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Gene Inactivation Causes Both Impaired and Enhanced Gene Expression and Inverse Regulation of IL-12 p40 and p35 mRNAs in Macrophages1
* School of Life Sciences, Wellcome Trust Biocentre, University of Dundee, Dundee, Scotland; and
Consiglio Nazionale delle Ricerche Cellular and Molecular Pharmacology Center, Department of Medical Pharmacology, University of Milan, Milan, Italy
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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is believed to play a fundamental
role in regulating activated macrophage functions. However, the
molecular mechanisms and the target genes involved have been, so far,
poorly characterized, partly due to the difficulty of reproducibly
obtaining homogeneous and abundant primary macrophage populations. In
this study, we describe the generation and characterization of
immortalized macrophage-like cell lines from C/EBP-deficient and
wild-type mice. Using these cells, we were able to identify a number of
genes involved in activated macrophage functions whose induction was
affected in the C/EBP-/- cells. IFN-
/LPS-dependent
induction of IL-6, IL-1, TNF-
, inducible NO synthase, and
plasminogen activator inhibitor-1 mRNAs was variably impaired, while
IL-12 p40, RANTES and macrophage inflammatory protein-1 mRNAs were
up-regulated in the absence of C/EBP. The differential mRNA
expression correlated with differential transcription levels of the
corresponding genes, and was in most cases confirmed in primary
macrophage populations. Moreover, in sharp contrast to the enhanced
induction of IL-12 p40 mRNA, C/EBP-/- primary
macrophages derived from both the bone marrow and the peritoneal
cavity displayed totally defective expression of IL-12 p35 mRNA.
Therefore, the IL-12 p35 gene represents a novel
obligatory target for C/EBP in macrophages and this may explain the
defective production of bioactive IL-12 and the impaired Th1 responses
of C/EBP-deficient mice to Candida albicans infection
observed in previous work. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, a member of the C/EBP
family of leucine zipper transcription factors (see Ref. 2
and references therein).
There are many indications that C/EBP may be an important regulator
of macrophage activities. Its expression is strongly induced during the
differentiation of lymphoid cells into macrophages (3, 4),
where C/EBP is by far the predominant C/EBP isoform and is further
induced by activating stimuli such as bacterial LPS. Many genes
encoding for cytokines and other macrophage inflammatory mediators
carry on their promoters C/EBP sites that have been demonstrated to be
important for their expression. Examples include the cytokines IL-6,
TNF-, IL-1, G-CSF, and IL-12 p40 (5, 6, 7, 8, 9), the
chemokines IL-8, macrophage inflammatory protein
(MIP)6-1, monocyte
chemoattractant protein-1, and RANTES (5, 10, 11, 12),
and the genes encoding lysozyme, cyclooxygenase (COX)-2, and
inducible NO synthase (iNOS) (13, 14, 15). Moreover,
expression of C/EBP in M1 cells triggers the induction of MIP1,
osteopontin, and CD14 mRNAs (10), while its ectopic
expression in the lymphoblastoid cell line P388 confers LPS
inducibility to the IL-6 and monocyte chemoattractant
protein-1 genes (11).
C/EBP-deficient mice exhibited defective immune responses consistent
with defective macrophage functions. Impaired Th1 responses to
Candida albicans infection correlated with undetectable
levels of circulating bioactive IL-12 and with impaired NO production
by splenic macrophages (16). Macrophages isolated from the
mutant mice failed to kill intracellular bacteria and displayed
defective anti-tumoral activity (17). Bactericidal
activity could be partially restored by treatment with G-CSF, whose
production is defective in C/EBP-/-
macrophages (17, 18). In contrast to the high number of
genes that can be activated by C/EBP, only two genes whose induction
is defective in the absence of this factor have been so far identified,
G-CSF and Mincle (17, 19). In an
effort to extend our understanding of the role of C/EBP in
determining macrophage functions, and to provide a cellular system
where the molecular mechanisms regulating the transcriptional induction
of C/EBP target genes could be analyzed in detail, we generated
immortalized macrophage-like cell lines from the spleens of
C/EBP-deficient and wild-type mice making use of the
myc-transducing mouse retrovirus, VN-11 (20).
