Ikappa Bbeta Interacts with the Retinoid X Receptor and Inhibits Retinoid-dependent Transactivation in Lipopolysaccharide-treated Cells*

Soon-Young NaDagger , Han-Jong Kim§, Soo-Kyung Lee§, Hueng-Sik Choi, Doe-Sun Napar , Mi-Ock Lee**, Mirra ChungDagger Dagger §§, David D. MooreDagger Dagger ¶¶, and Jae Woon Lee§||

From the Dagger  Department of Biology, the § College of Pharmacy, and the  Hormone Research Center, Chonnam National University, Kwangju, 500-757 Korea, the par  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 Dagger Dagger  Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts 02114

    ABSTRACT
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Procedures
Results & Discussion
References

To elucidate the molecular action of the NFkappa B inhibitor Ikappa Bbeta , we isolated a number of Ikappa Bbeta interactors using the yeast two-hybrid system. These include the retinoid X receptor (RXR), whose interaction with Ikappa Bbeta 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 Ikappa Bbeta accumulates in the nucleus only in cells stimulated with lipopolysaccharide or other inducers that result in prolonged activation of NFkappa B. Consistent with this, cotransfection with Ikappa Bbeta specifically repressed the 9-cis-RA-induced transcriptional activities of RXR in an lipopolysaccharide-dependent manner. These results suggest a novel Ikappa Bbeta -mediated antagonism between the signaling pathways of NFkappa B and RXR.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results & Discussion
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The transcription factor NFkappa B is important for the inducible expression of a wide variety of cellular and viral genes (1, 2). NFkappa B is composed of homo- and heterodimeric complexes of members of the Rel/NFkappa 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 Ikappa Bbeta inhibitors, which include Ikappa Balpha , Ikappa Bbeta , Ikappa Bgamma , Bcl-3, p105, and p100 (3). In the majority of cells, NFkappa B exists in an inactive form in the cytoplasm, bound to the inhibitory Ikappa B proteins. Treatment of cells with various inducers results in the degradation of Ikappa B proteins. The bound NFkappa B is released and translocates to the nucleus, where it activates appropriate target genes. Ikappa Balpha is degraded in response to all of the known inducers of NFkappa B, whereas Ikappa Bbeta is degraded only when cells are stimulated with inducers such as lipopolysaccharide (LPS)1 and interleukin-1 that cause persistent activation of NFkappa B (4). Following degradation of the initial pool of Ikappa Bbeta in response to LPS or interleukin-1, newly synthesized Ikappa Bbeta accumulates in the nucleus as an unphosphorylated protein that forms a stable complex with NFkappa B and prevents it from binding to newly synthesized Ikappa Balpha (5-7).

To understand the molecular action of Ikappa Bbeta , we exploited the yeast two-hybrid system (8) to isolate a series of cDNAs encoding proteins that specifically interact with Ikappa Bbeta . 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-Ikappa Bbeta interactions are stimulated by the RXR ligand 9-cis-RA and that cotransfection with Ikappa Bbeta specifically represses the 9-cis-RA-induced transcriptional activities of RXR in an LPS-dependent manner. These results are consistent with a novel Ikappa Bbeta -mediated antagonism between the signaling pathways of NFkappa B and RXR.

    EXPERIMENTAL PROCEDURES
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Plasmids-- Polymerase chain reaction-amplified fragments encoding Ikappa Bbeta , 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/Ikappa Bbeta Delta 1, Ikappa Bbeta Delta 2, and Ikappa Bbeta Delta 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, TRDelta , TR459 and TR-ABC are as described (11-13). To express GST fusions, polymerase chain reaction-amplified fragments encoding full-length Ikappa Bbeta and Ikappa Bbeta Delta 1 were cloned into EcoRI and XhoI restriction sites of pGEX4T (Pharmacia Biotech Inc.). For mammalian expressions, full-length Ikappa Balpha and Ikappa Bbeta were constructed into the CDM8 vector. The expression vector for RXR, the reporter construct TREpal-CAT, and the transfection indicator construct pRSV-beta -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 Ikappa Bbeta 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-Ikappa Bbeta Delta 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-beta -galactosidase construct. A number of leucine-independent colonies harboring cDNAs encoding candidate LexA-Ikappa Bbeta Delta 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-Ikappa Bbeta Delta 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 beta -gal reporter plasmid, SH/18-34 (8). Plate and liquid assays of beta -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 beta -galactosidase expression vector pRSV-beta -gal and 100 ng of a reporter gene TREpal-CAT along with 10 ng of RXRalpha and increasing amounts (10-200 ng) of Ikappa Bbeta 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 Ikappa Bbeta 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 beta -galactosidase expression. Similar results were obtained in more than two similar experiments.

