(Received for publication, May 23, 1997)
From the Department of Internal Medicine, Pulmonary and Critical Care Section, Yale University School of Medicine, New Haven, Connecticut 06520
We previously reported the cloning of a cDNA
for IBR (for I
B-related) from human lung alveolar epithelial
cells. I
BR is related to the I
B proteins that function as
regulators of the NF-
B family of transcription factors. Here, we
investigated the consequence of I
BR overexpression on the expression
of NF-
B-regulated chemokine genes in lung alveolar epithelial cells.
Chemokines play an important role in many inflammatory diseases such as
asthma and AIDS. Overexpression of I
BR in the lung cells resulted in a rapid 50-100-fold greater production of the RANTES
(regulated upon activation, normal
T expressed and presumably
secreted) protein upon cytokine-induction compared with
control cells. I
BR overexpression, however, did not enhance
interleukin-8 or MIP-1
gene expression, despite the fact that the
expression of all three chemokine genes are regulated by NF-
B. The
up-regulation of RANTES gene expression resulting from overexpression
of I
BR correlated with increased amounts of a unique RANTES-
B
binding activity and decreased binding of p50 homodimers. Previous
studies have shown that p50 homodimers function as repressors of
certain
B sites. Our studies suggest that I
BR can aid activation
of select NF-
B-regulated genes by sequestering p50
homodimers.
We previously described the cloning of a cDNA for IBR (for
I
B-related) from human lung alveolar epithelial cells (1). I
BR
belongs to the I
B family of proteins that function as cytoplasmic retention proteins for the Rel-NF-
B transcription factors and serve
to regulate their functions (2, 3). Several members of the I
B family
have now been identified that, in vertebrates, include I
B
,
I
B
, I
B
, I
BR, Bcl-3, p105, and p100 (reviewed in Refs. 2
and 3). So far, five members of the NF-
B family have been described
that are c-Rel, NF-
B1 (p50), NF-
B2 (p52), RelA (p65), and RelB
(reviewed in Refs. 4-6). Most members of the NF-
B family can form
homo- or heterodimers (except for RelB, which only forms heterodimers
with p50 or p52) that bind slightly different
B motifs (2-6). The
existence of multiple homo- and heterodimeric forms of NF-
B-Rel
proteins in a given cell type requires tight regulation of their
activity that appears to be achieved via differential interactions with
different I
B proteins. For example, both I
B
and I
B
inhibit the DNA binding activity of p50-p65 heterodimers but not that
of the p50 homodimer (2, 3). Bcl-3, on the other hand, interacts with
(p50)2 or (p52)2 and has been reported to
behave as a transactivator (2, 3). Because gene knockout studies
indicate that the different NF-
B-Rel proteins are not functionally
redundant (2-6), it may be speculated that the I
B proteins, which
regulate their functions, also play distinct roles in the cell.
NF-B proteins control the expression of multiple genes involved in
inflammatory processes such as those encoding pro-inflammatory cytokines and chemokines (4-6). Chemokines are low molecular weight
proteins secreted in response to inflammatory cytokines and play a
critical role in leukocyte trafficking. Recently, the C-C chemokines
RANTES, MIP1
, and MIP1
were found to suppress HIV-11 infection (7, 8). The
suppressive activity of MIP1
, MIP1
, and RANTES for HIV-1
infection combined with the identification of CCR5 (the cell surface
receptor for these chemokines) as a coreceptor for HIV-1 entry (9-13)
has led to the proposal that these chemokines may have therapeutic
benefits in blocking or delaying virus infection.
RANTES, originally isolated as a cDNA from CD8+ T cells (14), also
plays an important role in the chemotaxis of cells such as eosinophils
(15, 16), mast cells (17, 18), and CD45 RO+ memory T cells
(19), which predominate in allergic inflammation. In humans, an
up-regulation of RANTES message was observed in the airways of
asthmatics (20-22) and in a local endobronchial allergen challenge
model, a significant correlation between RANTES concentration and
eosinophil number was observed at the challenge site (23). In a murine
model of endotoxemia, significant levels of RANTES mRNA and protein
were detected in lung homogenates of mice following intraperitoneal
administration of endotoxin and RANTES mRNA was detected as early
as 1-2 h post endotoxin stimulation in the lung (24). Furthermore, the
RANTES protein was immunolocalized to the alveolar epithelium in this
setting and was implicated in macrophage recruitment in the lungs (24).