We report in this study the generation and characterization of these
cells as macrophages. Quantitative analysis of the expression of
candidate targets identified a group of genes whose transcriptional
induction was variably defective in the mutant cells including a novel
C/EBP target, the p35 subunit of IL-12. Several genes were, in
contrast, more efficiently activated in the absence of C/EBP, thus
underlying the complexity of the role played by this factor in
controlling transcription from different promoters.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Primary spleen cultures were derived from
C/EBP-/- or C/EBP+/+ littermate mice of
a mixed genetic background (16), plated on Petri dishes in
RPMI 1640 medium supplemented with 10% heat-inactivated FCS and
infected with fresh supernatant harvested from subconfluent N-11 cells
as described (20). The resulting macrophage cultures were
cloned by limiting dilution. Southern blot analysis was as previously
described (16). Cells were stimulated with 100 U/ml of
IFN- (kindly provided by G. Garotta, Ares-Serono, Geneva,
Switzerland) and with 100 ng/ml of LPS (Escherichia coli
serotype 026:B6; Sigma-Aldrich, Poole, U.K.) for the times
indicated.
Resident peritoneal macrophages and bone marrow cells were collected
from C/EBP-/- and C/EBP+/+ mice as
described (21) and treated with 100 U/ml of IFN- for
16 h, followed by 1 µg/ml of LPS for 4 h before RNA
extraction.
To generate the revertant cells, a plasmid containing a 4.8-kb C/EBP
genomic fragment was coelectroporated into 107 K4
cells with the plasmid pZeo SV2+ (Invitrogen,
Groningen, The Netherlands). Resistant colonies were pooled and cloned
by limiting dilution.
RNA extraction, Northern and slot blot analysis, semiquantitative RT-PCR
Total RNA was prepared using the RNeasy Midi kit (Qiagen, Crawley, U.K.). Total RNA (20 or 5 µg) was analyzed by Northern blot or slot blot, respectively, as previously described (22). The signals were quantified by phosphorimager analysis and normalized to GAPDH as an internal control.
For semiquantitative RT-PCR, cDNA was synthesized using the reverse transcription system and oligo(dT) from Promega (Madison, WI) and amplified with specific oligonucleotides (23). PCR products were stained with SYBR Gold nucleic acid gel stain (Molecular Probes, Leiden, The Netherlands) and quantified by phosphorimager analysis.
Nuclear extracts and Western blot analysis
Nuclear extracts were prepared as previously described
(24) and snap frozen at -80°C. In Western blot
experiments, 50 µg were used with anti-C/EBP (14AA),
anti-C/EBP (C-19), anti-C/EBP
(C-22), and
anti-C/EBP
(C-22) rabbit polyclonal Abs (Santa Cruz
Biotechnology, Santa Cruz, CA).
Flow cytometry analysis
Live cells (3 x 105) were incubated
with the Abs in PBS supplemented with 1% FCS and 0.02% sodium azide
for 1 h on ice, washed, and incubated with a FITC-conjugated
F(ab') goat anti-rat IgG (Jackson ImmunoResearch
Laboratories, West Grove, PA). MHC class II molecules were directly
detected with a FITC-conjugated mouse anti-mouse
I-Ab mAb (BD PharMingen, San Diego, CA). Rat
anti-mouse MOMA-2 was purchased from Serotec (Oxford, U.K.).
F4/80 (25), Mac1 (26), Mac2
(27), scavenger receptor (28), FcRII
(29), sialoadhesin (30), MOMA-1
(31), and macrosialin (32) rat anti-mouse
Abs were kindly provided by Dr. P. Crocker (Dundee, U.K.). For CD11c
detection, biotinylated N418 Ab (33) was kindly provided
by Dr. C. Watts (Dundee, U.K.) and was detected with FITC-conjugated
streptavidin (BD PharMingen).
Nuclear run-on assays
A total of 60 x 106
cells/sample were harvested and lysed as for nuclear protein extraction
(see Nuclear extracts and Western blot analysis).
Sucrose was added to a final concentration of 1.5 M and nuclei were
collected by ultracentrifugation over a 2 M sucrose cushion,
resuspended in nuclear freezing buffer (34) and stored at
-80°C until use. Nuclear run-on was performed essentially as
described (34). Equal amounts of radioactivity (
5
x 106 cpm/ml) were used to hybridize dot blots
containing 10 µg of linear plasmid cDNA for at least 36 h. Blots
were visualized by autoradiography, quantified using a phosphorimager
and values were normalized to GAPDH values.
Statistical analysis
Results were analyzed by the Analysis of Variable Test using the Statview computer program (Abacus Concepts, Berkeley, CA). A p value of <0.05 was considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-deficient and
sufficient macrophage cell lines.
