunol. 164:5352.B, and c-Ets family members and transcriptional regulation of the cell-specific and inducible macrophage inflammatory protein 1 immediate-early gene. Mol. Cell. Biol. 13:5276.B/Rel binding sites in the major histocompatibility complex class II invariant chain promoter is dependent on cell-specific binding of different NF-B/Rel subunits. Mol. Cell. Biol. 14:2926.B-like enhancer binding activity which displaces constitutive factors. EMBO J. 8:3793.[Medline]
on the intercellular adhesion molecule-1 promoter. Cell Growth Differ. 9:949.[Abstract]
-mediated matrix metalloproteinase-9 expression in mesangial cells by NF-B and AP-1. Am. J. Physiol. 270:F123.B and cell-cycle regulation: the cyclin connection. Cytokine Growth Factor Rev. 12:73.[Medline]
B in preventing TNF--induced cell death. Science 274:782.-induced apoptosis by NF-B. Science 274:787.B. Science 274:784.B, cancer, and apoptosis. Biochem. Pharmacol. 60:1085.[Medline]
B-regulated X-chromosome-linked iap gene expression protects endothelial cells from tumor necrosis factor -induced apoptosis. J. Exp. Med. 188:211./CCL20 and CC chemokine receptor 6 in psoriasis. J. Immunol. 164:6621.-chemokine. Blood 89:3315. from lung dendritic cells. J. Exp. Med. 186:825. and MIP-3. J. Immunol. 158:1033.[Abstract]
interferon induction found in the livers of mice causes endotoxic shock. Infect. Immun. 63:3966.[Abstract]
B. J. Exp. Med. 183:1829.B determines between apoptosis and proliferation in hepatocytes during liver regeneration. Am. J. Physiol. Gastrointest. Liver Physiol. 278:G173.B in vivo. Radiat. Res. 151:703.[Medline]
B inhibits tumor necrosis factor--induced human vascular smooth muscle proliferation. Circ. Res. 84:867.B activation prevents cell death. Cell 87:565.[Medline]
2 with concomitant reduction of cytokine production. Infect. Immun. 63:4812.[Abstract]
-deficient mice by a concomitant reduction of TNF-, IL-12, and IL-18 production. J. Immunol. 162:1049.- and Fas ligand-mediated hepatotoxic pathways in endotoxin-induced liver injury in mice. J. Immunol. 159:3961.[Abstract]
B-dependent gene expression mediated by the p75 tumor necrosis factor receptor. J. Interferon Cytokine Res. 15:171.[Medline]
B and induction of human cytomegalovirus enhancer. J. Biol. Chem. 269:7785.B controls expression of inhibitor IB : evidence for an inducible autoregulatory pathway. Science 259:1912.This article has been cited by other articles:
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