In other studies, the cytokine TNF was shown to induce RANTES
mRNA at 16 h post-stimulation in lung epithelial cells (25).
Although lung-derived RANTES has been implicated in the recruitment of
eosinophils in the lungs of asthmatics, little is known at the
molecular level about mechanisms that control RANTES gene expression in
lung cells. Clearly, an understanding of such mechanisms could be
useful in the design of drugs to combat asthma on the one hand and for
developing strategies for suppressing HIV-1 infection via
overproduction of RANTES on the other.
Here we show that overexpression of IBR can selectively up-regulate
cytokine-induced expression of the RANTES gene in lung epithelial
cells. Furthermore, this up-regulation of RANTES gene expression is
associated with increased binding of a unique NF-
B complex to the
RANTES
B region and decreased binding of p50 homodimers.
The oligonucleotide
GACTACAAGGACGACGATGACAAA encoding the FLAG octapeptide epitope
(DYKDDDDK; IBI/Kodak) was inserted immediately downstream of the codon
for amino acid 479 of IBR using polymerase chain reaction
techniques. The FLAG epitope-tagged I
BR cDNA was subcloned into
pcDNA3 (Invitrogen) for expression under the control of the
cytomegalovirus promoter in eukaryotic cells and for in vitro transcription with T7 polymerase. The cloning was confirmed by coupled in vitro transcription-translation reactions
using T7 polymerase and the TNT rabbit reticulocyte system (Promega). The in vitro translated product was immunoprecipitated with
anti-FLAG monoclonal Ab (M2; IBI/Kodak) and analyzed by SDS-PAGE and
autoradiography. This plasmid and the insert-free vector pcDNA3
were subsequently used to generate the stable transfectants A549/I
BR
and A549/vector, respectively. Briefly, A549 cells (ATCC) were
maintained in Dulbecco's modified Eagle's medium/Ham's F-12 medium
(Life Technologies, Inc.) supplemented with 10% fetal bovine serum
(Life Technologies, Inc.). Cells were transfected using the
calcium-phosphate coprecipitation procedure (26), and transfected cells
were selected by using 500 µg/ml of active G418 (Life Technologies,
Inc.) in culture medium 48 h after transfection. G418-resistant
clones were picked after 2 weeks, and pooled clones were used for all
analyses.
For transient transfection
assays, cells were transfected by the the calcium-phosphate
coprecipitation procedure (26). The wt RANTES promoter-reporter
(luciferase) plasmid contained promoter sequences between 897
(StuI) and +54 (KpnI) (derived from a
961/+54 construct obtained from A. M. Krensky and co-workers (27)), and
the mutant
897/+54 plasmid was constructed by swapping a fragment
(
181/+54) containing mutations in the A and B NF-
B sites (28) with
the corresponding wt fragment. 16 h after transfection, cells were
washed, suspended in serum-free medium, and treated with recombinant
hIL-1
(5 ng/ml) or hTNF
(50 ng/ml) and harvested 6 h later
for assaying
-galactosidase and luciferase activity. Luciferase
assays were performed using the Luciferase Assay System of Promega
(Wisconsin), and the luciferase activity was measured in the LB 9501 luminometer of Berthold, Inc. (Germany).
-Galactosidase assays were
performed using chlorophenyl
-D-galactoside as
substrate.
For Northern blot
analyses, cells were either left untreated or treated with recombinant
hIL-1 (5 ng/ml; R & D Systems) or recombinant hTNF
(50 ng/ml;
R & D Systems) for the indicated times in serum-free medium. The
culture supernatants were collected and clarified by centrifugation at
12,000 × g for 10 min, and the supernatants were
stored at
70 °C until further use for protein estimation by ELISA.