Primary splenic cultures from C/EBP-/-
mice and littermate wild-type controls (16) were
immortalized by infection with VN-11 (20) followed by
limiting dilution cloning. Individual mutant (K1, K3, K4, K7) and
wild-type (W2, W3) clones were expanded and clone W3 was further
subcloned into subclones W3B and W3E. The genotypes were confirmed by
genomic Southern blot as described (16) (Fig. 1
A). Characterization of the
insertion point by Southern blot analysis using a c-myc
probe indicated that, with the exception of K3 and K1 that displayed a
similar pattern, all W and K clones arose from independent insertion
events (data not shown).
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and LPS was analyzed by Northern blot with a
C/EBP cDNA probe. C/EBP mRNA was already present in untreated
wild-type cells and was induced at similar levels in all clones
analyzed while no signal was detected in the mutant clones (Fig. 1
B). Comparable induction was obtained with LPS alone, while
treatment with IFN- only elicited almost no mRNA increase (data not
shown). Nuclear protein extracts from two representative
C/EBP-/- or C/EBP+/+ clones (K4 and W2)
were then analyzed by Western blot to detect the levels of different
C/EBP family members (Fig. 2A). A clear inducible signal
for C/EBP was detected in extracts from the wild-type cells only.
C/EBP was abundantly present in both cell types and similarly
decreased upon IFN-/LPS treatment. The levels of C/EBP and
were similar in K4 and W2 cells before treatment. Both proteins
appeared to be induced in the treated mutant cells while very poor
induction was detected in the wild-type cell line, thus raising
the possibility that compensatory induction of C/EBP and might
take place in the absence of C/EBP. Moreover, as C/EBP is known
to be induced by LPS in several monocyte and macrophage cells lines
(35), failure to detect its activation in our wild-type
cells might imply an intrinsic defect in these cells. Therefore, we
analyzed C/EBP and mRNA levels in primary bone marrow-derived
(BMM) and peritoneal macrophages (PM). To our surprise, we could not
detect any induction of the C/EBP mRNA in either cell type,
suggesting that perhaps in these primary cells C/EBP mRNA is not
regulated by IFN-/LPS (Fig. 2B). A modest induction was
in contrast detected in the mutant cells, where upon treatment the net
levels of C/EBP mRNA were significantly higher in both BMM and PM
derived from the mutant mice as compared with the wild types. C/EBP
mRNA levels were not induced by IFN-/LPS treatment and were in
general very low, in agreement with the knowledge that this family
member is primarily expressed in granulocytic populations (36, 37). However, interestingly, C/EBP-deficient PM displayed
significantly higher levels of C/EBP mRNA than their wild-type
counterparts. These results indicate that indeed both C/EBP and
C/EBP can be up-regulated in both immortalized and primary
C/EBP-/- macrophages, likely acting to
partially compensate for C/EBP functions.
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into the mutant cells to generate a revertant
cell line was achieved by stably transfecting a mouse C/EBP genomic
clone known to confer correct regulation in transgenic mice
(38), as several attempts at using strong viral promoters
had failed. Although even the best-expressing clone
(r-/- clone 16) featured only partially rescued
C/EBP expression at both the mRNA and protein level (Fig. 1C and data not shown), it was nevertheless included as a
control in most of the following experiments.
Characterization of the C/EBP-/-,
r-/- and C/EBP+/+ cell lines as
macrophages
To establish whether the isolated cell lines displayed macrophage
phenotype, all four C/EBP-/- clones, the
r-/- cells and one wild-type clone (W2) were
subjected to cytofluorimetric analysis. Data from representative
experiments are shown for K4, W2, and
r-/- cells in Fig. 3. Table I summarizes the results obtained with
all clones and all markers analyzed. All cells were positive for the
macrophage markers F4/80, Mac1, Mac2, FcRII, scavenger receptor,
MOMA-2, macrosialin, and CD11c (Fig. 3 and Table I), while MOMA-1, a
marker present only on certain macrophage subsets, was negative in all
cells. The levels of Mac2 and FcRII were slightly lower in the
mutant cells. Although this might be the result of reduced gene
expression in the absence of C/EBP, at present we cannot rule out
differential staining efficiency or other nonspecific phenomena. MHC
class II expression was constitutive in the wild-type cells and could
be slightly further induced by IFN- treatment. In contrast, it was
barely detectable in the untreated C/EBP-/-
cell lines where it could be strongly induced by IFN-, similarly to
what was previously reported for VN-11-immortalized macrophages
(39). The revertant r-/- cells
displayed an intermediate phenotype, with appreciable MHC class II
expression in basal conditions and induced levels comparable to those
of both -/- and +/+ cells. CD11c was, in contrast, already expressed
in untreated cells and it was not further induced by IFN-. This is
in agreement with a macrophage phenotype, because this marker is
inducible only in dendritic cells (40).