    RESULTS AND DISCUSSION
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Abstract
Introduction
Procedures
Results & Discussion
References

Genetic Selection for Ikappa Bbeta Interacting cDNA Clones in Yeast-- The validity of the yeast two-hybrid system (8) in isolating Ikappa Bbeta interactors was tested by examining interaction properties of Ikappa Bbeta with various components of the NFkappa B complex in yeast. As expected from the published results (1-4), Ikappa Bbeta Delta 1, a N-terminal deletion mutant of Ikappa Bbeta (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 Ikappa Bbeta and Ikappa Bbeta Delta 1.2 Full-length Ikappa Bbeta was a transcriptional activator in yeast when fused to a heterologous DNA binding domain, whereas Ikappa Bbeta Delta 1 was transcriptionally inert.2 Thus, we used Ikappa Bbeta Delta 1 as a bait to isolate Ikappa Bbeta interactors in the yeast two-hybrid system. One of the strongest Ikappa Bbeta 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 Ikappa Bbeta (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, Ikappa Bbeta -mediated cross-talk between NFkappa B and nuclear receptor signaling pathways.


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Fig. 1.   Schematic representation of chimeras consisting of the transactivation domain B42 fused to full-length Bcl-3, full-length Ikappa Bbeta , or N-terminal deletion mutants of Ikappa Bbeta . Ankyrin repeats of each protein are stippled, and amino acid motifs of LXXLL (16, 17) are indicated as arrows. Numbers represent amino acids of Bcl-3 or Ikappa Bbeta included in each chimera.

                              
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Table I
Interactions between various components of the NFkappa B complex in yeast
The indicated B42 and LexA plasmids were transformed into a yeast strain containing an appropriate beta -galactosidase reporter gene. At least six separate transformants from each transformation were transferred to indicator plates containing 5-bromo-4-chloro-3-indolyl beta -D-galactopyranoside, and reproducible results were obtained using colonies from a separate transformation. W, white colonies (no interaction); B, blue colonies after 1 day (strong interaction); LB, blue colonies after 3 days (weak interaction).

Ligand-stimulated Interactions of Ikappa Bbeta and RXR/TR-- The interaction of Ikappa Bbeta with receptors was further characterized using the yeast two-hybrid system and GST pull down assays. As shown in Table II, the full-length Ikappa Bbeta 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 Ikappa Bbeta and Bcl-3 failed to interact with glucocorticoid receptor. As shown in Fig. 1, Ikappa Bbeta contains six ankyrin repeats that constitute the interaction interface with the Rel homology domain of NFkappa B, whereas Bcl-3 contains seven ankyrin repeats. Among these, ankyrin repeats 1, 5, and 6 of Ikappa Bbeta 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 Ikappa Bbeta capable of interacting with the receptors retain the last two LXXLL motifs (Fig. 1). Thus, we tested whether Ikappa Bbeta 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 Ikappa Bbeta proteins, suggesting that the interaction interface may involve the AF-2 domain of nuclear receptors and the LXXLL motifs in the ankyrin repeats of Ikappa Bbeta .

                              
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Table II
Ikappa Bbeta Interacts with RXR and TR in Yeast
The indicated B42 and LexA plasmids were transformed into a yeast strain containing an appropriate beta -galactosidase reporter gene. The LexA fusions to GR, RXR, TR, and TR459 include sequences from the C terminus of the DNA-binding domain of the various receptors to their C termini as described (10, 11, 13). The respective ligands for GR, RXR, and TR were added to the plates as described (10). At least six separate transformants from each transformation were transferred to indicator plates containing 5-bromo-4-chloro-3-indolyl beta -D-galactopyranoside, and reproducible results were obtained using colonies from a separate transformation. W, white colonies (no interaction); B, blue colonies after 1 day (strong interaction); LB, blue colonies after 3 days (weak interaction).

To further characterize these interactions in vitro, GST fusions to Ikappa Bbeta and Ikappa Bbeta Delta 1 were expressed, purified, and tested for interaction with various in vitro translated receptor constructs. These include the ligand binding domains of RXR and TR (RXR-LBD and TR-LBD), the ABC domains of TR (TR-ABC), and full-length TRs deleted or point-mutated for the AF2 domain (TRDelta and TR459, respectively). As shown in Fig. 2, Ikappa Bbeta and Ikappa Bbeta Delta 1 interacted weakly with TR-LBD and RXR-LBD in the absence of ligand. In agreement with the yeast results, however, interactions of Ikappa Bbeta with TR-LBD or RXR-LBD were significantly enhanced in the presence of each cognate ligand. Ikappa Bbeta Delta 1 behaved similarly. In contrast, the AF2 mutants TRDelta and TR459 as well as TR-ABC showed relatively weak and hormone-independent interaction with Ikappa Bbeta and Ikappa Bbeta Delta 1. Thus, these results confirm the importance of the AF-2 for the ligand-dependent interactions and also suggest an additional ligand-independent interaction interface at the N-terminal ABC domains.