Total RNA was prepared from the cell pellets by treatment with Trizol
(Life Technologies, Inc.) according to the instructions of the
manufacturer. Total RNA (15 µg) from each sample was fractionated on
a formaldehyde agarose gel, transferred to nylon membrane, and
cross-linked to the membrane by UV light (Stratalinker;
Stratagene). Prehybridization of the membranes was performed with
QuikHyb (Stratagene) at 50 °C for 2 h. Fragments derived from
the RANTES gene, the IL-8 gene, the I
BR gene and glyceraldehyde
3-phosphate dehydrogenase gene were labeled with [
-32P]dCTP using a Random Primed DNA labeling kit
(Boehringer Mannheim) and used as probes to detect expression of the
corresponding mRNAs. Hybridizations with individual probes were
carried out with ~3 × 106 cpm/ml of hybridization
buffer (QuikHyb). The blots were washed twice for 15 min at room
temperature in 2 × SSC/0.1% SDS and once for 15 min at 42 °C
in 0.2 × SSC/0.1% SDS. The RANTES probe was a 410-base pair
EcoRI/HindIII fragment of the RANTES cDNA
(14), the IL-8 probe was a 250-base pair cDNA fragment (29), and
the MIP1
probe was an EcoRI fragment of the MIP1
cDNA (30).
For the preparation
of nuclear extracts, cells were harvested and suspended in ice-cold
phosphate-buffered saline. The cells were collected by centrifugation
and suspended in ice-cold buffer A (10 mM Hepes, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 1 mM
dithiothreitol, and a mixture of protease inhibitors (Boehringer
Mannheim); 100 µl per 2 × 107 cells). After
incubation of on ice for 10 min, the mixture was diluted with an equal
volume of buffer A containing 0.4% Nonidet P-40 and immediately
centrifuged at 800 × g for 1 min at 4 °C. The
pelleted nuclei were washed in buffer A-Nonidet P-40 and suspended in
50 µl of buffer B (20 mM Hepes, pH 7.9, 420 mM NaCl, 1 mM EDTA, 1 mM
dithiothreitol, 25% glycerol, and a mixture of protease inhibitors). The nuclei were incubated with shaking for 15 min at 4 °C and centrifuged at 14,000 × g at 4 °C. The supernatants
were aliquoted and quickly frozen on dry ice and stored at 70 °C
until further use. The experiments were repeated with different batches
of nuclear extracts to ensure reproducibility of data. Binding
reactions and gel electrophoresis (using 6% native polyacrylamide
gels) were performed as described previously (1).
Cells in 6-cm
plates were washed and starved for 30 min in serum-free Dulbecco's
modified Eagle's medium/Ham's F-12 medium without methionine or
cysteine. Cells were labeled in 2 ml of the same medium with Express
Label (NEN Life Science Products) containing
[35S]methionine and cysteine at 100 µCi/ml for 3 h
at 37 °C. Cells were washed, and 35S-labeled nuclear and
cytoplasmic extracts were prepared. Equal amounts of counts from
nuclear and cytoplasmic fractions were diluted 10-fold in buffer
containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl,
10% glycerol, 0.1% Triton X-100, 0.5 mM EDTA, and a
mixture of protease inhibitors (Boehringer Mannheim). The extracts were then precleared by incubation with fixed Staphylococcus
aureus cells (Zysorbin; Zymed) for 2 h at 4 °C. The
mixtures were centrifuged, and immunoprecipitations were performed with
the supernatants using anti-FLAG Ab M2 coupled Sepharose (IBI/Kodak) or
with Sepharose coupled control IgG1 by overnight incubation at 4 °C.
The beads were washed four times in the same buffer, suspended in
2 × SDS-PAGE sample buffer and analyzed by electrophoresis on
4-20% gradient gels (Bio-Rad). In the sequential
immunoprecipitation-Western blot analyses, unlabeled nuclear and
cytoplasmic extracts were subjected to immunoprecipitations with the
anti-FLAG antibody, and the immunoprecipitates were separated by
SDS-PAGE and transferred to polyvinylidene difluoride membrane. The
membrane was probed with different anti-NF-B antibodies, and
antibody interactions were detected by chemiluminiscence with
horseradish peroxidase-conjugated secondary antibody and ECL reagent
(Amersham Corp.).