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Differential gene expression in +/+ vs -/- macrophages
To characterize the expression of a representative panel of
candidate C/EBP target genes, total RNA extracted from the
C/EBP-/-, C/EBP+/+, and revertant
cells, either untreated or treated with IFN- and LPS, was subjected
to slot blot analysis using cDNA probes corresponding to the indicated
genes (Fig. 4). Although only the data
for K4, W2, and r-/- cells are shown, the
cytokine mRNA levels were analyzed with similar results in all clones
(data not shown). Among the RNAs analyzed, lysozyme, M-CSF, MIP-1
(Fig. 4), scavenger receptor, and M-CSFR (data not shown) were
expressed at similar levels in the three lines, thus suggesting that
these genes do not require C/EBP for their transcription. The RNAs
corresponding to the inflammatory cytokines IL-1, TNF-, and IL-6
as well as those encoding iNOS and plasminogen activator inhibitor
(PAI-1) were considerably less induced in the absence of C/EBP. Only
partial rescue was obtained by expression of C/EBP in the revertant
cells. Unexpectedly another group of genes, IL-12 p40,
RANTES, and MIP-1, appeared to be up-regulated
in the mutant cells. In this case, expression of C/EBP in the
revertant cells was sufficient to completely normalize the mRNA
levels.
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priming, suggesting differential regulatory mechanisms (Fig. 5). As expected, IFN- pretreatment
strongly enhanced both iNOS and TNF- mRNA induction. However, while
TNF- induction was similarly impaired in the mutant cells under both
conditions, it was mainly the further increase elicited by IFN-
priming that was defective in the case of iNOS mRNA. IL-1 and IL-6
were efficiently induced by both LPS alone and IFN-/LPS and
similarly impaired in the absence of C/EBP. LPS alone was a more
potent inducer of RANTES and MIP1 mRNAs than LPS/IFN-. The
IFN--induced inhibition appeared to be mediated by C/EBP because
little or no decrease was detected in the mutant cells. IL-12 p40
induction was reported to be increased by IFN- only at short times
after LPS treatment, while at longer times it was inhibited
(41). In our wild-type cells, LPS alone did induce p40
mRNA more efficiently that LPS/IFN- 8 h after treatment and, in
contrast to what was observed upon IFN- priming, no significant
up-regulation was observed in the mutant cells. Therefore, C/EBP
appeared to be involved in the IFN--mediated down-regulation of
p40.
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). C/EBP transcription was induced by
2.5- to 3-fold upon IFN-/LPS treatment of wild-type cells.
Transcription of the RNAs corresponding to TNF- and iNOS was less
induced in the absence of C/EBP, while it was induced at higher
levels in the case of IL-12 p40, RANTES, and MIP1. Therefore, the
defective mRNA induction of these genes in the mutant cells can be
accounted for at least partly by impaired transcription of the
corresponding genes. In contrast, we could not detect differential
transcription in the case of IL-6 and PAI-1. Altered mRNA levels may
also be due to differential mRNA stability. However, we observed that
the stability of the IL-6, TNF-, and IL-12 p40 mRNAs was comparable
between the C/EBP-/- and C/EBP+/+ cells
(data not shown). Therefore, it is not clear why we did not obtain
differential transcription rates in the case of IL-6.
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To further validate our system, we assessed gene expression
in primary BMM and PM from C/EBP-/- and
C/EBP+/+ mice. Cells were treated with IFN-/LPS and
total RNA was extracted and analyzed by either slot blot (BMM, Fig. 7A) or semiquantitative RT-PCR
(PM, Fig. 7B). Among the genes analyzed, IL-1 mRNA was
less induced in C/EBP-/- PM but not BMM,
where its induction was very weak in the wild-type cells as well. This
might reflect intrinsic differences between the two populations that
were not further investigated. PAI-1 and RANTES mRNA levels were
comparable in both BMM and PM from C/EBP-/- and
C/EBP+/+ mice, thus suggesting no real dependence on
C/EBP. In contrast, TNF-, IL-6, iNOS, IL-12 p40, and MIP1
mRNAs were expressed differentially in C/EBP-/- and
C/EBP+/+ primary macrophages. The differences correlated
very closely with those detected in the cell lines, thus confirming
that C/EBP is an important player in regulating the induction of
these genes.