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Fig. 2.   GST pull down assays. RXR-LBD, TR-LBD, TRDelta , TR-ABC, TR459, and luciferase labeled with [35S]methionine by in vitro translation were incubated with glutathione beads containing GST alone, GST fusion to Ikappa Bbeta , or GST fusion to Ikappa Bbeta Delta 1. Results with TR459 and TR-ABC were overexposed relative to others. Under similar conditions, the band intensities of TRDelta , TR459, and TR-ABC are comparable with each other. (-) indicates no hormone added, and (+) indicates addition of 10-7 M 9-cis-RA or T3. For TR-ABC, interactions only in the absence of hormone were executed.

Cotransfections of Ikappa Bbeta Repress the 9-cis-RA-induced Transcriptional Activities of RXR in an LPS-dependent Manner-- To assess the functional consequences of these interactions, Ikappa Bbeta 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 Ikappa Bbeta 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 Ikappa Bbeta or Ikappa Balpha to inhibit transactivation by p65.3 However, addition of 2 µg/ml LPS specifically inhibited 9-cis-RA-induced transcription in an Ikappa Bbeta dose-dependent manner, with cotransfection of 200 ng of Ikappa Bbeta decreasing transcriptional activities to near background levels (Fig. 3). In contrast, cotransfection of Ikappa Bbeta 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, Ikappa Bbeta did not significantly affect beta -galactosidase expression of the transfection indicator construct pRSV-beta -gal in the presence or the absence of LPS or 9-cis-RA (data not shown). These results are consistent with the proposal that Ikappa Bbeta translocation into the nucleus is dependent on stimulation by chronic inducers such as LPS (5-7) and suggests that only this nuclear Ikappa Bbeta is capable of interacting with nuclear receptors. These results along with the yeast and in vitro interaction results suggest that this nuclear Ikappa Bbeta may mask the AF-2 domain of nuclear receptors from interacting with receptor coactivators. Alternatively, Ikappa Bbeta bound to the AF-2 domain may have more direct inhibitory interactions with the transcriptional machinery. Consistent with this, LexA/Ikappa Bbeta was a transcriptional activator in yeast, whereas LexA/Ikappa Bbeta Delta 1 was not, suggesting the existence of an autonomous transactivation domain at the N terminus of Ikappa Bbeta (amino acids 1-173).2 In addition, full-length Ikappa Bbeta also showed specific binding to a novel transcription cofactor we recently isolated.4


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Fig. 3.   Effects of Ikappa Bbeta cotransfection on the transcriptional activities of RXR. CV1 cells were transfected with beta -galactosidase expression vector and increasing amounts of Ikappa Bbeta expression vectors along with a reporter gene TREpal-CAT and RXRalpha , as indicated. Cells were left unstimulated (A) or stimulated with 2 µg/ml LPS (B). Open boxes indicate no hormone added; closed boxes indicate 9-cis-RA added in the transfection media. Normalized CAT expressions from triplicate samples is presented relative to the transactivation observed in the presence of 9-cis-RA without Ikappa Bbeta added.

In conclusion, we have identified RXR as an Ikappa Bbeta interactor and shown that this interaction decreases RXR-driven transcriptional activities in an LPS-dependent manner. This antagonism is in marked contrast to the glucocorticoid-dependent inhibition of NFkappa B activities, in which glucocorticoids increase the synthesis of Ikappa Balpha , which should then sequester NFkappa B in an inactive cytoplasmic form (19). However, it was recently suggested that glucocorticoid-induced Ikappa Balpha synthesis and inhibition of NFkappa B activity are two separable biochemical processes (20). Accordingly, glucocorticoid-mediated inhibition of NFkappa B activity may involve other mechanisms such as the Ikappa Bbeta -RXR interactions described here. The antagonistic interaction is consistent with the fact that LPS is one of the best known pro-inflammatory agents (21), whereas retinoids are anti-inflammatory (22-24). Thus, exploration of these interactions may lead to new insights into mechanisms of inflammatory signal transduction pathways and possibly the development of new anti-inflammatory agents.

    ACKNOWLEDGEMENT

We thank Dr. Ron Evans for Gal4-VP16 and Gal4-TKLuc.

    FOOTNOTES

* 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; beta -gal, beta -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.

    REFERENCES
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Abstract
Introduction
Procedures
Results & Discussion
References

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