To address the effect of IBR on NF-
B-mediated chemokine gene
regulation in lung epithelial cells, we established A549 clones stably
expressing epitope-tagged I
BR, named A549/I
BR. I
BR was epitope-tagged to distinguish the protein expressed from the expression vector from other endogenous I
B proteins. A pool of stable
transfectants were incubated with IL-1
or TNF
for various lengths
of time and analyzed for expression of different chemokine mRNAs by
Northern blotting techniques. In control A549/vector cells, TNF
induced a very low level of RANTES mRNA expression at 24 h
post-stimulation, and the level of induction with IL-1
was barely
detectable even after 24 h (Fig. 1).
In contrast, in A549/I
BR cells, both TNF
and IL-1
caused an
up-regulation of RANTES gene expression as early as 2 h
post-stimulation, which continued to increase even at 24 h after
stimulation. We then tested whether I
BR overexpression specifically
up-regulated RANTES gene expression or the expression of other
NF-
B-regulated genes such as IL-8 (31-35) and MIP-1
(macrophage inflammatory
protein-1
) (36, 37) as well. As illustrated in Fig.
1A, IL-8 gene expression induced by either IL-1
or TNF
was partly inhibited in A549/I
BR cells. We also studied the effect
of I
BR overexpression on the expression of MIP1
, whose induced
expression is barely detectable in A549 cells. Overexpression of I
BR
failed to up-regulate IL-1
- or TNF
-induced MIP1
expression in
these cells (data not shown). We tested the culture supernatants for
the presence of RANTES and IL-8 proteins. As shown in Fig.
1B, the protein data were in good agreement with the
Northern analyses. At 6 h post-stimulation with IL-1
or TNF
, A549/I
BR cells produced 50- and 100-fold more RANTES than the control cells. The results of these experiments showed that I
BR preferentially up-regulated RANTES gene expression in A549 cells.
To determine whether the IBR-mediated up-regulation of
cytokine-induced RANTES mRNA expression was associated with
activation of the RANTES promoter, A549/vector and A549/I
BR cells
were transiently transfected with a reporter gene (luciferase)
construct containing ~900 nucleotides from the 5
-flanking region
immediately upstream of the transcription start site of the RANTES gene
(27, 28). The 5
-flanking region of the RANTES gene contains two
NF-
B-binding sites in tandem, termed A and B (see Fig. 3 and Refs.
27 and 28). In A549/vector cells, the RANTES promoter was activated by
IL-1
or TNF
between 1.5-2-fold (Fig.
2). In contrast, in A549/I
BR cells,
IL-1
and TNF
activated the RANTES promoter between 7-8- and
8-10-fold, respectively (Fig. 2). Mutations in the A and B sites in
the context of the 900-base pair promoter abolished reporter gene
activity in both cell types. The difference between the level of RANTES
mRNA expression and the fold induction of the RANTES promoter in
cytokine-induced A549/I
BR cells can be attributed to the stability
of RANTES mRNA. RANTES mRNA lacks the destabilizing AUUUA
sequence in its 3
-untranslated region (14). This probably explains why
RANTES mRNA induced by TNF
or IFN-
was reduced by only 30%
after incubation for 4 h in the presence of actinomycin D (25).
IL-8, on the other hand has a half-life of less than 1 h in
epithelial cells (38). Because mRNA destabilization appears to play
an insignificant role in the regulation of RANTES production, it stands
to reason that an up-regulation of RANTES promoter activity by IL-1 or
TNF in A549/I
BR cells would cause a significant difference in
steady-state levels of RANTES mRNA and protein levels between the
I
BR-overexpressing cells and the control cells.
We next examined the possibility that the IBR-mediated up-regulation
of RANTES promoter activity was associated with altered binding of
protein(s) to the RANTES
B region. Toward this end, EMSAs were
performed with nuclear fractions of cytokine-induced A549/I
BR cells
and A549/vector cells and a radiolabeled 32-base pair double stranded
oligonucleotide containing the two RANTES NF-
B sites as the probe.