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The finding that IL-12 p40 mRNA was more efficiently induced in
both immortalized and primary C/EBP-/-
macrophages was surprising, as previous work had shown that
C/EBP-/- mice displayed defective production
of bioactive IL-12 (16). Because bioactive IL-12 is made
of two subunits encoded by two distinct genes, p40 and
p35 (42, 43), we attempted to assess the levels
of p35 mRNA in the immortalized cell lines but failed to detect any
signal even by RT-PCR (data not shown). However, analysis of the RNA
from primary macrophages showed that IL12 p35 mRNA, not detectable in
untreated cells, was strongly induced by IFN-/LPS treatment in
macrophages derived from the wild-type mice (Fig. 7C). In
contrast, no signal was ever detected in either BMM or PM derived from
the mutant mice. This result identifies for the first time the
IL-12 p35 gene as a C/EBP target, and may explain the
defective production of bioactive IL-12 in the mutant mice even in the
presence of abundant p40 subunits.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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/LPS and Ag presentation. The immortalized
C/EBP-/- and C/EBP+/+ macrophages
display indeed all markers of bona fide macrophages and are fully able
to respond to LPS. It might be argued that the analysis of only a few
independent clones cannot account for the complexity of functionally
heterogeneous macrophage populations, and that lineage, rather than
genotype differences, might generate the distinct phenotypes detected
in wild-type and mutant cell lines. However, the gene expression
profiles of the mutant and wild-type cell lines correlated well with
those of two different primary macrophage populations derived from
C/EBP-deficient and wild-type mice. This indicates that, although
clearly not representative of all possible macrophage populations, at
least for the features analyzed, the differences between K and W cells
lines truly reflect physiological differences between wild-type and
mutant macrophages. Moreover, even low expression of C/EBP in the
revertant cells could partially or totally re-establish wild-type-like
gene expression, a further indication that the phenotype described is
really due to the absence of this factor. These cells are amenable to
transient transfections (24) and can be easily maintained
in culture, thus representing a useful tool to study in detail the
molecular mechanisms through which C/EBP and other members of the
C/EBP family regulate transcription of different responsive genes in
macrophages.
Our results show how several genes that had been reported to be induced
by C/EBP do not require this factor for their expression
(lysozyme, MIP1, M-CSF,
M-CSFR, and scavenger receptor). This observation
underscores the importance of gene targeting experiments to confirm
whether specific transcription factors are physiologically involved in
the regulation of distinct gene promoters. Moreover, it indicates that
the C/EBP-/- cells maintain an intact
responsiveness to LPS, as also confirmed by our finding that both
NF-
B activation and CREB phosphorylation induced by LPS were normal
in the mutant cells (24, 44).
Only two genes whose expression is almost totally abolished in primary
macrophages derived from C/EBP-/- mice had
so far been identified, G-CSF and Mincle
(17, 19). However, impaired cellular functions are not
necessarily caused by totally defective gene expression. Our results
indicate that the expression of IL-1, TNF-,
IL-6, and iNOS, all genes involved in
differentiated macrophage functions, is partially impaired in
C/EBP-/- macrophages and that expression of
IL-12 p35 is completely defective. In addition, we have subsequently
found that expression of the COX-2 gene and production of
PGE2 is profoundly impaired in the mutant cells (24). In
contrast, several genes (IL-12 p40, RANTES, and
MIP1) are more efficiently induced in response to IFN-
and LPS in the absence of C/EBP. The effects of the absence of
C/EBP on the different genes analyzed varied remarkably. For
example, both TNF- and iNOS required IFN- priming for full
activation in the wild-type cells. However, while TNF- expression
was defective in the mutant cells under both conditions, iNOS levels
were equivalent in the two cell types in response to LPS alone but
failed to be further up-regulated by IFN- priming in the absence of
C/EBP. Of the other genes analyzed, IL-1 and IL-6 induction was
equally affected in the mutant cells in response to both LPS alone or
IFN-/LPS, while failure to undergo IFN--mediated down-regulation
appeared to be responsible for the up-regulation of IL-12 p40, RANTES,
and MIP1 mRNAs. Taken together, these data are in agreement with the
highly differential composition of macrophage-specific promoters that
emerges clearly from functional studies (5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15).
