From the Department of Biology, the
§ College of Pharmacy, and the ¶ Hormone Research
Center, Chonnam National University, Kwangju, 500-757 Korea, the
Department of Biochemistry, College of Medicine, University of
Ulsan, Seoul 138-040, Korea, ** Department of Microbiology, College
of Medicine, Yonsei University, Seoul 120-752, Korea, the
Department of Molecular Biology,
Massachusetts General Hospital, Boston, Massachusetts 02114
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ABSTRACT |
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To elucidate the molecular action of the NFB
inhibitor I
B
, we isolated a number of I
B
interactors using
the yeast two-hybrid system. These include the retinoid X receptor
(RXR), whose interaction with I
B
is significantly stimulated by
the RXR ligand 9-cis-retinoic acid, as shown in the yeast system as
well as the glutathione S-transferase pull down assays. RXR
is a nuclear protein, whereas I
B
accumulates in the nucleus only
in cells stimulated with lipopolysaccharide or other inducers that
result in prolonged activation of NF
B. Consistent with this,
cotransfection with I
B
specifically repressed the
9-cis-RA-induced transcriptional activities of RXR in an
lipopolysaccharide-dependent manner. These results suggest
a novel I
B
-mediated antagonism between the signaling pathways of
NF
B and RXR.
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INTRODUCTION |
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The transcription factor NFB is important for the inducible
expression of a wide variety of cellular and viral genes (1, 2). NF
B
is composed of homo- and heterodimeric complexes of members of the
Rel/NF
B family of polypeptides. In vertebrates, this family
comprises p50, p65 (RelA), c-Rel, p52, and RelB. These proteins share a
300-amino acid region, known as the Rel homology domain, which binds to
DNA and mediates homo- and heterodimerization. This domain also is the
target of the I
B
inhibitors, which include I
B
, I
B
,
I
B
, Bcl-3, p105, and p100 (3). In the majority of cells, NF
B
exists in an inactive form in the cytoplasm, bound to the inhibitory
I
B proteins. Treatment of cells with various inducers results in the
degradation of I
B proteins. The bound NF
B is released and
translocates to the nucleus, where it activates appropriate target
genes. I
B
is degraded in response to all of the known inducers of
NF
B, whereas I
B
is degraded only when cells are stimulated
with inducers such as lipopolysaccharide (LPS)1 and interleukin-1 that
cause persistent activation of NF
B (4). Following degradation of the
initial pool of I
B
in response to LPS or interleukin-1, newly
synthesized I
B
accumulates in the nucleus as an unphosphorylated
protein that forms a stable complex with NF
B and prevents it from
binding to newly synthesized I
B
(5-7).
To understand the molecular action of IB
, we exploited the yeast
two-hybrid system (8) to isolate a series of cDNAs encoding proteins that specifically interact with I
B
. Interestingly, these
include retinoid X receptor (RXR), a member of the nuclear hormone
receptors that comprise a large family of ligand-dependent transcription factors, bind as homodimers or heterodimers to their cognate DNA elements, and regulate genes involved in critical aspects
of cell proliferation, differentiation, and homeostasis (9). Herein, we
show that the RXR-I
B
interactions are stimulated by the RXR
ligand 9-cis-RA and that cotransfection with I
B
specifically represses the 9-cis-RA-induced transcriptional activities of RXR in an
LPS-dependent manner. These results are consistent with a
novel I
B
-mediated antagonism between the signaling pathways of
NF
B and RXR.
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EXPERIMENTAL PROCEDURES |
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Plasmids--
Polymerase chain reaction-amplified fragments
encoding IB
, Bcl-3, p50, and p65 were cloned into
EcoRI and SalI restriction sites of the LexA
fusion vector pEG202PL or EcoRI and XhoI
restriction sites of the B42 fusion vector pJG4-5 (8).
B42/I
B
1, I
B
2, and I
B
3 were previously
isolated as independent isolates of Trip9 in the yeast two-hybrid
screening as described (10). Plasmids encoding LexA fusions to
glucocorticoid receptor, RXR, TR, and TR459 as well as T7 vectors to
express RXR-LBD, TR-LBD, TR
, TR459 and TR-ABC are as described
(11-13). To express GST fusions, polymerase chain reaction-amplified
fragments encoding full-length I
B
and I
B
1 were cloned
into EcoRI and XhoI restriction sites of pGEX4T (Pharmacia Biotech Inc.). For mammalian expressions, full-length I
B
and I
B
were constructed into the CDM8 vector. The
expression vector for RXR, the reporter construct TREpal-CAT, and the
transfection indicator construct pRSV-
-gal are as described (12,
13). The expression vector for Gal4-VP16 and the reporter construct Gal4-TKLuc were as described (14).