Although IL-1
and TNF
caused induction of NF-
B DNA binding
activity in both cell types, the DNA-protein complexes formed with the
activated proteins were not identical with nuclear extracts prepared
from the two cell types (Fig. 3). As
illustrated in Fig. 3, the binding intensity of complex I was equivalent in the two cell types. The intensity of complex II was
2-3-fold greater with nuclear extracts of A549/vector cells compared
with that observed with A549/I
BR nuclear extracts. However, the
intensity of complex III was at least 10-15-fold (as judged by
densitometric scanning) greater with nuclear extracts of A549/I
BR cells compared with that observed with extracts of control cells (Fig.
3, lower panel). Two additional minor complexes migrating more slowly than complex I were also obtained with extracts of both
cell types. All of these complexes were specific because an excess of
the same unlabeled oligonucleotide efficiently competed for formation
of the complexes, whereas an oligonucleotide containing mutations in
the
B sites failed to compete (Fig. 3, lanes 6 and 7, respectively). Because different members of the
NF-
B-Rel family could potentially interact with the
B motifs, we
used specific antibodies to determine the composition of the different
polypeptide complexes. Formation of complex I was affected with both
anti-p50 and anti-p65 antibodies, suggesting that it contained the
classic p50-p65 heterodimer (lanes 8 and 9).
Complex II and complex III formation were both affected only by the
anti-p50 antibody (lane 8). The complex migrating
immediately after (slower than) complex I was affected only by the
anti-p65 antibody (lane 9). This complex may contain a p65
homodimer. None of the complexes were inhibited/supershifted by the
antibodies against c-Rel, p52, and RelB (lanes 10-12). Also, I
BR itself did not appear to be present in any of the
complexes because a monoclonal antibody against the FLAG epitope (Ab
M2; IBI/Kodak) had no effect on the complexes (lane 13).
The ability of the anti-p50 antibody to affect both complex II and
complex III formation and the selective augmentation of complex III
formation with the A549/IBR nuclear extracts prompted us to further
analyze the nature of both II and III. Toward this end, we first tested
whether recombinant p50 (Promega) could bind to the RANTES
B region.
The recombinant p50 was derived from a full-length cDNA encoding
453 amino acids (39) that was identical to the human p50 cDNA
described previously (40). As shown in Fig.
4A, p50 homodimers bound to
the RANTES
B region. As expected, the (p50)2-DNA complex
could be supershifted with the anti-p50 antibody but not with the
anti-p65 (used as control) antibody (Fig. 4A). Also, in
accordance with previous findings (31, 34), recombinant p50 did not
interact with the IL-8
B site (Fig. 4A). Interestingly,
the (p50)2-RANTES
B complex comigrated with complex II
(compare lanes 1, 4, and 5 in Fig.
4A).
Because both complexes II and III could not contain (p50)2 and the data in Fig. 4A suggested that complex II contained (p50)2, we further investigated the composition of the faster migrating complex III with two different anti-p50 antibodies. Although the first antibody (114x; Santa Cruz Biotech) recognizes sequences mapping within the nuclear localization signal (NLS) region of p50, the second antiserum (1191x; Santa Cruz Biotech) was raised against the NH2-terminal of p50. Both antibodies supershifted the RANTES DNA-(p50)2 complex (Fig. 4B). However, the anti-NLS antibody but not the anti-NH2-terminal antibody supershifted complex III (Fig. 4B). The latter antibody did not inhibit complex I (p50-p65) formation either (Fig. 4B). This was consistent with previous reports showing weaker effects of some p50-specific antisera on p50 heterodimers than on p50 homodimers (32, 41). These results demonstrated that complex III did not consist of p50 homodimers.