Consequently, the specific role of C/EBP in regulating transcription
of the different genes will vary according to specific promoter
composition and relative affinity for specific sites of different C/EBP
homo- or heterodimers. Moreover, although all genes analyzed in this
study have been reported to carry functional C/EBP sites on their
promoters, their defective expression in the mutant cells might also be
caused indirectly by the absence of C/EBP, for example through
alteration of the levels or activities of distinct transcription
factors. Defective induction of most genes analyzed was more pronounced
at later time points (4 and 8 h after treatment), in agreement
with the 4-h peak of C/EBP induction detected in our cells. However,
C/EBP is already abundantly present before any treatment, and we
have recently shown that it is directly required for transcriptional
induction of the COX-2 promoter even in the early phases (i.e., 1
h after treatment), before significant accumulation of newly
synthesized factors can occur (44). Further studies
analyzing the transcriptional activity of individual promoters in the
mutant cells as well as the occupancy of distinct regulatory sites by
the different C/EBP family members will be instrumental in clarifying
the molecular mechanisms underlying positive and negative regulation by
C/EBP.
Of particular interest was the observation that transcription of the
IL-12 p35 and p40 genes appears to be inversely
regulated by C/EBP. p40 was initially thought to be the regulated
subunit of IL-12 (45). However, more recent work has
demonstrated that the synthesis of p35 mRNA is also highly regulated
and that p35, being much less abundant than p40, is actually the
rate-limiting factor in the production of the heterodimer (41, 46, 47). The murine p35 promoter contains several putative C/EBP
sites (48). Recently, transcription of the p35
gene in CD8+ dendritic cells has been shown to
require the NF-B family member c-Rel (47). Our finding,
that in macrophages p35 requires C/EBP for its
transcriptional induction, extends the understanding of p35
gene regulation and identifies a novel important target gene for
C/EBP. The dependence of p35 from C/EBP may explain
the defective production of circulating bioactive IL-12 in C.
albicans-infected C/EBP-/- mice,
providing a molecular mechanism for the defective Th1 responses
occurring in these mice (16). In contrast to p35, p40 was
more efficiently transcribed in the mutant cells. Because the p40
homodimer is known to act as a receptor antagonist (49),
the inverse regulation of p35 and p40 production might represent a
distinct mean of regulating IL-12 bioactivity by C/EBP. This factor
might, on one side, stimulate the production of the bioactive
heterodimer by up-regulating p35 transcription while at the same time
contributing to maintain p40 transcription within levels compatible
with low homodimer production. A similar reciprocal regulatory
mechanism for IL-12 biosynthesis has been proposed to occur in
dendritic cells in response to IL-4 (50). Interestingly,
p40 has been recently shown to dimerize with a novel cytokine named p19
(51). This novel heterodimer (p60 or IL-23) displays both
overlapping and unique functions with respect to IL-12. It will be
interesting to analyze the consequences of higher p40 production on the
synthesis of bioactive p60/IL-23 in the
C/EBP-/- mice.
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Acknowledgments |
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Footnotes |
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2 Current address: MRC HGU, Western General Hospital, Crewe Road, EH4 2XU, Edinburgh, U.K.
3 Current address: Department of Genetics, Biology, and Biochemistry, University of Turin, 10126 Turin, Italy.
4 Current address: Department of Medical Pharmacology, University of Milan, 20122 Milan, Italy.
5 Address correspondence and reprint requests to Dr. Valeria Poli, School of Life Sciences, Wellcome Trust Biocentre, University of Dundee, Dow Street, Dundee DD1 5EH Scotland. E-mail address: v.poli{at}dundee.ac.uk
6 Abbreviations used in this paper: MIP, macrophage inflammatory protein; iNOS, inducible NO synthase; BMM, bone marrow-derived macrophage; PM, peritoneal macrophage; PAI, plasminogen activator inhibitor; COX, cyclooxygenase.
Received for publication July 2, 2001. Accepted for publication February 13, 2002.
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References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
B synergistically activate transcription of the inflammatory cytokines, interleukin 6 and interleukin 8. Proc. Natl. Acad. Sci. USA 90:10193. regulation of the tumor necrosis factor gene. J. Clin. Invest. 94:1449.