Yeast Two-hybrid Screening of cDNA Library--
Candidate
IB
interacting clones were isolated from a mouse liver cDNA
library (13) using the yeast two-hybrid system as described (8), with
slight modifications. Approximately 106 primary yeast
transformants of a derivative of EGY48 expressing the LexA-I
B
1
were generated from an initial transformation with the cDNA library
with selection for Trp auxotrophy. Library transformed cells were
pooled and selected for Leu auxotrophy and expression of the
LexA-
-galactosidase construct. A number of leucine-independent
colonies harboring cDNAs encoding candidate LexA-I
B
1
interactors were obtained. The cDNA library plasmids were recovered
from appropriate yeast strains, propagated in Escherichia coli, and reintroduced into EGY48 derivatives expressing LexA alone, LexA-I
B
1, or other LexA chimeras to confirm specific interaction. Finally, identities of isolated cDNAs were determined by DNA sequencing.
Yeast Two-hybrid Test--
For the yeast two-hybrid tests,
plasmids encoding LexA fusions and B42 fusions were cotransformed into
Saccharomyces cerevisiae EGY48 strain containing the -gal
reporter plasmid, SH/18-34 (8). Plate and liquid assays of
-gal
expression were carried out as described (10, 13). Similar results were
obtained in more than two similar experiments.
GST Pull Down Assays-- The GST fusions or GST alone was expressed in E. coli, bound to glutathione-Sepharose-4B beads (Pharmacia), and incubated with labeled receptors or luciferase expressed by in vitro translation by using the TNT-coupled transcription-translation system, with conditions as described by the manufacturer (Promega, Madison, WI). Specifically bound proteins were eluted from beads with 40 mM reduced glutathione in 50 mM Tris (pH 8.0) and analyzed by SDS-polyacrylamide gel electorphoresis and autoradiography as described (12).
Cell Culture and Transfections--
CV1 cells were grown in
24-well plates with medium supplemented with 10% charcoal-stripped
serum for 24 h and transfected with 150 ng of -galactosidase
expression vector pRSV-
-gal and 100 ng of a reporter gene TREpal-CAT
along with 10 ng of RXR
and increasing amounts (10-200 ng) of
I
B
expression vectors. For control experiments, Gal4-TKLuc and
Gal4-VP16 (14) replaced TREpal-CAT and RXR, respectively. Total amounts
of expression vectors were kept constant by adding decreasing amounts
of the CDM8 expression vector to transfections containing increasing amounts of the I
B
vector. After 12 h, cells were washed and refed with Dulbecco's modified Eagle's medium containing 10%
charcoal-stripped fetal bovine serum. After 12 h, cells were left
unstimulated or stimulated with 2 µg/ml LPS either in the presence or
the absence of 10
7 M 9-cis-RA. Cells were
harvested 24 h later, and CAT or luciferase activity was assayed
as described (15), and the results were normalized to the
-galactosidase expression. Similar results were obtained in more
than two similar experiments.
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RESULTS AND DISCUSSION |
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Genetic Selection for IB
Interacting cDNA Clones in
Yeast--
The validity of the yeast two-hybrid system (8) in
isolating I
B
interactors was tested by examining interaction
properties of I
B
with various components of the NF
B complex in
yeast. As expected from the published results (1-4), I
B
1, a
N-terminal deletion mutant of I
B
(depicted in Fig.
1), interacted specifically with p65 but
not with p50. Similarly, p50 interacted with p65, Bcl-3, and p50,
whereas p65 did not interact with Bcl-3 or p65 (Table
I). In addition, Bcl-3 readily interacted
with I
B
and I
B
1.2 Full-length
I
B
was a transcriptional activator in yeast when fused to a
heterologous DNA binding domain, whereas I
B
1 was transcriptionally inert.2 Thus, we used I
B
1 as a
bait to isolate I
B
interactors in the yeast two-hybrid system.
One of the strongest I
B
interactors isolated from a mouse liver
cDNA library (13) encoded the RXR sequences from the third cysteine
residue of the first zinc finger motif within the DNA-binding domain to
the C termini. This result is consistent with our previous report in
which I
B
(initially referred to as Trip9) was first identified as
one of a series of TR or RXR interacting proteins (10). These results
immediately raised a possibility of a novel, I
B
-mediated
cross-talk between NF
B and nuclear receptor signaling pathways.