Because our Northern analyses and ELISAs showed that IBR
overexpression actually slightly inhibited IL-8 gene expression, we
tested the ability of the IL-8
B site to compete in the EMSA. At a
100-fold molar excess, the wt IL-8
B sequence did compete for
formation of complex III, albeit incompletely (Fig. 4C,
lane 4). The binding of the p50-p65 heterodimer (complex I)
was only partially competed for by the wt IL-8
B sequence (Fig. 4C,
lane 4). This was in accordance with previous reports
showing the preferential binding of p65-c-rel heterodimers rather than
p50-p65 heterodimers to the IL-8
B site (31, 34, 35), and as shown
previously (31, 34), the binding of the p50 homodimers (complex II) was not competed out by the wt IL-8
B sequence. The formation of complex
III with the RANTES probe could not be competed out with 200-fold
excess of oligonucleotides containing consensus binding sequences for a
few additional families of transcription factors, C/EBP, Oct, CREB/ATF,
AP-1, and AP-2 (data not shown). Also, the mobility of complex III is
far lower than what might be expected of an NF-
B-HMG-I(Y) dimeric
complex (42). Taken together, it appears that complex I is a
heterodimer of p50-p65 and complex II contains p50 homodimers. Complex
III may contain a homodimer of a p50-related protein or more likely a
heterodimer containing p50 and an as yet unidentified Rel protein, a
splice variant of a Rel protein, or a non-Rel protein. It is unlikely
that this complex contains any of the known splice variants of p65
(p65
, p65
2, or p65
3), all of which are larger in size than
p50. A TNF
-inducible complex of similar mobility (faster than p50
homodimers) and with reactivity only to anti-p50 antiserum but not to
any other Rel-specific antisera was previously detected in primary murine embryonal fibroblasts (41).
Because the RANTES B site contains two NF-
B sites (A and B) (27,
28), we also examined the involvement of the individual sites for
formation of the different complexes using two mutant probes. One of
these mutants contained a mutant A site and a wt B site (mAwtB),
whereas the other contained a wt A site and a mutant B site (wtAmB). As
shown in Fig. 4D, both probes formed complex I, although the
binding to wtAmB was slightly better. Also, whereas the intensity of
complex III appeared to be the same with either probe, it was less than
that obtained with the complete wt probe (Fig. 4D, compare
lanes 2 and 3 with lane 1). Complex II
containing (p50)2, however, was only formed with the mAwtB
probe (lane 2), whereas the p65 homodimeric complex was formed only with the wtAmB probe (lane 3). Thus, both A and
B sites need to be intact for stabilization of complex III.
Collectively, the EMSAs showed that overexpression of I
BR decreases
formation of complex II but augments formation of complex III.
To determine whether the observed decrease in binding of
(p50)2 to the RANTES probe with nuclear extracts prepared
from A549/IBR cells stemmed from the ability of I
BR to interact
with p50 in vivo, we investigated the subcellular
distribution of I
BR in A549 cells. Untreated A549/I
BR cells
expressing FLAG epitope-tagged I
BR protein were metabolically
labeled with [35S]methionine, and the cell lysates were
fractionated into cytoplasmic and nuclear fractions. The fractions were
subjected to immunoprecipitation with the anti-FLAG monoclonal Ab M2
(IBI/Kodak). Two specific bands were detected on the SDS gel resulting
from immunoprecipitations of the cytoplasmic fraction. The identity of
the upper band was confirmed to be I
BR in several Western blot
experiments using a polyclonal antisereum against I
BR (1). No
specific bands were immunoprecipitated from the nuclear fraction of the
A549/I
BR cells. These results showed that I
BR predominantly
resides in the cytoplasm. To determine the identity of the
coimmunoprecipitated band from the cytoplasmic fraction, we performed
sequential immunoprecipitation-Western blot experiments. In these
experiments, cytoplasmic extracts of untreated A549/I
BR cells were
immunoprecipitated with M2. The immunoprecipitate was divided into
three parts and subjected to Western blot analysis with antibodies
against three different NF-
B-Rel proteins. The results of this
experiment suggested that I
BR preferentially interacts with p50
(Fig. 5B). Use of a different anti-p50 antibody that works well in Western blot analysis (BioMol) also gave identical results (data not shown). A similar
immunoprecipitation-Western blot experiment was also simultaneously
carried out with untransfected A549 cells using a rabbit anti-I
BR
antibody (1). The results were essentially similar except that less p50
was immunoprecipitated from untransfected A549 cells (data not shown).
This was what we expected because the endogenous I
BR is not
expressed in sufficient amounts to sequester all (p50)2,
which is probably why, as all of our data suggest, these cells make
much less RANTES than the I
BR-overexpressing cells.