(IL-1) gene by mycobacterial components and lipopolysaccharide is mediated by two nuclear factor-IL6 motifs. Mol. Cell. Biol. 13:3831.B and NF-IL6 binding elements in the TNF response regions of the G-CSF promoter. Blood 83:2469., osteopontin and CD14 in a monocytic leukemia cell line. Int. Immunol. 10:1825. and lipopolysaccharide. Proc. Natl. Acad. Sci. USA 90:9730.-deficient mice. EMBO J. 14:1932.[Medline]
is essential for inducible expression of the cox-2 gene in macrophages but not in fibroblasts. J. Biol. Chem. 276:40769. receptor monoclonal Ab. J. Exp. Med. 164:474., -, and - in supporting the lipopolysaccharide-induced transcription of IL-6 and monocyte chemoattractant protein-1. J. Immunol. 160:2334. is preferentially up-regulated during granulocytic differentiation and its functional versatility is determined by alternative use of promoters and differential splicing. Proc. Natl. Acad. Sci. USA 94:6462., is upregulated during granulocytic, but not monocytic differentiation. Blood 90:2591.) gene can direct high-level, position-independent, copy number-dependent expression in multiple tissues in transgenic mice. Nucleic Acids Res. 22:756. of lipopolysaccharide-inducible p35 and p40 genes. Blood 86:646. (C/EBP) and C/EBP transcription factors. J. Biol. Chem. 276:48693., and IL-1 production in human myeloid leukemia cell lines and peripheral blood mononuclear cells. Blood 83:1847.This article has been cited by other articles:
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 J. Worm, J. Stenvang, A. Petri, K. S. Frederiksen, S. Obad, J. Elmen, M. Hedtjarn, E. M. Straarup, J. B. Hansen, and S. Kauppinen Silencing of microRNA-155 in mice during acute inflammatory response leads to derepression of c/ebp Beta and down-regulation of G-CSF Nucleic Acids Res., September 1, 2009; 37(17): 5784 - 5792. [Abstract] [Full Text] [PDF]
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 Y.-C. Lu, I. Kim, E. Lye, F. Shen, N. Suzuki, S. Suzuki, S. Gerondakis, S. Akira, S. L. Gaffen, W.-C. Yeh, et al. Differential Role for c-Rel and C/EBP{beta}/{delta} in TLR-Mediated Induction of Proinflammatory Cytokines J. Immunol., June 1, 2009; 182(11): 7212 - 7221. [Abstract] [Full Text] [PDF]
|
|
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|
C. A. Wells, J. A. Salvage-Jones, X. Li, K. Hitchens, S. Butcher, R. Z. Murray, A. G. Beckhouse, Y.-L.-S. Lo, S. Manzanero, C. Cobbold, et al. The Macrophage-Inducible C-Type Lectin, Mincle, Is an Essential Component of the Innate Immune Response to Candida albicans J. Immunol., June 1, 2008; 180(11): 7404 - 7413. [Abstract] [Full Text] [PDF]
|
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 |
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 H. Li, P. Gade, S. C. Nallar, A. Raha, S. K. Roy, S. Karra, J. K. Reddy, S. P. Reddy, and D. V. Kalvakolanu The Med1 Subunit of Transcriptional Mediator Plays a Central Role in Regulating CCAAT/Enhancer-binding Protein-{beta}-driven Transcription in Response to Interferon-{gamma} J. Biol. Chem., May 9, 2008; 283(19): 13077 - 13086. [Abstract] [Full Text] [PDF]
|
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 T. Akagi, T. Saitoh, J. O'Kelly, S. Akira, A. F. Gombart, and H. P. Koeffler Impaired response to GM-CSF and G-CSF, and enhanced apoptosis in C/EBP{beta}-deficient hematopoietic cells Blood, March 15, 2008; 111(6): 2999 - 3004. [Abstract] [Full Text] [PDF]
|
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M. E. Pennini, Y. Liu, J. Yang, C. M. Croniger, W. H. Boom, and C. V. Harding CCAAT/Enhancer-Binding Protein beta and {delta} Binding to CIITA Promoters Is Associated with the Inhibition of CIITA Expression in Response to Mycobacterium tuberculosis 19-kDa Lipoprotein J. Immunol., November 15, 2007; 179(10): 6910 - 6918. [Abstract] [Full Text] [PDF]
|