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Ligand-stimulated Interactions of IB
and RXR/TR--
The
interaction of I
B
with receptors was further characterized using
the yeast two-hybrid system and GST pull down assays. As shown in Table
II, the full-length I
B
and three
N-terminal deletion mutants (depicted in Fig. 1) corresponding to
independent isolates of Trip9 (10) all interacted strongly with TR or
RXR in a hormone-stimulated manner. In contrast, the related Bcl-3 protein interacted relatively weakly only with RXR in a
9-cis-RA-stimulated manner. Both I
B
and Bcl-3 failed to interact
with glucocorticoid receptor. As shown in Fig. 1, I
B
contains six
ankyrin repeats that constitute the interaction interface with the Rel
homology domain of NF
B, whereas Bcl-3 contains seven ankyrin
repeats. Among these, ankyrin repeats 1, 5, and 6 of I
B
and
ankyrin repeat 3 of Bcl-3 contain a single amino acid motif
LXXLL, recently shown to be an interaction interface for the
nuclear hormone receptors (16, 17). This motif apparently mediates the
ligand-dependent interaction of the AF-2 transactivation
domain of the receptors with transcription cofactors such as RIP-140,
SRC-1, and CBP (16, 17). Consistent with this, all of the N-terminal
deletion mutants of I
B
capable of interacting with the receptors
retain the last two LXXLL motifs (Fig. 1). Thus, we tested
whether I
B
and the N-terminal deletion mutants interact with
LexA/TR459, a mutant TR-LBD that lacks AF-2 function but retains wild
type affinity for thyroid hormone (T3) (10, 11). This mutation blocked
interaction with all of the I
B
proteins, suggesting that the
interaction interface may involve the AF-2 domain of nuclear receptors
and the LXXLL motifs in the ankyrin repeats of I
B
.
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Cotransfections of IB
Repress the 9-cis-RA-induced
Transcriptional Activities of RXR in an LPS-dependent
Manner--
To assess the functional consequences of these
interactions, I
B
was cotransfected into CV1 cells along with an
RXR expression vector and a reporter construct controlled by TREpal,
which is transactivated by RXR-RXR homodimers as well as various
receptor heterodimers (18). Increasing amounts of cotransfected
I
B
had no significant effect on transcriptional activities of the TREpal-driven reporter, either in the presence or the absence of
9-cis-RA (Fig. 3). Similarly, RXR did not
affect the ability of either I
B
or I
B
to inhibit
transactivation by p65.3
However, addition of 2 µg/ml LPS specifically inhibited
9-cis-RA-induced transcription in an I
B
dose-dependent manner,
with cotransfection of 200 ng of I
B
decreasing transcriptional
activities to near background levels (Fig. 3). In contrast,
cotransfection of I
B
did not affect the transcriptional activity
of Gal4-VP16, either in the presence or the absence of LPS, as assessed
using the Gal4-TKLuc reporter construct (14) (data not shown).
Similarly, I
B
did not significantly affect
-galactosidase
expression of the transfection indicator construct pRSV-
-gal in the
presence or the absence of LPS or 9-cis-RA (data not shown). These
results are consistent with the proposal that I
B
translocation
into the nucleus is dependent on stimulation by chronic inducers such
as LPS (5-7) and suggests that only this nuclear I
B
is capable
of interacting with nuclear receptors. These results along with the
yeast and in vitro interaction results suggest that this
nuclear I
B
may mask the AF-2 domain of nuclear receptors from
interacting with receptor coactivators. Alternatively, I
B
bound
to the AF-2 domain may have more direct inhibitory interactions with
the transcriptional machinery. Consistent with this, LexA/I
B
was
a transcriptional activator in yeast, whereas LexA/I
B
1 was
not, suggesting the existence of an autonomous transactivation domain
at the N terminus of I
B
(amino acids 1-173).2 In
addition, full-length I
B
also showed specific binding to a novel
transcription cofactor we recently
isolated.4
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ACKNOWLEDGEMENT |
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We thank Dr. Ron Evans for Gal4-VP16 and Gal4-TKLuc.
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FOOTNOTES |
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* This work was supported by Ministry of Education, Korea Grant GE 97-113 (to M.-O. L.), Ministry of Science and Technology, Korea Grant 08-01-13 (to D.-S. N. and J. W. L.), Korea Scence and Engineering Foundation Grant HRC (to J. W. L. and H.-S. C.), and National Institutes of Health Grant R01 DK43382 (to D. D. M.)The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§§ Present address; Dept. of Gerontology, Beth Israel Hospital, Boston, MA 02115.
¶¶ Present address; Dept. of Cell Biology, Baylor College of Medicine, Houston, TX 77030.
|| To whom correspondence should be addressed. Tel.: 82-62-520-7423; Fax: 82-62-522-5654; E-mail: jlee{at}chonnam.chonnam.ac.kr.
1
The abbreviations used are: LPS,
lipopolysaccharide; RXR, retinoid X receptor; TR, thyroid hormone
receptor; GST, glutathione S-transferase; -gal,
-galactosidase; CAT, chloramphenical acetyltransferase; RA, retinoic
acid.
2 S. Y. Na and J. W. Lee, unpublished observations.
3 M. Chung and D. D. Moore, unpublished observations.
4 H. J. Kim, S. K. Lee, S. Y. Na, H. S. Choi, and J. W. Lee, manuscript in preparation.
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REFERENCES |
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