In our previous report we demonstrated that recombinant IBR inhibits
the DNA binding activity of (p50)2 in EMSAs (1). Our
present data suggest that in intact cells I
BR preferentially interacts with p50. Earlier studies have shown that among the various
NF-
B-Rel dimers, (p50)2 possesses the highest affinity for the NF-
B site present in the Ig
enhancer (43). This probably explains why complex III can only be detected in I
BR-overexpressing cells; by sequestering (p50)2, I
BR causes a shift in the
stoichiometry of complex III to complex II from ~1:2 in control cells
to ~10:1 in I
BR-overexpressing cells (see Figs. 3 and
4A). In control cells, occupancy of the B site in the RANTES
NF-
B region by (p50)2 can be expected to exert a
negative regulatory influence on RANTES gene expression because
(p50)2 has been shown to act as a dominant repressor of
transcription from certain
B sites (44-46). By permitting increased
binding of complex III, I
BR allows the RANTES gene to override the
competitive repressor activity of coexisting (p50)2. This
model is particularly attractive in intact cells where the various
NF-
B dimers are in competition to bind limiting amounts of target
DNA unlike in EMSAs where a large excess of the probe is present. A
similar mechanism, involving a switch from inhibitory p50 homodimers to
p50-p65 activators, was proposed in the induction of IL-2 gene
expression in activated CD4+ T cells (46). In addition to
less competition from inhibitory (p50)2, synergy between
complex III and complex I can also be envisaged as a component in the
up-regulation of RANTES gene expression in A549/I
BR cells. In our
previous studies we showed that I
BR added in vitro to
nuclear extracts inhibited the DNA binding activity of p50-p65
heterodimers in EMSAs. In these studies, nuclear extracts prepared from
control cells and I
BR-overproducing cells displayed similar binding
of the p50-p65 heterodimer to the RANTES
B region. The difference in
the two results can be best explained by the fact that the p50-p65
heterodimer in vivo is normally sequestered by I
B
and
I
B
via the p65 subunit of the complex and therefore unavailable
for interaction with excess I
BR in A549/I
BR cells.
Although IBR overexpression resulted in an up-regulation of RANTES
gene expression, it did not stimulate IL-8 gene expression, which is
also regulated by NF-
B. It is important to note in this regard that
the p50 homodimer does not bind the IL-8
B site as this study and
previous studies show (31, 34), and therefore the IL-8 promoter should
not be subjected to repression by this dimer. Furthermore, the
expression of the IL-8 gene has been shown to require not only the
NF-
B site in the promoter but also the adjacent C/EBP site. It is
likely that the synergism between the C/EBP site-binding protein
C/EBP
(NF-IL6) and the p65-c-Rel heterodimer, which is the major
IL-8
B binding activity (31, 34), is greater than that between
C/EBP
and the proteins constituting complex III, and therefore the
increased availability of the latter does not up-regulate IL-8 gene
expression.
The NF-B region of the RANTES promoter is important for its
induction in activated T lymphocytes (28). Our experiments indicate
that this region is also critical for RANTES promoter activation in
lung alveolar epithelial cells. However, T cells express the RANTES
gene only around 3-5 days after activation (28) in contrast to
detectable expression in lung epithelial cells ~20-24 h after
stimulation that can be shifted to as early as 2 h
post-stimulation by overexpression of I
BR. It appears that an
up-regulation of RANTES gene expression in lung epithelial cells
involves the formation of a unique NF-
B-Rel complex (complex III)
with the RANTES
B site. Our data suggest that overexpression of
I
BR sequesters (p50)2, which may facilitate the binding
of the unique complex to the RANTES
B region. This complex was not detected in EMSAs with nuclear extracts of activated T cells (28). Therefore, the expression of the RANTES gene might follow different kinetics through the involvement of different DNA-binding proteins. In
inflammatory disease conditions such as asthma and endotoxemia, where
an up-regulation of RANTES gene expression has been reported in lung
epithelial cells (20-22, 24), the expression of I
BR and formation
of complex III need to be investigated in the disease processes. The
present system will allow us to further characterize complex III, which
might provide a tool for the specific modulation of RANTES gene
expression in different diseases.
We thank A. M. Krensky for the kind gift of the RANTES reporter plasmids, J. A. Elias and N. H. Ruddle for critically reading the manuscript, and Z. Zhou for assistance with ELISAs.