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S. Uematsu, T. Kaisho, T. Tanaka, M. Matsumoto, M. Yamakami, H. Omori, M. Yamamoto, T. Yoshimori, and S. Akira The C/EBPbeta Isoform 34-kDa LAP Is Responsible for NF-IL-6-Mediated Gene Induction in Activated Macrophages, but Is Not Essential for Intracellular Bacteria Killing J. Immunol., October 15, 2007; 179(8): 5378 - 5386. [Abstract] [Full Text] [PDF]
|
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|
A. Schwegmann, R. Guler, A. J. Cutler, B. Arendse, W. G. C. Horsnell, A. Flemming, A. H. Kottmann, G. Ryan, W. Hide, M. Leitges, et al. Protein kinase C {delta} is essential for optimal macrophage-mediated phagosomal containment of Listeria monocytogenes PNAS, October 9, 2007; 104(41): 16251 - 16256. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Csoka, Z. H. Nemeth, L. Virag, P. Gergely, S. J. Leibovich, P. Pacher, C.-X. Sun, M. R. Blackburn, E. S. Vizi, E. A. Deitch, et al. A2A adenosine receptors and C/EBP{beta} are crucially required for IL-10 production by macrophages exposed to Escherichia coli Blood, October 1, 2007; 110(7): 2685 - 2695. [Abstract] [Full Text] [PDF]
|
|
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 |
|
 M. Yamamoto, S. Uematsu, T. Okamoto, Y. Matsuura, S. Sato, H. Kumar, T. Satoh, T. Saitoh, K. Takeda, K. J. Ishii, et al. Enhanced TLR-mediated NF-IL6-dependent gene expression by Trib1 deficiency J. Exp. Med., September 3, 2007; 204(9): 2233 - 2239. [Abstract] [Full Text] [PDF]
|
|
|
|
|
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T.-H. Pham, S. Langmann, L. Schwarzfischer, C. El Chartouni, M. Lichtinger, M. Klug, S. W. Krause, and M. Rehli CCAAT Enhancer-binding Protein beta Regulates Constitutive Gene Expression during Late Stages of Monocyte to Macrophage Differentiation J. Biol. Chem., July 27, 2007; 282(30): 21924 - 21933. [Abstract] [Full Text] [PDF]
|
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|
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A. Zwergal, M. Quirling, B. Saugel, K. C. Huth, C. Sydlik, V. Poli, D. Neumeier, H. W. L. Ziegler-Heitbrock, and K. Brand C/EBPbeta Blocks p65 Phosphorylation and Thereby NF-{kappa}B-Mediated Transcription in TNF-Tolerant Cells J. Immunol., July 1, 2006; 177(1): 665 - 672. [Abstract] [Full Text] [PDF]
|
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T. Ito, H. Kanzler, O. Duramad, W. Cao, and Y.-J. Liu Specialization, kinetics, and repertoire of type 1 interferon responses by human plasmacytoid predendritic cells Blood, March 15, 2006; 107(6): 2423 - 2431. [Abstract] [Full Text] [PDF]
|
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 P. Mukhopadhyay, M. A. Ali, A. Nandi, P. Carreon, H. Choy, and D. Saha The Cyclin-Dependent Kinase 2 Inhibitor Down-regulates Interleukin-1{beta}-Mediated Induction of Cyclooxygenase-2 Expression in Human Lung Carcinoma Cells Cancer Res., February 1, 2006; 66(3): 1758 - 1766. [Abstract] [Full Text] [PDF]
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 A. S. Dusso, A. J. Brown, and E. Slatopolsky Vitamin D Am J Physiol Renal Physiol, July 1, 2005; 289(1): F8 - F28. [Abstract] [Full Text] [PDF]
|
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Q. Meng, A. Raha, S. Roy, J. Hu, and D. V. Kalvakolanu IFN-{gamma}-Stimulated Transcriptional Activation by IFN-{gamma}-Activated Transcriptional Element-Binding Factor 1 Occurs via an Inducible Interaction with CAAAT/Enhancer-Binding Protein-{beta} J. Immunol., May 15, 2005; 174(10): 6203 - 6211. [Abstract] [Full Text] [PDF]
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 M. N. Bradley, L. Zhou, and S. T. Smale C/EBP{beta} Regulation in Lipopolysaccharide-Stimulated Macrophages Mol. Cell. Biol., July 15, 2003; 23(14): 4841 - 4858. [Abstract] [Full Text] [PDF]
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, statistically
significant difference between untreated and treated -/- cells
(p < 0.05). u/t, untreated; T, IFN-
